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Sample records for preq1 riboswitch aptamer

  1. All-atom molecular dynamics insights on preQ1 riboswitch aptamer

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    Gong, Zhou; Zhao, Yunjie; Chen, Changjun; Xiao, Yi

    2012-02-01

    Recently, a series of experiments have focused on two types of preQ1 riboswitch with known smallest aptamer. One of them is from Bacillus subtilis, which have been discussed before. The other one comes from T. tengcongensis, and Jenkins et al recently release its crystal structure in both ligand-bound and free state. These two types of riboswitch aptamer have similar structures but totally different functions. Consequently, contrast studies of these two preQ1 riboswitches will help us to understand the regulation function of riboswitch better. Here, we study the dynamical properties of two types of preQ1 riboswitches using molecular dynamics simulation. We find that the unfolding pathway of the two preQ1 aptamer domains in bound state are both hierarchical and have an intermediate state. We believe that such conformation would be a good candidate structure for ligand binding. On the other hand, in the absent of ligand, the preQ1 riboswitch from Bacillus subtilis can only form the stable state with P1-L3 triplex, while the preQ1 riboswitch from T. tengcongensis can form the conformation with pseudoknot shape. We suggest that such intermediate structures may perform regulation functions in the absent of ligand.

  2. Insights into ligand binding to PreQ1 Riboswitch Aptamer from molecular dynamics simulations.

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    Zhou Gong

    Full Text Available Riboswitches play roles in transcriptional or translational regulation through specific ligand binding of their aptamer domains. Although a number of ligand-bound aptamer complex structures have been solved, it is important to know ligand-free conformations of the aptamers in order to understand the mechanism of specific binding by ligands. In this paper, preQ1 riboswitch aptamer domain from Bacillus subtilis is studied by overall 1.5 μs all-atom molecular dynamics simulations We found that the ligand-free aptamer has a stable state with a folded P1-L3 and open binding pocket. The latter forms a cytosine-rich pool in which the nucleotide C19 oscillates between close and open positions, making it a potential conformation for preQ1 entrance. The dynamic picture further suggests that the specific recognition of preQ1 by the aptamer domain is not only facilitated by the key nucleotide C19 but also aided and enhanced by other cytosines around the binding pocket. These results should help to understand the details of preQ1 binding.

  3. Comparison of a preQ1 riboswitch aptamer in metabolite-bound and free states with implications for gene regulation.

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    Jenkins, Jermaine L; Krucinska, Jolanta; McCarty, Reid M; Bandarian, Vahe; Wedekind, Joseph E

    2011-07-15

    Riboswitches are RNA regulatory elements that govern gene expression by recognition of small molecule ligands via a high affinity aptamer domain. Molecular recognition can lead to active or attenuated gene expression states by controlling accessibility to mRNA signals necessary for transcription or translation. Key areas of inquiry focus on how an aptamer attains specificity for its effector, the extent to which the aptamer folds prior to encountering its ligand, and how ligand binding alters expression signal accessibility. Here we present crystal structures of the preQ(1) riboswitch from Thermoanaerobacter tengcongensis in the preQ(1)-bound and free states. Although the mode of preQ(1) recognition is similar to that observed for preQ(0), surface plasmon resonance revealed an apparent K(D) of 2.1 ± 0.3 nm for preQ(1) but a value of 35.1 ± 6.1 nm for preQ(0). This difference can be accounted for by interactions between the preQ(1) methylamine and base G5 of the aptamer. To explore conformational states in the absence of metabolite, the free-state aptamer structure was determined. A14 from the ceiling of the ligand pocket shifts into the preQ(1)-binding site, resulting in "closed" access to the metabolite while simultaneously increasing exposure of the ribosome-binding site. Solution scattering data suggest that the free-state aptamer is compact, but the "closed" free-state crystal structure is inadequate to describe the solution scattering data. These observations are distinct from transcriptional preQ(1) riboswitches of the same class that exhibit strictly ligand-dependent folding. Implications for gene regulation are discussed.

  4. Structural analysis of a class III preQ1 riboswitch reveals an aptamer distant from a ribosome-binding site regulated by fast dynamics.

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    Liberman, Joseph A; Suddala, Krishna C; Aytenfisu, Asaminew; Chan, Dalen; Belashov, Ivan A; Salim, Mohammad; Mathews, David H; Spitale, Robert C; Walter, Nils G; Wedekind, Joseph E

    2015-07-07

    PreQ1-III riboswitches are newly identified RNA elements that control bacterial genes in response to preQ1 (7-aminomethyl-7-deazaguanine), a precursor to the essential hypermodified tRNA base queuosine. Although numerous riboswitches fold as H-type or HLout-type pseudoknots that integrate ligand-binding and regulatory sequences within a single folded domain, the preQ1-III riboswitch aptamer forms a HLout-type pseudoknot that does not appear to incorporate its ribosome-binding site (RBS). To understand how this unusual organization confers function, we determined the crystal structure of the class III preQ1 riboswitch from Faecalibacterium prausnitzii at 2.75 Å resolution. PreQ1 binds tightly (KD,app 6.5 ± 0.5 nM) between helices P1 and P2 of a three-way helical junction wherein the third helix, P4, projects orthogonally from the ligand-binding pocket, exposing its stem-loop to base pair with the 3' RBS. Biochemical analysis, computational modeling, and single-molecule FRET imaging demonstrated that preQ1 enhances P4 reorientation toward P1-P2, promoting a partially nested, H-type pseudoknot in which the RBS undergoes rapid docking (kdock ∼ 0.6 s(-1)) and undocking (kundock ∼ 1.1 s(-1)). Discovery of such dynamic conformational switching provides insight into how a riboswitch with bipartite architecture uses dynamics to modulate expression platform accessibility, thus expanding the known repertoire of gene control strategies used by regulatory RNAs.

  5. Computational study of unfolding and regulation mechanism of preQ1 riboswitches.

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    Zhou Gong

    Full Text Available Riboswitches are novel RNA regulatory elements. Each riboswitch molecule consists of two domains: aptamer and express platform. The three-dimensional (3D structure of the aptamer domain, depending on ligand binding or not, controls that of the express platform, which then switches on or off transcriptional or translational process. Here we study the two types of preQ(1 riboswitch aptamers from T. Tengcongensis (denoted as Tte preQ(1 riboswitch for short below and Bacillus subtilis (denoted as Bsu preQ(1 riboswitch for short below, respectively. The free-state 3D structure of the Tte preQ(1 riboswitch is the same as its bound state but the Bsu preQ(1 riboswitch is not. Therefore, it is very interesting to investigate how these riboswitches realize their different regulation functions. We simulated the unfolding of these two aptamers through all-atom molecular dynamic simulation and found that they have similar unfolding or folding pathways and ligand-binding processes. The main difference between them is the folding intermediate states. The similarity and difference of their unfolding or folding dynamics may suggest their similar regulation mechanisms and account for their different functions, respectively. These results are also useful to understand the regulation mechanism of other riboswitches with free-state 3D structures similar to their bound states.

  6. Structural determinants for ligand capture by a class II preQ1 riboswitch.

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    Kang, Mijeong; Eichhorn, Catherine D; Feigon, Juli

    2014-02-11

    Prequeuosine (preQ1) riboswitches are RNA regulatory elements located in the 5' UTR of genes involved in the biosynthesis and transport of preQ1, a precursor of the modified base queuosine universally found in four tRNAs. The preQ1 class II (preQ1-II) riboswitch regulates preQ1 biosynthesis at the translational level. We present the solution NMR structure and conformational dynamics of the 59 nucleotide Streptococcus pneumoniae preQ1-II riboswitch bound to preQ1. Unlike in the preQ1 class I (preQ1-I) riboswitch, divalent cations are required for high-affinity binding. The solution structure is an unusual H-type pseudoknot featuring a P4 hairpin embedded in loop 3, which forms a three-way junction with the other two stems. (13)C relaxation and residual dipolar coupling experiments revealed interhelical flexibility of P4. We found that the P4 helix and flanking adenine residues play crucial and unexpected roles in controlling pseudoknot formation and, in turn, sequestering the Shine-Dalgarno sequence. Aided by divalent cations, P4 is poised to act as a "screw cap" on preQ1 recognition to block ligand exit and stabilize the binding pocket. Comparison of preQ1-I and preQ1-II riboswitch structures reveals that whereas both form H-type pseudoknots and recognize preQ1 using one A, C, or U nucleotide from each of three loops, these nucleotides interact with preQ1 differently, with preQ1 inserting into different grooves. Our studies show that the preQ1-II riboswitch uses an unusual mechanism to harness exquisite control over queuosine metabolism.

  7. Structural, functional, and taxonomic diversity of three preQ1 riboswitch classes.

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    McCown, Phillip J; Liang, Jonathan J; Weinberg, Zasha; Breaker, Ronald R

    2014-07-17

    Previously, two riboswitch classes have been identified that sense and respond to the hypermodified nucleobase called prequeuosine1 (preQ1). The enormous expansion of available genomic DNA sequence data creates new opportunities to identify additional representatives of the known riboswitch classes and to discover novel classes. We conducted bioinformatics searches on microbial genomic DNA data sets to discover numerous additional examples belonging to the two previously known riboswitch classes for preQ1 (classes preQ1-I and preQ1-II), including some structural variants that further restrict ligand specificity. Additionally, we discovered a third preQ1-binding riboswitch class (preQ1-III) that is structurally distinct from previously known classes. These findings demonstrate that numerous organisms monitor the concentrations of this modified nucleobase by exploiting one or more riboswitch classes for this widespread compound.

  8. Structure of a class II preQ1 riboswitch reveals ligand recognition by a new fold.

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    Liberman, Joseph A; Salim, Mohammad; Krucinska, Jolanta; Wedekind, Joseph E

    2013-06-01

    PreQ1 riboswitches regulate genes by binding the pyrrolopyrimidine intermediate preQ1 during the biosynthesis of the essential tRNA base queuosine. We report what is to our knowledge the first preQ1-II riboswitch structure at 2.3-Å resolution, which uses a previously uncharacterized fold to achieve effector recognition at the confluence of a three-way helical junction flanking a pseudoknotted ribosome-binding site. The results account for translational control mediated by the preQ1-II riboswitch class and expand the known repertoire of ligand-binding modes used by regulatory RNAs.

  9. Dynamics Correlation Network for Allosteric Switching of PreQ1 Riboswitch

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    Wang, Wei; Jiang, Cheng; Zhang, Jinmai; Ye, Wei; Luo, Ray; Chen, Hai-Feng

    2016-01-01

    Riboswitches are a class of metabolism control elements mostly found in bacteria. Due to their fundamental importance in bacteria gene regulation, riboswitches have been proposed as antibacterial drug targets. Prequeuosine (preQ1) is the last free precursor in the biosynthetic pathway of queuosine that is crucial for translation efficiency and fidelity. However, the regulation mechanism for the preQ1 riboswitch remains unclear. Here we constructed fluctuation correlation network based on all-atom molecular dynamics simulations to reveal the regulation mechanism. The results suggest that the correlation network in the bound riboswitch is distinctly different from that in the apo riboswitch. The community network indicates that the information freely transfers from the binding site of preQ1 to the expression platform of the P3 helix in the bound riboswitch and the P3 helix is a bottleneck in the apo riboswitch. Thus, a hypothesis of “preQ1-binding induced allosteric switching” is proposed to link riboswitch and translation regulation. The community networks of mutants support this hypothesis. Finally, a possible allosteric pathway of A50-A51-A52-U10-A11-G12-G56 was also identified based on the shortest path algorithm and confirmed by mutations and network perturbation. The novel fluctuation network analysis method can be used as a general strategy in studies of riboswitch structure-function relationship. PMID:27484311

  10. Single transcriptional and translational preQ1 riboswitches adopt similar pre-folded ensembles that follow distinct folding pathways into the same ligand-bound structure

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    Suddala, Krishna C.; Rinaldi, Arlie J.; Feng, Jun; Mustoe, Anthony M.; Eichhorn, Catherine D.; Liberman, Joseph A.; Wedekind, Joseph E.; Al-Hashimi, Hashim M.; Brooks, Charles L.; Walter, Nils G.

    2013-01-01

    Riboswitches are structural elements in the 5′ untranslated regions of many bacterial messenger RNAs that regulate gene expression in response to changing metabolite concentrations by inhibition of either transcription or translation initiation. The preQ1 (7-aminomethyl-7-deazaguanine) riboswitch family comprises some of the smallest metabolite sensing RNAs found in nature. Once ligand-bound, the transcriptional Bacillus subtilis and translational Thermoanaerobacter tengcongensis preQ1 riboswitch aptamers are structurally similar RNA pseudoknots; yet, prior structural studies have characterized their ligand-free conformations as largely unfolded and folded, respectively. In contrast, through single molecule observation, we now show that, at near-physiological Mg2+ concentration and pH, both ligand-free aptamers adopt similar pre-folded state ensembles that differ in their ligand-mediated folding. Structure-based Gō-model simulations of the two aptamers suggest that the ligand binds late (Bacillus subtilis) and early (Thermoanaerobacter tengcongensis) relative to pseudoknot folding, leading to the proposal that the principal distinction between the two riboswitches lies in their relative tendencies to fold via mechanisms of conformational selection and induced fit, respectively. These mechanistic insights are put to the test by rationally designing a single nucleotide swap distal from the ligand binding pocket that we find to predictably control the aptamers′ pre-folded states and their ligand binding affinities. PMID:24003028

  11. Rational Re-engineering of a Transcriptional Silencing PreQ1 Riboswitch.

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    Wu, Ming-Cheng; Lowe, Phillip T; Robinson, Christopher J; Vincent, Helen A; Dixon, Neil; Leigh, James; Micklefield, Jason

    2015-07-22

    Re-engineered riboswitches that no longer respond to cellular metabolites, but that instead can be controlled by synthetic molecules, are potentially useful gene regulatory tools for use in synthetic biology and biotechnology fields. Previously, extensive genetic selection and screening approaches were employed to re-engineer a natural adenine riboswitch to create orthogonal ON-switches, enabling translational control of target gene expression in response to synthetic ligands. Here, we describe how a rational targeted approach was used to re-engineer the PreQ1 riboswitch from Bacillus subtilis into an orthogonal OFF-switch. In this case, the evaluation of just six synthetic compounds with seven riboswitch mutants led to the identification of an orthogonal riboswitch-ligand pairing that effectively repressed the transcription of selected genes in B. subtilis. The streamlining of the re-engineering approach, and its extension to a second class of riboswitches, provides a methodological platform for the creation of new orthogonal regulatory components for biotechnological applications including gene functional analysis and antimicrobial target validation and screening.

  12. Urea Induced Denaturation of Pre-Q1 Riboswitch

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    Yoon, Jeseong; Thirumalai, Devarajan; Hyeon, Changbong

    2013-01-01

    Urea, a polar molecule with a large dipole moment, not only destabilizes the folded RNA structures, but can also enhance the folding rates of large ribozymes. Unlike the mechanism of urea-induced unfolding of proteins, which is well understood, the action of urea on RNA has barely been explored. We performed extensive all atom molecular dynamics (MD) simulations to determine the molecular underpinnings of urea-induced RNA denaturation. Urea displays its denaturing power in both secondary and tertiary motifs of the riboswitch (RS) structure. Our simulations reveal that the denaturation of RNA structures is mainly driven by the hydrogen bonds and stacking interactions of urea with the bases. Through detailed studies of the simulation trajectories, we found that geminate pairs between urea and bases due to hydrogen bonds and stacks persist only ~ (0.1-1) ns, which suggests that urea-base interaction is highly dynamic. Most importantly, the early stage of base pair disruption is triggered by penetration of water molecules into the hydrophobic domain between the RNA bases. The infiltration of water into the narrow space between base pairs is critical in increasing the accessibility of urea to transiently disrupted bases, thus allowing urea to displace inter base hydrogen bonds. This mechanism, water-induced disruption of base-pairs resulting in the formation of a "wet" destabilized RNA followed by solvation by urea, is the exact opposite of the two-stage denaturation of proteins by urea. In the latter case, initial urea penetration creates a dry-globule, which is subsequently solvated by water penetration leading to global protein unfolding. Our work shows that the ability to interact with both water and polar, non-polar components of nucleotides makes urea a powerful chemical denaturant for nucleic acids.

  13. Molecular mechanism for preQ1-II riboswitch function revealed by molecular dynamics

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    Aytenfisu, Asaminew H.; Liberman, Joseph A.; Wedekind, Joseph E.; Mathews, David H.

    2015-01-01

    Riboswitches are RNA molecules that regulate gene expression using conformational change, affected by binding of small molecule ligands. A crystal structure of a ligand-bound class II preQ1 riboswitch has been determined in a previous structural study. To gain insight into the dynamics of this riboswitch in solution, eight total molecular dynamic simulations, four with and four without ligand, were performed using the Amber force field. In the presence of ligand, all four of the simulations demonstrated rearranged base pairs at the 3′ end, consistent with expected base-pairing from comparative sequence analysis in a prior bioinformatic analysis; this suggests the pairing in this region was altered by crystallization. Additionally, in the absence of ligand, three of the simulations demonstrated similar changes in base-pairing at the ligand binding site. Significantly, although most of the riboswitch architecture remained intact in the respective trajectories, the P3 stem was destabilized in the ligand-free simulations in a way that exposed the Shine–Dalgarno sequence. This work illustrates how destabilization of two major groove base triples can influence a nearby H-type pseudoknot and provides a mechanism for control of gene expression by a fold that is frequently found in bacterial riboswitches. PMID:26370581

  14. Molecular mechanism for preQ1-II riboswitch function revealed by molecular dynamics.

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    Aytenfisu, Asaminew H; Liberman, Joseph A; Wedekind, Joseph E; Mathews, David H

    2015-11-01

    Riboswitches are RNA molecules that regulate gene expression using conformational change, affected by binding of small molecule ligands. A crystal structure of a ligand-bound class II preQ1 riboswitch has been determined in a previous structural study. To gain insight into the dynamics of this riboswitch in solution, eight total molecular dynamic simulations, four with and four without ligand, were performed using the Amber force field. In the presence of ligand, all four of the simulations demonstrated rearranged base pairs at the 3' end, consistent with expected base-pairing from comparative sequence analysis in a prior bioinformatic analysis; this suggests the pairing in this region was altered by crystallization. Additionally, in the absence of ligand, three of the simulations demonstrated similar changes in base-pairing at the ligand binding site. Significantly, although most of the riboswitch architecture remained intact in the respective trajectories, the P3 stem was destabilized in the ligand-free simulations in a way that exposed the Shine-Dalgarno sequence. This work illustrates how destabilization of two major groove base triples can influence a nearby H-type pseudoknot and provides a mechanism for control of gene expression by a fold that is frequently found in bacterial riboswitches.

  15. The structural basis for recognition of the PreQ0 metabolite by an unusually small riboswitch aptamer domain.

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    Spitale, Robert C; Torelli, Andrew T; Krucinska, Jolanta; Bandarian, Vahe; Wedekind, Joseph E

    2009-04-24

    Riboswitches are RNA elements that control gene expression through metabolite binding. The preQ(1) riboswitch exhibits the smallest known ligand-binding domain and is of interest for its economical organization and high affinity interactions with guanine-derived metabolites required to confer tRNA wobbling. Here we present the crystal structure of a preQ(1) aptamer domain in complex with its precursor metabolite preQ(0). The structure is highly compact with a core that features a stem capped by a well organized decaloop. The metabolite is recognized within a deep pocket via Watson-Crick pairing with C15. Additional hydrogen bonds are made to invariant bases U6 and A29. The ligand-bound state confers continuous helical stacking throughout the core fold, thus providing a platform to promote Watson-Crick base pairing between C9 of the decaloop and the first base of the ribosome-binding site, G33. The structure offers insight into the mode of ribosome-binding site sequestration by a minimal RNA fold stabilized by metabolite binding and has implications for understanding the molecular basis by which bacterial genes are regulated.

  16. Ligand binding by the tandem glycine riboswitch depends on aptamer dimerization but not double ligand occupancy.

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    Ruff, Karen M; Strobel, Scott A

    2014-11-01

    The glycine riboswitch predominantly exists as a tandem structure, with two adjacent, homologous ligand-binding domains (aptamers), followed by a single expression platform. The recent identification of a leader helix, the inclusion of which eliminates cooperativity between the aptamers, has reopened the debate over the purpose of the tandem structure of the glycine riboswitch. An equilibrium dialysis-based assay was combined with binding-site mutations to monitor glycine binding in each ligand-binding site independently to understand the role of each aptamer in glycine binding and riboswitch tertiary interactions. A series of mutations disrupting the dimer interface was used to probe how dimerization impacts ligand binding by the tandem glycine riboswitch. While the wild-type tandem riboswitch binds two glycine equivalents, one for each aptamer, both individual aptamers are capable of binding glycine when the other aptamer is unoccupied. Intriguingly, glycine binding by aptamer-1 is more sensitive to dimerization than glycine binding by aptamer-2 in the context of the tandem riboswitch. However, monomeric aptamer-2 shows dramatically weakened glycine-binding affinity. In addition, dimerization of the two aptamers in trans is dependent on glycine binding in at least one aptamer. We propose a revised model for tandem riboswitch function that is consistent with these results, wherein ligand binding in aptamer-1 is linked to aptamer dimerization and stabilizes the P1 stem of aptamer-2, which controls the expression platform.

  17. Sequence-dependent folding landscapes of adenine riboswitch aptamers

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    Lin, Jong-Chin; Hyeon, Changbong; Thirumalai, D.

    Prediction of the functions of riboswitches requires a quantitative description of the folding landscape so that the barriers and time scales for the conformational change in the switching region in the aptamer can be estimated. Using a combination of all atom molecular dynamics and coarse-grained model simulations we studied the response of adenine (A) binding add and pbuE A-riboswitches to mechanical force. The two riboswitches contain a structurally similar three-way junction formed by three paired helices, P1, P2, and P3, but carry out different functions. Using pulling simulations, with structures generated in MD simulations, we show that after P1 rips the dominant unfolding pathway in add A-riboswitch is the rupture of P2 followed by unraveling of P3. In the pbuE A-riboswitch, after P1 unfolds P3 ruptures ahead of P2. The order of unfolding of the helices, which is in accord with single molecule pulling experiments, is determined by the relative stabilities of the individual helices. Our results show that the stability of isolated helices determines the order of assembly and response to force in these non-coding regions. We use the simulated free energy profile for pbuE A-riboswitch to estimate the time scale for allosteric switching, which shows that this riboswitch is under kinetic control lending additional support to the conclusion based on single molecule pulling experiments. A consequence of the stability hypothesis is that a single point mutation (U28C) in the P2 helix of the add A-riboswitch, which increases the stability of P2, would make the folding landscapes of the two riboswitches similar. This prediction can be tested in single molecule pulling experiments.

  18. Tuning riboswitch-mediated gene regulation by rational control of aptamer ligand binding properties.

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    Rode, Ambadas B; Endoh, Tamaki; Sugimoto, Naoki

    2015-01-12

    Riboswitch-mediated control of gene expression depends on ligand binding properties (kinetics and affinity) of its aptamer domain. A detailed analysis of interior regions of the aptamer, which affect the ligand binding properties, is important for both understanding natural riboswitch functions and for enabling rational design of tuneable artificial riboswitches. Kinetic analyses of binding reaction between flavin mononucleotide (FMN) and several natural and mutant aptamer domains of FMN-specific riboswitches were performed. The strong dependence of the dissociation rate (52.6-fold) and affinity (100-fold) on the identities of base pairs in the aptamer stem suggested that the stem region, which is conserved in length but variable in base-pair composition and context, is the tuning region of the FMN-specific aptamer. Synthetic riboswitches were constructed based on the same aptamer domain by rationally modifying the tuning regions. The observed 9.31-fold difference in the half-maximal effective concentration (EC50) corresponded to a 11.6-fold difference in the dissociation constant (K(D)) of the aptamer domains and suggested that the gene expression can be controlled by rationally adjusting the tuning regions.

  19. Transcriptional pausing coordinates folding of the aptamer domain and the expression platform of a riboswitch.

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    Perdrizet, George A; Artsimovitch, Irina; Furman, Ran; Sosnick, Tobin R; Pan, Tao

    2012-02-28

    Riboswitches are cis-acting elements that regulate gene expression by affecting transcriptional termination or translational initiation in response to binding of a metabolite. A typical riboswitch is made of an upstream aptamer domain and a downstream expression platform. Both domains participate in the folding and structural rearrangement in the absence or presence of its cognate metabolite. RNA polymerase pausing is a fundamental property of transcription that can influence RNA folding. Here we show that pausing plays an important role in the folding and conformational rearrangement of the Escherichia coli btuB riboswitch during transcription by the E. coli RNA polymerase. This riboswitch consists of an approximately 200 nucleotide, coenzyme B12 binding aptamer domain and an approximately 40 nucleotide expression platform that controls the ribosome access for translational initiation. We found that transcriptional pauses at strategic locations facilitate folding and structural rearrangement of the full-length riboswitch, but have minimal effect on the folding of the isolated aptamer domain. Pausing at these regulatory sites blocks the formation of alternate structures and plays a chaperoning role that couples folding of the aptamer domain and the expression platform. Pausing at strategic locations may be a general mechanism for coordinated folding and conformational rearrangements of riboswitch structures that underlie their response to environmental cues.

  20. Ligand Binding and Conformational Changes in the Purine-Binding Riboswitch Aptamer Domains

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    Noeske, Jonas; Buck, Janina; Wöhnert, Jens; Schwalbe, Harald

    Riboswitches are highly structured mRNA elements that regulate gene expression upon specific binding of small metabolite molecules. The purine-binding riboswitches bind different purine ligands by forming both canonical Watson—Crick and non-canonical intermolecular base pairs, involving a variety of hydrogen bonds between the riboswitch aptamer domain and the purine ligand. Here, we summarize work on the ligand binding modes of both purine-binding aptamer domains, their con-formational characteristics in the free and ligand-bound forms, and their ligand-induced folding. The adenine- and guanine-binding riboswitch aptamer domains display different conformations in their free forms, despite nearly identical nucleotide loop sequences that form a loop—loop interaction in the ligand-bound forms. Interestingly, the stability of helix II is crucial for the formation of the loop—loop interaction in the free form. A more stable helix II in the guanine riboswitch leads to a preformed loop—loop interaction in its free form. In contrast, a less stable helix II in the adenine riboswitch results in a lack of this loop—loop interaction in the absence of ligand and divalent cations.

  1. Thermodynamic and kinetic characterization of ligand binding to the purine riboswitch aptamer domain.

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    Gilbert, Sunny D; Stoddard, Colby D; Wise, Sarah J; Batey, Robert T

    2006-06-09

    Riboswitches are cis-acting genetic regulatory elements found commonly in bacterial mRNAs that consist of a metabolite-responsive aptamer domain coupled to a regulatory switch. Purine riboswitches respond to intracellular concentrations of either adenine or guanine/hypoxanthine to control gene expression. The aptamer domain of the purine riboswitch contains a pyrimidine residue (Y74) that forms a Watson-Crick base-pairing interaction with the bound purine nucleobase ligand that discriminates between adenine and guanine. We sought to understand the structural basis of this specificity and the mechanism of ligand recognition by the purine riboswitch. Here, we present the 2,6-diaminopurine-bound structure of a C74U mutant of the xpt-pbuX guanine riboswitch, along with a detailed thermodynamic and kinetic analysis of nucleobase recognition by both the native and mutant riboswitches. These studies demonstrate clearly that the pyrimidine at position 74 is the sole determinant of purine riboswitch specificity. In addition, the mutant riboswitch binds adenine and adenine derivatives well compared with the guanine-responsive riboswitch. Under our experimental conditions, 2,6-diaminopurine binds the RNA with DeltaH=-40.3 kcal mol(-1), DeltaS=-97.6 cal mol(-1)K(-1), and DeltaG=-10.73 kcal mol(-1). A kinetic determination of the slow rate (0.15 x 10(5)M(-1)s(-1) and 2.1 x 10(5)mM(-1)s(-1) for 2-aminopurine binding the adenine-responsive mutant riboswitch and 7-deazaguanine-binding guanine riboswitch, respectively) of association under varying experimental conditions allowed us to propose a mechanism for ligand recognition by the purine riboswitch. A conformationally dynamic unliganded state for the binding pocket is stabilized first by the Watson-Crick base pairing between the ligand and Y74, and by the subsequent ordering of the J2/3 loop, enclosing the ligand within the three-way junction.

  2. In vitro analysis of riboswitch-Spinach aptamer fusions as metabolite-sensing fluorescent biosensors.

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    Kellenberger, Colleen A; Hammond, Ming C

    2015-01-01

    The development of fluorescent biosensors has been motivated by the interest to monitor and measure the levels of specific metabolites in live cells in real time. Common approaches include fusing a protein-based receptor to fluorescent proteins or synthesizing a small molecule reactive probe. Natural metabolite-sensing riboswitches also have been used in reporter-based systems that take advantage of ligand-dependent regulation of downstream gene expression. More recently, it has been shown that RNA-based fluorescent biosensors can be generated by fusing a riboswitch aptamer to the in vitro selected Spinach aptamer, which binds a cell-permeable and conditionally fluorescent molecule. Here, we describe methods to design, prepare, and analyze riboswitch-Spinach aptamer fusion RNAs for ligand-dependent activation of fluorescence in vitro. Examples of procedures to measure fluorescence activation, ligand binding selectivity and affinity, and binding kinetics are given for a cyclic di-GMP-responsive biosensor. The relative ease of in vitro RNA synthesis and purification should make this method accessible to other researchers interested in developing riboswitch-based fluorescent biosensors.

  3. Riboswitch-Mediated Aptamer Binding for Imaging and Therapy (RABIT): A Novel Technique to Selectively Target an Intracelluar Ligand Specific for Ovarian Cancer

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    2015-12-01

    Award Number: W81XWH-12-1-0554 TITLE: Riboswitch- Mediated Aptamer Binding for Imaging and Therapy (RABIT): A Novel Technique to Selectively...ADDRESS. 1. REPORT DATE December 2015 2. REPORT TYPE Final 3. DATES COVERED 15Sep2012 - 14Sep2015 4. TITLE AND SUBTITLE Riboswitch- Mediated Aptamer...As proof of concept that an aptamer can mediate cell uptake into cultured cells , ES2 ovarian cancer cells were exposed to a DNA EpCAM aptamer

  4. DNA-rescuable allosteric inhibition of aptamer II ligand affinity by aptamer I element in the shortened Vibrio cholerae glycine riboswitch.

    Science.gov (United States)

    Sherman, Eileen M; Elsayed, Galal; Esquiaqui, Jackie M; Elsayed, Mohammed; Brinda, Bryan; Ye, Jing-Dong

    2014-12-01

    Glycine riboswitches contain two aptamers and turn on the expression of downstream genes in bacteria. Although full-length glycine riboswitches were shown to exhibit no glycine-binding cooperativity, the truncated glycine riboswitches were confirmed to bind two glycine molecules cooperatively. Thorough understanding of the ligand-binding cooperativity may shed light on the molecular basis of the cooperativity and help design novel intricate biosensing genetic circuits for application in synthetic biology. A previously proposed sequential model does not readily provide explanation for published data showing a deleterious mutation in the first aptamer inhibiting the glycine binding of the second one. Using the glycine riboswitch from Vibrio cholerae as a model system, we have identified a region in the first aptamer that modulates the second aptamer function especially in the shortened glycine riboswitch. Importantly, this modulation can be rescued by the addition of a complementary oligodeoxynucleotide, demonstrating the feasibility of developing this system into novel genetic circuits that sense both glycine and a DNA signal.

  5. Observation of long-range tertiary interactions during ligand binding by the TPP riboswitch aptamer.

    Science.gov (United States)

    Duesterberg, Van K; Fischer-Hwang, Irena T; Perez, Christian F; Hogan, Daniel W; Block, Steven M

    2015-12-28

    The thiamine pyrophosphate (TPP) riboswitch is a cis-regulatory element in mRNA that modifies gene expression in response to TPP concentration. Its specificity is dependent upon conformational changes that take place within its aptamer domain. Here, the role of tertiary interactions in ligand binding was studied at the single-molecule level by combined force spectroscopy and Förster resonance energy transfer (smFRET), using an optical trap equipped for simultaneous smFRET. The 'Force-FRET' approach directly probes secondary and tertiary structural changes during folding, including events associated with binding. Concurrent transitions observed in smFRET signals and RNA extension revealed differences in helix-arm orientation between two previously-identified ligand-binding states that had been undetectable by spectroscopy alone. Our results show that the weaker binding state is able to bind to TPP, but is unable to form a tertiary docking interaction that completes the binding process. Long-range tertiary interactions stabilize global riboswitch structure and confer increased ligand specificity.

  6. A minimalist biosensor: Quantitation of cyclic di-GMP using the conformational change of a riboswitch aptamer.

    Science.gov (United States)

    Kellenberger, Colleen A; Sales-Lee, Jade; Pan, Yuchen; Gassaway, Madalee M; Herr, Amy E; Hammond, Ming C

    2015-01-01

    Cyclic di-GMP (c-di-GMP) is a second messenger that is important in regulating bacterial physiology and behavior, including motility and virulence. Many questions remain about the role and regulation of this signaling molecule, but current methods of detection are limited by either modest sensitivity or requirements for extensive sample purification. We have taken advantage of a natural, high affinity receptor of c-di-GMP, the Vc2 riboswitch aptamer, to develop a sensitive and rapid electrophoretic mobility shift assay (EMSA) for c-di-GMP quantitation that required minimal engineering of the RNA.

  7. Crystallographic analysis of small ribozymes and riboswitches.

    Science.gov (United States)

    Lippa, Geoffrey M; Liberman, Joseph A; Jenkins, Jermaine L; Krucinska, Jolanta; Salim, Mohammad; Wedekind, Joseph E

    2012-01-01

    Ribozymes and riboswitches are RNA motifs that accelerate biological reactions and regulate gene expression in response to metabolite recognition, respectively. These RNA molecules gain functionality via complex folding that cannot be predicted a priori, and thus requires high-resolution three-dimensional structure determination to locate key functional attributes. Herein, we present an overview of the methods used to determine small RNA structures with an emphasis on RNA preparation, crystallization, and structure refinement. We draw upon examples from our own research in the analysis of the leadzyme ribozyme, the hairpin ribozyme, a class I preQ(1) riboswitch, and variants of a larger class II preQ(1) riboswitch. The methods presented provide a guide for comparable investigations of noncoding RNA molecules including a 48-solution, "first choice" RNA crystal screen compiled from our prior successes with commercially available screens.

  8. E88, a new cyclic-di-GMP class I riboswitch aptamer from Clostridium tetani, has a similar fold to the prototypical class I riboswitch, Vc2, but differentially binds to c-di-GMP analogs.

    Science.gov (United States)

    Luo, Yiling; Chen, Bin; Zhou, Jie; Sintim, Herman O; Dayie, T Kwaku

    2014-03-04

    C-di-GMP has emerged as a ubiquitous second messenger, which regulates the transition between sessile and motile lifestyles and virulence factor expression in many pathogenic bacteria using both RNA riboswitches and protein effectors. We recently showed that two additional class I c-di-GMP riboswitch aptamers (Ct-E88 and Cb-17B) bind c-di-GMP with nanomolar affinity, and that Ct-E88 RNA binds 2'-F-c-di-GMP 422 times less tightly than class I Vc2 RNA. Based on sequence comparison, it was concluded that the global folds of Ct-E88 and Vc2 RNAs were similar and that differences in ligand binding were probably due to differences in binding site architectures. Herein, we utilized EMSA, aptamer sensing spinach modules, SAXS and 1D NMR titration to study the conformational transitions of Ct-E88. We conclude that whereas the global folds of the bound states of Vc2 and Ct-E88 RNAs are similar, the unbound states are different and this could explain differences in ligand affinities between these class I c-di-GMP riboswitches.

  9. ITC analysis of ligand binding to preQ₁ riboswitches.

    Science.gov (United States)

    Liberman, Joseph A; Bogue, Jarrod T; Jenkins, Jermaine L; Salim, Mohammad; Wedekind, Joseph E

    2014-01-01

    Riboswitches regulate genes by binding to small-molecule effectors. Isothermal titration calorimetry (ITC) provides a label-free method to quantify the equilibrium association constant, K(A), of a riboswitch interaction with its cognate ligand. In addition to probing affinity and specific chemical contributions that contribute to binding, ITC can be used to measure the thermodynamic parameters of an interaction (ΔG, ΔH, and ΔS), in addition to the binding stoichiometry (N). Here, we describe methods developed to measure the binding affinity of various preQ1 riboswitch classes for the pyrrolopyrimidine effector, preQ1. Example isotherms are provided along with a review of various preQ1-II (class 2) riboswitch mutants that were interrogated by ITC to quantify the energetic contributions of specific interactions visualized in the crystal structure. Protocols for ITC are provided in sufficient detail that the reader can reproduce experiments independently, or develop derivative methods suitable for analyzing novel riboswitch-ligand binding interactions.

  10. Monitoring ribosomal frameshifting as a platform to screen anti-riboswitch drug candidates.

    Science.gov (United States)

    Yu, Chien-Hung; Olsthoorn, René C L

    2015-01-01

    Riboswitches are regions within mRNAs that can regulate downstream expression of genes through metabolite-induced alteration of their secondary structures. Due to the significant association of bacterial essential or virulence genes, bacterial riboswitches have become promising targets for development of putative antibacterial drugs. However, most of the screening systems to date are based on in vitro or bacterial systems, lacking the possibility to preobserve the adverse effects to the host's translation machinery. This chapter describes a novel screening method based on monitoring the riboswitch-induced -1 ribosomal frameshifting (-1 FS) efficiency in a mammalian cell-free lysate system using preQ1 class-I (preQ1-I) riboswitches as model target.

  11. Identification and cloning of four riboswitches from Burkholderia pseudomallei strain K96243

    Science.gov (United States)

    Munyati-Othman, Noor; Fatah, Ahmad Luqman Abdul; Piji, Mohd Al Akmarul Fizree Bin Md; Ramlan, Effirul Ikhwan; Raih, Mohd Firdaus

    2015-09-01

    Structured RNAs referred as riboswitches have been predicted to be present in the genome sequence of Burkholderia pseudomallei strain K96243. Four of the riboswitches were identified and analyzed through BLASTN, Rfam search and multiple sequence alignment. The RNA aptamers belong to the following riboswitch classifications: glycine riboswitch, cobalamin riboswitch, S-adenosyl-(L)-homocysteine (SAH) riboswitch and flavin mononucleotide (FMN) riboswitch. The conserved nucleotides for each aptamer were identified and were marked on the secondary structure generated by RNAfold. These riboswitches were successfully amplified and cloned for further study.

  12. Idiosyncratically tuned switching behavior of riboswitch aptamer domains revealed by comparative small-angle X-ray scattering analysis

    Energy Technology Data Exchange (ETDEWEB)

    Baird, Nathan J.; Ferré-D' Amaré, Adrian R. (HHMI)

    2010-05-25

    Riboswitches are structured mRNA elements that regulate gene expression upon binding specific cellular metabolites. It is thought that the highly conserved metabolite-binding domains of riboswitches undergo conformational change upon binding their cognate ligands. To investigate the generality of such a mechanism, we employed small-angle X-ray scattering (SAXS). We probed the nature of the global metabolite-induced response of the metabolite-binding domains of four different riboswitches that bind, respectively, thiamine pyrophosphate (TPP), flavin mononucleotide (FMN), lysine, and S-adenosyl methionine (SAM). We find that each RNA is unique in its global structural response to metabolite. Whereas some RNAs exhibit distinct free and bound conformations, others are globally insensitive to the presence of metabolite. Thus, a global conformational change of the metabolite-binding domain is not a requirement for riboswitch function. It is possible that the range of behaviors observed by SAXS, rather than being a biophysical idiosyncrasy, reflects adaptation of riboswitches to the regulatory requirements of their individual genomic context.

  13. Riboswitch Detection Using Profile Hidden Markov Models

    Directory of Open Access Journals (Sweden)

    Krishnamachari A

    2009-10-01

    Full Text Available Abstract Background Riboswitches are a type of noncoding RNA that regulate gene expression by switching from one structural conformation to another on ligand binding. The various classes of riboswitches discovered so far are differentiated by the ligand, which on binding induces a conformational switch. Every class of riboswitch is characterized by an aptamer domain, which provides the site for ligand binding, and an expression platform that undergoes conformational change on ligand binding. The sequence and structure of the aptamer domain is highly conserved in riboswitches belonging to the same class. We propose a method for fast and accurate identification of riboswitches using profile Hidden Markov Models (pHMM. Our method exploits the high degree of sequence conservation that characterizes the aptamer domain. Results Our method can detect riboswitches in genomic databases rapidly and accurately. Its sensitivity is comparable to the method based on the Covariance Model (CM. For six out of ten riboswitch classes, our method detects more than 99.5% of the candidates identified by the much slower CM method while being several hundred times faster. For three riboswitch classes, our method detects 97-99% of the candidates relative to the CM method. Our method works very well for those classes of riboswitches that are characterized by distinct and conserved sequence motifs. Conclusion Riboswitches play a crucial role in controlling the expression of several prokaryotic genes involved in metabolism and transport processes. As more and more new classes of riboswitches are being discovered, it is important to understand the patterns of their intra and inter genomic distribution. Understanding such patterns will enable us to better understand the evolutionary history of these genetic regulatory elements. However, a complete picture of the distribution pattern of riboswitches will emerge only after accurate identification of riboswitches across genomes

  14. Structural Basis of Cooperative Ligand Binding by the Glycine Riboswitch

    OpenAIRE

    Butler, Ethan B.; Xiong, Yong; Wang, Jimin; Strobel, Scott A.

    2011-01-01

    The glycine riboswitch regulates gene expression through the cooperative recognition of its amino acid ligand by a tandem pair of aptamers. A 3.6Å crystal structure of the tandem riboswitch from the glycine permease operon of Fusobacterium nucleatum reveals the glycine binding sites and an extensive network of interactions, largely mediated by asymmetric A-minor contacts, that serve to communicate ligand binding status between the aptamers. These interactions provide a structural basis for ho...

  15. Base ionization and ligand binding: how small ribozymes and riboswitches gain a foothold in a protein world.

    Science.gov (United States)

    Liberman, Joseph A; Wedekind, Joseph E

    2011-06-01

    Genome sequencing has produced thousands of nonprotein coding (nc)RNA sequences including new ribozymes and riboswitches. Such RNAs are notable for their extraordinary functionality, which entails exquisite folding that culminates in biocatalytic or ligand-binding capabilities. Here we discuss advances in relating ncRNA form to function with an emphasis on base pK(a) shifting by the hairpin and hepatitis delta virus ribozymes. We then describe ligand binding by the two smallest riboswitches, which target preQ(1) and S-adenosyl-(l)-homocysteine, followed by an analysis of a second-messenger riboswitch that binds cyclic-di-GMP. Each riboswitch is then compared to a protein that binds the same ligand to contrast binding properties. The results showcase the breadth of functionality attainable from ncRNAs, as well as molecular features notable for antibacterial design.

  16. Structural Basis of Cooperative Ligand Binding by the Glycine Riboswitch

    Energy Technology Data Exchange (ETDEWEB)

    E Butler; J Wang; Y Xiong; S Strobel

    2011-12-31

    The glycine riboswitch regulates gene expression through the cooperative recognition of its amino acid ligand by a tandem pair of aptamers. A 3.6 {angstrom} crystal structure of the tandem riboswitch from the glycine permease operon of Fusobacterium nucleatum reveals the glycine binding sites and an extensive network of interactions, largely mediated by asymmetric A-minor contacts, that serve to communicate ligand binding status between the aptamers. These interactions provide a structural basis for how the glycine riboswitch cooperatively regulates gene expression.

  17. Microfluidic screening of electrophoretic mobility shifts elucidates riboswitch binding function.

    Science.gov (United States)

    Karns, Kelly; Vogan, Jacob M; Qin, Qian; Hickey, Scott F; Wilson, Stephen C; Hammond, Ming C; Herr, Amy E

    2013-02-27

    Riboswitches are RNA sensors that change conformation upon binding small molecule metabolites, in turn modulating gene expression. Our understanding of riboswitch regulatory function would be accelerated by a high-throughput, quantitative screening tool capable of measuring riboswitch-ligand binding. We introduce a microfluidic mobility shift assay that enables precise and rapid quantitation of ligand binding and subsequent riboswitch conformational change. In 0.3% of the time required for benchtop assays (3.2 versus 1020 min), we screen and validate five candidate SAM-I riboswitches isolated from thermophilic and cryophilic bacteria. The format offers enhanced resolution of conformational change compared to slab gel formats, quantitation, and repeatability for statistical assessment of small mobility shifts, low reagent consumption, and riboswitch characterization without modification of the aptamer structure. Appreciable analytical sensitivity coupled with high-resolution separation performance allows quantitation of equilibrium dissociation constants (K(d)) for both rapidly and slowly interconverting riboswitch-ligand pairs as validated through experiments and modeling. Conformational change, triplicate mobility shift measurements, and K(d) are reported for both a known and a candidate SAM-I riboswitch with comparison to in-line probing assay results. The microfluidic mobility shift assay establishes a scalable format for the study of riboswitch-ligand binding that will advance the discovery and selection of novel riboswitches and the development of antibiotics to target bacterial riboswitches.

  18. Riboswitch-Mediated Aptamer Binding for Imaging and Therapy (RABIT): A Novel Technique to Selectively Target an Intracellular Ligand Specific for Ovarian Cancer

    Science.gov (United States)

    2013-10-01

    iodine it will be possible to image the cancer cells (125I and 123I) and treat the cancer (131I). In contrast to other toxins , radioactive...also began a search for DNA rather than RNA aptamers to antipyrine. A modified SELEX approach was used, followed by next generation sequencing...oligonucleotides showing preferential PCR amplification, and used in a subsequent round of aptamer selection. After 5 rounds of SELEX selection the

  19. Computational Studies of the Binding of Ligands to the Guanine Riboswitch Aptamer%鸟嘌呤核糖开关识别配体小分子

    Institute of Scientific and Technical Information of China (English)

    马爱静; 扈国栋; 王吉华

    2016-01-01

    The Guanine Riboswitch is genetic regulatory element found in the non translation region loca‐ted of mRNA .It can be directly combined with organic metabolites to induces conformational changes to control gene expression .Recognition of them has highly specificity and selectivity .In this work ,we stud‐ied the nature of the Guanine Riboswitch and ligands by molecular dynamics simulation .Then ,the calcu‐lation of the relative binding free energy of them by Thermodynamic Integration .The study found that the binding pocket of Riboswitch form Waston -Crick base pairs with ligand and we learned that the affinities are different when the binding of different ligands to Riboswitch .These data indicate a more complicated relationship between the 6-position of the ligand and the RNA and The 2-position of the purine ligand is essential for the recognition .As an ideal target of new drugs ,the Guanine Riboswitch has an important role in the design and development of new drugs .%鸟嘌呤核糖开关是一类位于m RN A 的非翻译区,可直接结合有机代谢物,通过调整自身构象变化从而调控基因表达的m RN A元件。鸟嘌呤核糖开关识别配体具有高度特异性和选择性。用动力学模拟的方法,研究鸟嘌呤核糖开关特异识别配体小分子的本质和用热力学积分算法计算核糖开关与配体分子的相对结合自由能。研究发现,核糖开关的结合口袋与配体通过氢键形式相互作用,不同配体与核糖开关结合的亲和力不同。嘌呤配体2-号位对核糖开关的结合必不可少,6-号位对核糖开关的结合关系复杂。

  20. The Shine-Dalgarno sequence of riboswitch-regulated single mRNAs shows ligand-dependent accessibility bursts

    Science.gov (United States)

    Rinaldi, Arlie J.; Lund, Paul E.; Blanco, Mario R.; Walter, Nils G.

    2016-01-01

    In response to intracellular signals in Gram-negative bacteria, translational riboswitches--commonly embedded in messenger RNAs (mRNAs)--regulate gene expression through inhibition of translation initiation. It is generally thought that this regulation originates from occlusion of the Shine-Dalgarno (SD) sequence upon ligand binding; however, little direct evidence exists. Here we develop Single Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS) to investigate the ligand-dependent accessibility of the SD sequence of an mRNA hosting the 7-aminomethyl-7-deazaguanine (preQ1)-sensing riboswitch. Spike train analysis reveals that individual mRNA molecules alternate between two conformational states, distinguished by `bursts' of probe binding associated with increased SD sequence accessibility. Addition of preQ1 decreases the lifetime of the SD's high-accessibility (bursting) state and prolongs the time between bursts. In addition, ligand-jump experiments reveal imperfect riboswitching of single mRNA molecules. Such complex ligand sensing by individual mRNA molecules rationalizes the nuanced ligand response observed during bulk mRNA translation.

  1. Mutational analysis of structural elements in a class-I cyclic di-GMP riboswitch to elucidate its regulatory mechanism.

    Science.gov (United States)

    Inuzuka, Saki; Nishimura, Kei-Ichiro; Kakizawa, Hitoshi; Fujita, Yuki; Furuta, Hiroyuki; Matsumura, Shigeyoshi; Ikawa, Yoshiya

    2016-09-01

    The Vc2 riboswitch possesses an aptamer domain belonging to the class-I c-di-GMP riboswitch family. This domain has been analysed and the molecular mechanism by which it recognizes the c-di-GMP ligand has been elucidated. On the other hand, the regulatory mechanism of the full-length Vc2 riboswitch to control its downstream open reading frame (ORF) remains largely unknown. In this study, we performed in vivo reporter assays and in vitro biochemical analyses of the full-length riboswitch and its aptamer domain. We evaluated the results of in vivo and in vitro analyses to elucidate the regulatory mechanism of the Vc2 riboswitch. The present results suggest that recognition of c-di-GMP ligand by the Vc2 riboswitch aptamer domain downregulates expression of its downstream ORF primarily at the translational level.

  2. Control of alphavirus-based gene expression using engineered riboswitches.

    Science.gov (United States)

    Bell, Christie L; Yu, Dong; Smolke, Christina D; Geall, Andrew J; Beard, Clayton W; Mason, Peter W

    2015-09-01

    Alphavirus-based replicons are a promising nucleic acid vaccine platform characterized by robust gene expression and immune responses. To further explore their use in vaccination, replicons were engineered to allow conditional control over their gene expression. Riboswitches, comprising a ribozyme actuator and RNA aptamer sensor, were engineered into the replicon 3' UTR. Binding of ligand to aptamer modulates ribozyme activity and, therefore, gene expression. Expression from DNA-launched and VRP-packaged replicons containing riboswitches was successfully regulated, achieving a 47-fold change in expression and modulation of the resulting type I interferon response. Moreover, we developed a novel control architecture where riboswitches were integrated into the 3' and 5' UTR of the subgenomic RNA region of the TC-83 virus, leading to an 1160-fold regulation of viral replication. Our studies demonstrate that the use of riboswitches for control of RNA replicon expression and viral replication holds promise for development of novel and safer vaccination strategies.

  3. Riboswitch Structure: an Internal Residue Mimicking the Purine Ligand

    Energy Technology Data Exchange (ETDEWEB)

    Delfosse, V.; Bouchard, P; Bonneau, E; Dagenais, P; Lemay, J; Lafontaine, D; Legault, P

    2009-01-01

    The adenine and guanine riboswitches regulate gene expression in response to their purine ligand. X-ray structures of the aptamer moiety of these riboswitches are characterized by a compact fold in which the ligand forms a Watson-Crick base pair with residue 65. Phylogenetic analyses revealed a strict restriction at position 39 of the aptamer that prevents the G39-C65 and A39-U65 combinations, and mutational studies indicate that aptamers with these sequence combinations are impaired for ligand binding. In order to investigate the rationale for sequence conservation at residue 39, structural characterization of the U65C mutant from Bacillus subtilis pbuE adenine riboswitch aptamer was undertaken. NMR spectroscopy and X-ray crystallography studies demonstrate that the U65C mutant adopts a compact ligand-free structure, in which G39 occupies the ligand-binding site of purine riboswitch aptamers. These studies present a remarkable example of a mutant RNA aptamer that adopts a native-like fold by means of ligand mimicking and explain why this mutant is impaired for ligand binding. Furthermore, this work provides a specific insight into how the natural sequence has evolved through selection of nucleotide identities that contribute to formation of the ligand-bound state, but ensures that the ligand-free state remains in an active conformation.

  4. An Efficient Minimum Free Energy Structure-Based Search Method for Riboswitch Identification Based on Inverse RNA Folding.

    Directory of Open Access Journals (Sweden)

    Matan Drory Retwitzer

    Full Text Available Riboswitches are RNA genetic control elements that were originally discovered in bacteria and provide a unique mechanism of gene regulation. They work without the participation of proteins and are believed to represent ancient regulatory systems in the evolutionary timescale. One of the biggest challenges in riboswitch research is to find additional eukaryotic riboswitches since more than 20 riboswitch classes have been found in prokaryotes but only one class has been found in eukaryotes. Moreover, this single known class of eukaryotic riboswitch, namely the TPP riboswitch class, has been found in bacteria, archaea, fungi and plants but not in animals. The few examples of eukaryotic riboswitches were identified using sequence-based bioinformatics search methods such as a combination of BLAST and pattern matching techniques that incorporate base-pairing considerations. None of these approaches perform energy minimization structure predictions. There is a clear motivation to develop new bioinformatics methods, aside of the ongoing advances in covariance models, that will sample the sequence search space more flexibly using structural guidance while retaining the computational efficiency of sequence-based methods. We present a new energy minimization approach that transforms structure-based search into a sequence-based search, thereby enabling the utilization of well established sequence-based search utilities such as BLAST and FASTA. The transformation to sequence space is obtained by using an extended inverse RNA folding problem solver with sequence and structure constraints, available within RNAfbinv. Examples in applying the new method are presented for the purine and preQ1 riboswitches. The method is described in detail along with its findings in prokaryotes. Potential uses in finding novel eukaryotic riboswitches and optimizing pre-designed synthetic riboswitches based on ligand simulations are discussed. The method components are freely

  5. An Efficient Minimum Free Energy Structure-Based Search Method for Riboswitch Identification Based on Inverse RNA Folding.

    Science.gov (United States)

    Drory Retwitzer, Matan; Kifer, Ilona; Sengupta, Supratim; Yakhini, Zohar; Barash, Danny

    2015-01-01

    Riboswitches are RNA genetic control elements that were originally discovered in bacteria and provide a unique mechanism of gene regulation. They work without the participation of proteins and are believed to represent ancient regulatory systems in the evolutionary timescale. One of the biggest challenges in riboswitch research is to find additional eukaryotic riboswitches since more than 20 riboswitch classes have been found in prokaryotes but only one class has been found in eukaryotes. Moreover, this single known class of eukaryotic riboswitch, namely the TPP riboswitch class, has been found in bacteria, archaea, fungi and plants but not in animals. The few examples of eukaryotic riboswitches were identified using sequence-based bioinformatics search methods such as a combination of BLAST and pattern matching techniques that incorporate base-pairing considerations. None of these approaches perform energy minimization structure predictions. There is a clear motivation to develop new bioinformatics methods, aside of the ongoing advances in covariance models, that will sample the sequence search space more flexibly using structural guidance while retaining the computational efficiency of sequence-based methods. We present a new energy minimization approach that transforms structure-based search into a sequence-based search, thereby enabling the utilization of well established sequence-based search utilities such as BLAST and FASTA. The transformation to sequence space is obtained by using an extended inverse RNA folding problem solver with sequence and structure constraints, available within RNAfbinv. Examples in applying the new method are presented for the purine and preQ1 riboswitches. The method is described in detail along with its findings in prokaryotes. Potential uses in finding novel eukaryotic riboswitches and optimizing pre-designed synthetic riboswitches based on ligand simulations are discussed. The method components are freely available for use.

  6. Design of Artificial Riboswitches as Biosensors

    Directory of Open Access Journals (Sweden)

    Sven Findeiß

    2017-08-01

    Full Text Available RNA aptamers readily recognize small organic molecules, polypeptides, as well as other nucleic acids in a highly specific manner. Many such aptamers have evolved as parts of regulatory systems in nature. Experimental selection techniques such as SELEX have been very successful in finding artificial aptamers for a wide variety of natural and synthetic ligands. Changes in structure and/or stability of aptamers upon ligand binding can propagate through larger RNA constructs and cause specific structural changes at distal positions. In turn, these may affect transcription, translation, splicing, or binding events. The RNA secondary structure model realistically describes both thermodynamic and kinetic aspects of RNA structure formation and refolding at a single, consistent level of modelling. Thus, this framework allows studying the function of natural riboswitches in silico. Moreover, it enables rationally designing artificial switches, combining essentially arbitrary sensors with a broad choice of read-out systems. Eventually, this approach sets the stage for constructing versatile biosensors.

  7. Ligand-induced folding of the guanine-sensing riboswitch is controlled by a combined predetermined induced fit mechanism

    NARCIS (Netherlands)

    Ottink, O.M.; Rampersad, S.M.; Tessari, M.; Zaman, G.J.; Heus, H.A.; Wijmenga, S.S.

    2007-01-01

    All known guanine-sensing riboswitches regulate gene expression by specifically binding to guanine (G) or related analogs with high affinity to switch off transcription. The aptamers of this class of riboswitches are characterized by three helices (P1-P3), surrounding a central core of phylogenetica

  8. Basis for ligand discrimination between ON and OFF state riboswitch conformations: the case of the SAM-I riboswitch.

    Science.gov (United States)

    Boyapati, Vamsi Krishna; Huang, Wei; Spedale, Jessica; Aboul-Ela, Fareed

    2012-06-01

    Riboswitches are RNA elements that bind to effector ligands and control gene expression. Most consist of two domains. S-Adenosyl Methionine (SAM) binds the aptamer domain of the SAM-I riboswitch and induces conformational changes in the expression domain to form an intrinsic terminator (transcription OFF state). Without SAM the riboswitch forms the transcription ON state, allowing read-through transcription. The mechanistic link between the SAM/aptamer recognition event and subsequent secondary structure rearrangement by the riboswitch is unclear. We probed for those structural features of the Bacillus subtilis yitJ SAM-I riboswitch responsible for discrimination between the ON and OFF states by SAM. We designed SAM-I riboswitch RNA segments forming "hybrid" structures of the ON and OFF states. The choice of segment constrains the formation of a partial P1 helix, characteristic of the OFF state, together with a partial antiterminator (AT) helix, characteristic of the ON state. For most choices of P1 vs. AT helix lengths, SAM binds with micromolar affinity according to equilibrium dialysis. Mutational analysis and in-line probing confirm that the mode of SAM binding by hybrid structures is similar to that of the aptamer. Altogether, binding measurements and in-line probing are consistent with the hypothesis that when SAM is present, stacking interactions with the AT helix stabilize a partially formed P1 helix in the hybrids. Molecular modeling indicates that continuous stacking between the P1 and the AT helices is plausible with SAM bound. Our findings raise the possibility that conformational intermediates may play a role in ligand-induced aptamer folding.

  9. Riboswitch structure in the ligand-free state.

    Science.gov (United States)

    Liberman, Joseph A; Wedekind, Joseph E

    2012-01-01

    Molecular investigations of riboswitches bound to small-molecule effectors have produced a wealth of information on how these molecules achieve high affinity and specificity for a target ligand. X-ray crystal structures have been determined for the ligand-free state for representatives of the preQ₁-I, S-adenosylmethionine I, lysine, and glycine aptamer classes. These structures in conjunction with complimentary techniques, such as in-line probing, NMR spectroscopy, Förster resonance energy transfer, small-angle scattering, and computational simulations, have demonstrated that riboswitches adopt multiple conformations in the absence of ligand. Despite a number of investigations that support ligand-dependent folding, mounting evidence suggests that free-state riboswitches interact with their effectors in the sub-populations of largely prefolded states as embodied by the principle of conformational selection, which has been documented extensively for protein-mediated ligand interactions. Fundamental riboswitch investigations of the bound and free states have advanced our understanding of RNA folding, ligand recognition, and how these factors culminate in communication between an aptamer and its expression platform. An understanding of these topics is essential to comprehend riboswitch gene regulation at the molecular level, which has already provided a basis to understand the mechanism of action of natural antimicrobials.

  10. Riboswitches as hormone receptors: hypothetical cytokinin-binding riboswitches in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Downes Brian

    2010-10-01

    Full Text Available Abstract Background Riboswitches are mRNA elements that change conformation when bound to small molecules. They are known to be key regulators of biosynthetic pathways in both prokaryotes and eukaryotes. Presentation of the Hypothesis The hypothesis presented here is that riboswitches function as receptors in hormone perception. We propose that riboswitches initiate or integrate signaling cascades upon binding to classic signaling molecules. The molecular interactions for ligand binding and gene expression control would be the same as for biosynthetic pathways, but the context and the cadre of ligands to consider is dramatically different. The hypothesis arose from the observation that a compound used to identify adenine binding RNA sequences is chemically similar to the classic plant hormone, or growth regulator, cytokinin. A general tenet of the hypothesis is that riboswitch-binding metabolites can be used to make predictions about chemically related signaling molecules. In fact, all cell permeable signaling compounds can be considered as potential riboswitch ligands. The hypothesis is plausible, as demonstrated by a cursory review of the transcriptome and genome of the model plant Arabidopsis thaliana for transcripts that i contain an adenine aptamer motif, and ii are also predicted to be cytokinin-regulated. Here, one gene, CRK10 (for Cysteine-rich Receptor-like Kinase 10, At4g23180, contains an adenine aptamer-related sequence and is down-regulated by cytokinin approximately three-fold in public gene expression data. To illustrate the hypothesis, implications of cytokinin-binding to the CRK10 mRNA are discussed. Testing the hypothesis At the broadest level, screening various cell permeable signaling molecules against random RNA libraries and comparing hits to sequence and gene expression data bases could determine how broadly the hypothesis applies. Specific cases, such as CRK10 presented here, will require experimental validation of direct

  11. Force field dependence of riboswitch dynamics.

    Science.gov (United States)

    Hanke, Christian A; Gohlke, Holger

    2015-01-01

    Riboswitches are noncoding regulatory elements that control gene expression in response to the presence of metabolites, which bind to the aptamer domain. Metabolite binding appears to occur through a combination of conformational selection and induced fit mechanism. This demands to characterize the structural dynamics of the apo state of aptamer domains. In principle, molecular dynamics (MD) simulations can give insights at the atomistic level into the dynamics of the aptamer domain. However, it is unclear to what extent contemporary force fields can bias such insights. Here, we show that the Amber force field ff99 yields the best agreement with detailed experimental observations on differences in the structural dynamics of wild type and mutant aptamer domains of the guanine-sensing riboswitch (Gsw), including a pronounced influence of Mg2+. In contrast, applying ff99 with parmbsc0 and parmχOL modifications (denoted ff10) results in strongly damped motions and overly stable tertiary loop-loop interactions. These results are based on 58 MD simulations with an aggregate simulation time>11 μs, careful modeling of Mg2+ ions, and thorough statistical testing. Our results suggest that the moderate stabilization of the χ-anti region in ff10 can have an unwanted damping effect on functionally relevant structural dynamics of marginally stable RNA systems. This suggestion is supported by crystal structure analyses of Gsw aptamer domains that reveal χ torsions with high-anti values in the most mobile regions. We expect that future RNA force field development will benefit from considering marginally stable RNA systems and optimization toward good representations of dynamics in addition to structural characteristics.

  12. The regulation mechanism of yitJ and metF riboswitches

    Energy Technology Data Exchange (ETDEWEB)

    Gong, Sha; Wang, Yujie; Zhang, Wenbing, E-mail: wbzhang@whu.edu.cn [Department of Physics, Wuhan University, Wuhan, Hubei 430072 (China)

    2015-07-28

    Riboswitches which function at the transcriptional level are sensitive to cotranscriptional folding. Based on the recently proposed theory of cotranscriptional folding, we developed a transition node approximation method to effectively decrease the conformation space of long RNA chains. Our results indicate that this approximation is reliable for calculating the cotranscriptional folding kinetics of long mRNA chains. We theoretically studied the cotranscriptional folding behavior of the yitJ and metF riboswitches in the absence/presence of S-adenosylmethionine. Although the two S-box riboswitches have similar OFF-state structures and share common features of riboswitches operated at the transcriptional level, their regulation mechanisms are different. The yitJ riboswitch is regulated by a combination of thermodynamic and kinetic mechanisms, while the metF riboswitch is solely kinetically controlled. For the yitJ riboswitch, transcriptional pausing at the U-stretch directly following the terminator decreases the amount of ligand required to trigger the switch. The different regulation mechanisms and binding affinities of the two riboswitches result from the different lengths of the anti-terminator helix, which in yitJ is short and only disrupts helix P1 of the riboswitch aptamer, but in metF is long and breaks both the helices P1 and P4.

  13. The regulation mechanism of yitJ and metF riboswitches

    Science.gov (United States)

    Gong, Sha; Wang, Yujie; Zhang, Wenbing

    2015-07-01

    Riboswitches which function at the transcriptional level are sensitive to cotranscriptional folding. Based on the recently proposed theory of cotranscriptional folding, we developed a transition node approximation method to effectively decrease the conformation space of long RNA chains. Our results indicate that this approximation is reliable for calculating the cotranscriptional folding kinetics of long mRNA chains. We theoretically studied the cotranscriptional folding behavior of the yitJ and metF riboswitches in the absence/presence of S-adenosylmethionine. Although the two S-box riboswitches have similar OFF-state structures and share common features of riboswitches operated at the transcriptional level, their regulation mechanisms are different. The yitJ riboswitch is regulated by a combination of thermodynamic and kinetic mechanisms, while the metF riboswitch is solely kinetically controlled. For the yitJ riboswitch, transcriptional pausing at the U-stretch directly following the terminator decreases the amount of ligand required to trigger the switch. The different regulation mechanisms and binding affinities of the two riboswitches result from the different lengths of the anti-terminator helix, which in yitJ is short and only disrupts helix P1 of the riboswitch aptamer, but in metF is long and breaks both the helices P1 and P4.

  14. The impact of a ligand binding on strand migration in the SAM-I riboswitch.

    Directory of Open Access Journals (Sweden)

    Wei Huang

    Full Text Available Riboswitches sense cellular concentrations of small molecules and use this information to adjust synthesis rates of related metabolites. Riboswitches include an aptamer domain to detect the ligand and an expression platform to control gene expression. Previous structural studies of riboswitches largely focused on aptamers, truncating the expression domain to suppress conformational switching. To link ligand/aptamer binding to conformational switching, we constructed models of an S-adenosyl methionine (SAM-I riboswitch RNA segment incorporating elements of the expression platform, allowing formation of an antiterminator (AT helix. Using Anton, a computer specially developed for long timescale Molecular Dynamics (MD, we simulated an extended (three microseconds MD trajectory with SAM bound to a modeled riboswitch RNA segment. Remarkably, we observed a strand migration, converting three base pairs from an antiterminator (AT helix, characteristic of the transcription ON state, to a P1 helix, characteristic of the OFF state. This conformational switching towards the OFF state is observed only in the presence of SAM. Among seven extended trajectories with three starting structures, the presence of SAM enhances the trend towards the OFF state for two out of three starting structures tested. Our simulation provides a visual demonstration of how a small molecule (<500 MW binding to a limited surface can trigger a large scale conformational rearrangement in a 40 kDa RNA by perturbing the Free Energy Landscape. Such a mechanism can explain minimal requirements for SAM binding and transcription termination for SAM-I riboswitches previously reported experimentally.

  15. Dual-acting riboswitch control of translation initiation and mRNA decay.

    Science.gov (United States)

    Caron, Marie-Pier; Bastet, Laurène; Lussier, Antony; Simoneau-Roy, Maxime; Massé, Eric; Lafontaine, Daniel A

    2012-12-11

    Riboswitches are mRNA regulatory elements that control gene expression by altering their structure in response to specific metabolite binding. In bacteria, riboswitches consist of an aptamer that performs ligand recognition and an expression platform that regulates either transcription termination or translation initiation. Here, we describe a dual-acting riboswitch from Escherichia coli that, in addition to modulating translation initiation, also is directly involved in the control of initial mRNA decay. Upon lysine binding, the lysC riboswitch adopts a conformation that not only inhibits translation initiation but also exposes RNase E cleavage sites located in the riboswitch expression platform. However, in the absence of lysine, the riboswitch folds into an alternative conformation that simultaneously allows translation initiation and sequesters RNase E cleavage sites. Both regulatory activities can be individually inhibited, indicating that translation initiation and mRNA decay can be modulated independently using the same conformational switch. Because RNase E cleavage sites are located in the riboswitch sequence, this riboswitch provides a unique means for the riboswitch to modulate RNase E cleavage activity directly as a function of lysine. This dual inhibition is in contrast to other riboswitches, such as the thiamin pyrophosphate-sensing thiM riboswitch, which triggers mRNA decay only as a consequence of translation inhibition. The riboswitch control of RNase E cleavage activity is an example of a mechanism by which metabolite sensing is used to regulate gene expression of single genes or even large polycistronic mRNAs as a function of environmental changes.

  16. Structural insights into the interactions of xpt riboswitch with novel guanine analogues: a molecular dynamics simulation study.

    Science.gov (United States)

    Jain, Swapan S; Sonavane, Uddhavesh B; Uppuladinne, Mallikarjunachari V N; McLaughlin, Emily C; Wang, Weiqing; Black, Sheneil; Joshi, Rajendra R

    2015-01-01

    Ligand recognition in purine riboswitches is a complex process requiring different levels of conformational changes. Recent efforts in the area of purine riboswitch research have focused on ligand analogue binding studies. In the case of the guanine xanthine phosphoribosyl transferase (xpt) riboswitch, synthetic analogues that resemble guanine have the potential to tightly bind and subsequently influence the genetic expression of xpt mRNA in prokaryotes. We have carried out 25 ns Molecular Dynamics (MD) simulation studies of the aptamer domain of the xpt G-riboswitch in four different states: guanine riboswitch in free form, riboswitch bound with its cognate ligand guanine, and with two guanine analogues SJ1 and SJ2. Our work reveals novel interactions of SJ1 and SJ2 ligands with the binding core residues of the riboswitch. The ligands proposed in this work bind to the riboswitch with greater overall stability and lower root mean square deviations and fluctuations compared to guanine ligand. Reporter gene assay data demonstrate that the ligand analogues, upon binding to the RNA, lower the genetic expression of the guanine riboswitch. Our work has important implications for future ligand design and binding studies in the exciting field of riboswitches.

  17. Glycols modulate terminator stem stability and ligand-dependency of a glycine riboswitch.

    Science.gov (United States)

    Hamachi, Kokoro; Hayashi, Hikari; Shimamura, Miyuki; Yamaji, Yuiha; Kaneko, Ai; Fujisawa, Aruma; Umehara, Takuya; Tamura, Koji

    2013-08-01

    The Bacillus subtilis glycine riboswitch comprises tandem glycine-binding aptamers and a putative terminator stem followed by the gcvT operon. Gene expression is regulated via the sensing of glycine. However, we found that the riboswitch behaves in a "glycine-independent" manner in the presence of polyethylene glycol (PEG) and ethylene glycol. The effect is related to the formation of a terminator stem within the expression platform under such conditions. The results revealed that increasing PEG stabilized the structure of the terminator stem. By contrast, the addition of ethylene glycol destabilized the terminator stem. PEG and ethylene glycol have opposite effects on transcription as well as on stable terminator stem formation. The glycine-independency of the riboswitch and the effects of such glycols might shed light on the evolution of riboswitches.

  18. Functional roles of a tetraloop/receptor interacting module in a cyclic di-GMP riboswitch.

    Science.gov (United States)

    Fujita, Yuki; Tanaka, Takahiro; Furuta, Hiroyuki; Ikawa, Yoshiya

    2012-02-01

    Riboswitches are a class of structural RNAs that regulate transcription and translation through specific recognition of small molecules. Riboswitches are attractive not only as drug targets for novel antibiotics but also as modular tools for controlling gene expression. Sequence comparison of a class of riboswitches that sense cyclic di-GMP (type-I c-di-GMP riboswitches) revealed that this type of riboswitch frequently shows a GAAA loop/receptor interaction between P1 and P3 elements. In the crystal structures of a type-I c-di-GMP riboswitch from Vibrio cholerae (the Vc2 riboswitch), the GNRA loop/receptor interaction assembled P2 and P3 stems to organize a ligand-binding pocket. In this study, the functional importance of the GAAA loop-receptor interaction in the Vc2 riboswitch was examined. A series of variant Vc2 riboswitches with mutations in the GAAA loop/receptor interaction were assayed for their switching abilities. In mutants with mutations in the P2 GAAA loop, expression of the reporter gene was reduced to approximately 40% - 60% of that in the wild-type. However, mutants in which the P3 receptor motif was substituted with base pairs were as active as the wild-type. These results suggested that the GAAA loop/receptor interaction does not simply establish the RNA 3D structure but docking of P2 GAAA loop reduces the flexibility of the GAAA receptor motif in the P3 element. This mechanism was supported by a variant riboswitch bearing a theophylline aptamer module in P3 the structural rigidity of which could be modulated by the small molecule theophylline.

  19. A kissing loop is important for btuB riboswitch ligand sensing and regulatory control.

    Science.gov (United States)

    Lussier, Antony; Bastet, Laurène; Chauvier, Adrien; Lafontaine, Daniel A

    2015-10-30

    RNA-based genetic regulation is exemplified by metabolite-binding riboswitches that modulate gene expression through conformational changes. Crystal structures show that the Escherichia coli btuB riboswitch contains a kissing loop interaction that is in close proximity to the bound ligand. To analyze the role of the kissing loop interaction in the riboswitch regulatory mechanism, we used RNase H cleavage assays to probe the structure of nascent riboswitch transcripts produced by the E. coli RNA polymerase. By monitoring the folding of the aptamer, kissing loop, and riboswitch expression platform, we established the conformation of each structural component in the absence or presence of bound adenosylcobalamin. We found that the kissing loop interaction is not essential for ligand binding. However, we showed that kissing loop formation improves ligand binding efficiency and is required to couple ligand binding to the riboswitch conformational changes involved in regulating gene expression. These results support a mechanism by which the btuB riboswitch modulates the formation of a tertiary structure to perform metabolite sensing and regulate gene expression. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. A Kissing Loop Is Important for btuB Riboswitch Ligand Sensing and Regulatory Control*

    Science.gov (United States)

    Lussier, Antony; Bastet, Laurène; Chauvier, Adrien; Lafontaine, Daniel A.

    2015-01-01

    RNA-based genetic regulation is exemplified by metabolite-binding riboswitches that modulate gene expression through conformational changes. Crystal structures show that the Escherichia coli btuB riboswitch contains a kissing loop interaction that is in close proximity to the bound ligand. To analyze the role of the kissing loop interaction in the riboswitch regulatory mechanism, we used RNase H cleavage assays to probe the structure of nascent riboswitch transcripts produced by the E. coli RNA polymerase. By monitoring the folding of the aptamer, kissing loop, and riboswitch expression platform, we established the conformation of each structural component in the absence or presence of bound adenosylcobalamin. We found that the kissing loop interaction is not essential for ligand binding. However, we showed that kissing loop formation improves ligand binding efficiency and is required to couple ligand binding to the riboswitch conformational changes involved in regulating gene expression. These results support a mechanism by which the btuB riboswitch modulates the formation of a tertiary structure to perform metabolite sensing and regulate gene expression. PMID:26370077

  1. Aptamers in Bordeaux, 24-25 June 2016.

    Science.gov (United States)

    Toulmé, Jean-Jacques; Giangrande, Paloma H; Mayer, Günter; Suess, Beatrix; Ducongé, Frédéric; Sullenger, Bruce; de Franciscis, Vittorio; Darfeuille, Fabien; Peyrin, Eric

    2017-01-20

    The symposium covered the many different aspects of the selection and the characterization of aptamers as well as their application in analytical, diagnostic and therapeutic areas. Natural and artificial riboswitches were discussed. Recent advances for the design of mutated polymerases and of chemically modified nucleic acid bases that provide aptamers with new properties were presented. The power of aptamer platforms for multiplex analysis of biomarkers of major human diseases was described. The potential of aptamers for the treatment of cancer or cardiovascular diseases was also presented. Brief summaries of the lectures presented during the symposium are given in this report. A second edition of "Aptamers in Bordeaux" will take place on September 2017 (http://www.aptamers-in-bordeaux.com/).

  2. Aptamers in Bordeaux, 24–25 June 2016

    Directory of Open Access Journals (Sweden)

    Jean-Jacques Toulmé

    2017-01-01

    Full Text Available The symposium covered the many different aspects of the selection and the characterization of aptamers as well as their application in analytical, diagnostic and therapeutic areas. Natural and artificial riboswitches were discussed. Recent advances for the design of mutated polymerases and of chemically modified nucleic acid bases that provide aptamers with new properties were presented. The power of aptamer platforms for multiplex analysis of biomarkers of major human diseases was described. The potential of aptamers for the treatment of cancer or cardiovascular diseases was also presented. Brief summaries of the lectures presented during the symposium are given in this report. A second edition of “Aptamers in Bordeaux” will take place on September 2017 (http://www.aptamers-in-bordeaux.com/.

  3. Multiple metal-binding cores are required for metalloregulation by M-box riboswitch RNAs.

    Science.gov (United States)

    Wakeman, Catherine A; Ramesh, Arati; Winkler, Wade C

    2009-09-25

    Riboswitches are regulatory RNAs that control downstream gene expression in response to direct association with intracellular metabolites or metals. Typically, riboswitch aptamer domains bind to a single small-molecule metabolite. In contrast, an X-ray crystallographic structural model for the M-box riboswitch aptamer revealed the absence of an organic metabolite ligand but the presence of at least six tightly associated magnesiums. This observation agrees well with the proposed role of the M-box riboswitch in functioning as a sensor of intracellular magnesium, although additional nonspecific metal interactions are also undoubtedly required for these purposes. To gain greater functional insight into the metalloregulatory capabilities of M-box RNAs, we sought to determine whether all or a subset of the RNA-chelated magnesium ions were required for riboswitch function. To accomplish this task, each magnesium-binding site was simultaneously yet individually perturbed through random incorporation of phosphorothioate nucleotide analogues, and RNA molecules were investigated for their ability to fold in varying levels of magnesium. These data revealed that all of the magnesium ions observed in the structural model are important for magnesium-dependent tertiary structure formation. Additionally, these functional data revealed a new core of potential metal-binding sites that are likely to assist formation of key tertiary interactions and were previously unobserved in the structural model. It is clear from these data that M-box RNAs require specific binding of a network of metal ions for partial fulfillment of their metalloregulatory functions.

  4. Preparation and Crystallization of Riboswitches.

    Science.gov (United States)

    Peselis, Alla; Gao, Ang; Serganov, Alexander

    2016-01-01

    Recent studies have revealed that the majority of biological processes are controlled by noncoding RNAs. Among many classes of noncoding RNAs, metabolite-sensing segments of mRNAs called riboswitches are unique. Discovered over a decade ago in all three kingdoms of life, riboswitches specifically and directly interact with various metabolites and regulate expression of multiple genes, often associated with metabolism and transport of small molecules. Thus, riboswitches do not depend on proteins for binding to small molecules and play a role as both metabolite sensors and effectors of gene control. Riboswitches are typically located in the untranslated regions of mRNAs where they form alternative structures in the presence and absence of the ligand and modulate expression of genes through the formation of regulatory elements. To understand the mechanism of the riboswitch-driven gene control, it is important to elucidate how riboswitches interact with cognate and discriminate against non-cognate ligands. Here we outline the methodology to synthesize riboswitch RNAs and prepare riboswitch-ligand complexes for crystallographic and biochemical studies. The chapter describes how to design, prepare, and conduct crystallization screening of riboswitch-ligand complexes. The methodology was refined on crystallographic studies of several riboswitches and can be employed for other types of RNA molecules.

  5. The dynamic nature of RNA as key to understanding riboswitch mechanisms.

    Science.gov (United States)

    Haller, Andrea; Soulière, Marie F; Micura, Ronald

    2011-12-20

    Riboswitches are gene regulation elements within RNA that recognize specific metabolites. They predominantly occur in the untranslated leader regions of bacterial messenger RNA (mRNA). Upon metabolite binding to the aptamer domain, a structural change in the adjoining downstream expression platform signals "on" or "off" for gene expression. Researchers have achieved much progress in characterizing ligand-bound riboswitch states at the molecular level; an impressive number of high-resolution structures of aptamer-ligand complexes is now available. These structures have significantly contributed toward our understanding of how riboswitches interact with their natural ligands and with structurally related analogues. In contrast, relatively little is known about the nature of the unbound (apo) form of riboswitches. Moreover, the details of how changes in the aptamer domain are transduced into conformational changes in the decision-making expression platform remain murky. In this Account, we report on recent efforts aimed at the characterization of free states, ligand recognition, and ligand-induced folding in riboswitches. Riboswitch action is best approached as a cotranscriptional process, which implies sequential folding and release of the aptamer prior to the signaling of the expression platform. Thus, a complex interplay of several factors has to be taken into account, such as speed of transcription, transcriptional pausing, kinetics and thermodynamics of RNA structure formation, and kinetics and thermodynamics of ligand binding. The response mechanism appears to be best described as a process in which ligand recognition critically dictates the folding pathway of the nascent mRNA during its expression; the resulting structures determine the interactions with the transcriptional or translational apparatus. We discuss experimental methods that offer insight into the dynamics of the free riboswitch state. These include probing experiments, such as in-line and

  6. Structural Basis for Recognition of S-adenosylhomocysteine by Riboswitches

    Energy Technology Data Exchange (ETDEWEB)

    A Edwards; F Reyes; A Heroux; R Batey

    2011-12-31

    S-adenosyl-(L)-homocysteine (SAH) riboswitches are regulatory elements found in bacterial mRNAs that up-regulate genes involved in the S-adenosyl-(L)-methionine (SAM) regeneration cycle. To understand the structural basis of SAH-dependent regulation by RNA, we have solved the structure of its metabolite-binding domain in complex with SAH. This structure reveals an unusual pseudoknot topology that creates a shallow groove on the surface of the RNA that binds SAH primarily through interactions with the adenine ring and methionine main chain atoms and discriminates against SAM through a steric mechanism. Chemical probing and calorimetric analysis indicate that the unliganded RNA can access bound-like conformations that are significantly stabilized by SAH to direct folding of the downstream regulatory switch. Strikingly, we find that metabolites bearing an adenine ring, including ATP, bind this aptamer with sufficiently high affinity such that normal intracellular concentrations of these compounds may influence regulation of the riboswitch.

  7. Structural basis for recognition of S-adenosylhomocysteine by riboswitches

    Energy Technology Data Exchange (ETDEWEB)

    Edwards, A.L.; Heroux, A.; Reyes, F. E.; Batey, R. T.

    2010-11-01

    S-adenosyl-(L)-homocysteine (SAH) riboswitches are regulatory elements found in bacterial mRNAs that up-regulate genes involved in the S-adenosyl-(L)-methionine (SAM) regeneration cycle. To understand the structural basis of SAH-dependent regulation by RNA, we have solved the structure of its metabolite-binding domain in complex with SAH. This structure reveals an unusual pseudoknot topology that creates a shallow groove on the surface of the RNA that binds SAH primarily through interactions with the adenine ring and methionine main chain atoms and discriminates against SAM through a steric mechanism. Chemical probing and calorimetric analysis indicate that the unliganded RNA can access bound-like conformations that are significantly stabilized by SAH to direct folding of the downstream regulatory switch. Strikingly, we find that metabolites bearing an adenine ring, including ATP, bind this aptamer with sufficiently high affinity such that normal intracellular concentrations of these compounds may influence regulation of the riboswitch.

  8. Riboswitch finder—a tool for identification of riboswitch RNAs

    OpenAIRE

    Bengert, P.; Dandekar, T

    2004-01-01

    We describe a dedicated RNA motif search program and web server to identify RNA riboswitches. The Riboswitch finder analyses a given sequence using the web interface, checks specific sequence elements and secondary structure, calculates and displays the energy folding of the RNA structure and runs a number of tests including this information to determine whether high-sensitivity riboswitch motifs (or variants) according to the Bacillus subtilis type are present in the given RNA sequence. Batc...

  9. Nucleotides adjacent to the ligand-binding pocket are linked to activity tuning in the purine riboswitch.

    Science.gov (United States)

    Stoddard, Colby D; Widmann, Jeremy; Trausch, Jeremiah J; Marcano-Velázquez, Joan G; Knight, Rob; Batey, Robert T

    2013-05-27

    Direct sensing of intracellular metabolite concentrations by riboswitch RNAs provides an economical and rapid means to maintain metabolic homeostasis. Since many organisms employ the same class of riboswitch to control different genes or transcription units, it is likely that functional variation exists in riboswitches such that activity is tuned to meet cellular needs. Using a bioinformatic approach, we have identified a region of the purine riboswitch aptamer domain that displays conservation patterns linked to riboswitch activity. Aptamer domain compositions within this region can be divided into nine classes that display a spectrum of activities. Naturally occurring compositions in this region favor rapid association rate constants and slow dissociation rate constants for ligand binding. Using X-ray crystallography and chemical probing, we demonstrate that both the free and bound states are influenced by the composition of this region and that modest sequence alterations have a dramatic impact on activity. The introduction of non-natural compositions result in the inability to regulate gene expression in vivo, suggesting that aptamer domain activity is highly plastic and thus readily tunable to meet cellular needs.

  10. Role of lysine binding residues in the global folding of the lysC riboswitch.

    Science.gov (United States)

    Smith-Peter, Erich; Lamontagne, Anne-Marie; Lafontaine, Daniel A

    2015-01-01

    Riboswitches regulate gene expression by rearranging their structure upon metabolite binding. The lysine-sensing lysC riboswitch is a rare example of an RNA aptamer organized around a 5-way helical junction in which ligand binding is performed exclusively through nucleotides located at the junction core. We have probed whether the nucleotides involved in ligand binding play any role in the global folding of the riboswitch. As predicted, our findings indicate that ligand-binding residues are critical for the lysine-dependent gene regulation mechanism. We also find that these residues are not important for the establishment of key magnesium-dependent tertiary interactions, suggesting that folding and ligand recognition are uncoupled in this riboswitch for the formation of specific interactions. However, FRET assays show that lysine binding results in an additional conformational change, indicating that lysine binding may also participate in a specific folding transition. Thus, in contrast to helical junctions being primary determinants in ribozymes and rRNA folding, we speculate that the helical junction of the lysine-sensing lysC riboswitch is not employed as structural a scaffold to direct global folding, but rather has a different role in establishing RNA-ligand interactions required for riboswitch regulation. Our work suggests that helical junctions may adopt different functions such as the coordination of global architecture or the formation of specific ligand binding site.

  11. Design principles for riboswitch function.

    Directory of Open Access Journals (Sweden)

    Chase L Beisel

    2009-04-01

    Full Text Available Scientific and technological advances that enable the tuning of integrated regulatory components to match network and system requirements are critical to reliably control the function of biological systems. RNA provides a promising building block for the construction of tunable regulatory components based on its rich regulatory capacity and our current understanding of the sequence-function relationship. One prominent example of RNA-based regulatory components is riboswitches, genetic elements that mediate ligand control of gene expression through diverse regulatory mechanisms. While characterization of natural and synthetic riboswitches has revealed that riboswitch function can be modulated through sequence alteration, no quantitative frameworks exist to investigate or guide riboswitch tuning. Here, we combined mathematical modeling and experimental approaches to investigate the relationship between riboswitch function and performance. Model results demonstrated that the competition between reversible and irreversible rate constants dictates performance for different regulatory mechanisms. We also found that practical system restrictions, such as an upper limit on ligand concentration, can significantly alter the requirements for riboswitch performance, necessitating alternative tuning strategies. Previous experimental data for natural and synthetic riboswitches as well as experiments conducted in this work support model predictions. From our results, we developed a set of general design principles for synthetic riboswitches. Our results also provide a foundation from which to investigate how natural riboswitches are tuned to meet systems-level regulatory demands.

  12. Probing of Nascent Riboswitch Transcripts.

    Science.gov (United States)

    Chauvier, Adrien; Lafontaine, Daniel A

    2015-01-01

    The study of biologically significant and native structures is vital to characterize RNA-based regulatory mechanisms. Riboswitches are cis-acting RNA molecules that are involved in the biosynthesis and transport of cellular metabolites. Because riboswitches regulate gene expression by modulating their structure, it is vital to employ native probing assays to determine how native riboswitch structures perform highly efficient and specific ligand recognition. By employing RNase H probing, it is possible to determine the accessibility of specific RNA domains in various structural contexts. Herein, we describe how to employ RNase H probing to characterize nascent mRNA riboswitch molecules as a way to obtain information regarding the riboswitch regulation control mechanism.

  13. Screening for engineered neomycin riboswitches that control translation initiation.

    Science.gov (United States)

    Weigand, Julia E; Sanchez, Martin; Gunnesch, Ewald-Bernd; Zeiher, Sabrina; Schroeder, Renee; Suess, Beatrix

    2008-01-01

    Riboswitches are genetic control elements that regulate gene expression in a small molecule-dependent way. We developed a two-stage strategy of in vitro selection followed by a genetic screen and identified several artificial small molecule-binding riboswitches that respond to the aminoglycoside neomycin. Structure-function relationships and structural probing revealed that they adopt the general neomycin-binding motif. They display no sequence similarities to in vitro selected neomycin aptamers but contain parts of the decoding site that is the binding site for neomycin on the ribosomal RNA. We propose a model of a composed binding pocket of an internal loop as primary docking site and a terminal flaplike loop structure fixing neomycin in a sandwich-like manner. Such binding pockets characterized by multiple contacts between ligand and RNA are described for both natural and engineered riboswitches. We anticipate that combination of in vitro selection and in vivo screening is a useful strategy to identify RNA molecules with a desired functionality.

  14. Structural basis for molecular discrimination by a 3',3'-cGAMP sensing riboswitch.

    Science.gov (United States)

    Ren, Aiming; Wang, Xin C; Kellenberger, Colleen A; Rajashankar, Kanagalaghatta R; Jones, Roger A; Hammond, Ming C; Patel, Dinshaw J

    2015-04-07

    Cyclic dinucleotides are second messengers that target the adaptor STING and stimulate the innate immune response in mammals. Besides protein receptors, there are bacterial riboswitches that selectively recognize cyclic dinucleotides. We recently discovered a natural riboswitch that targets 3',3'-cGAMP, which is distinguished from the endogenous mammalian signal 2',3'-cGAMP by its backbone connectivity. Here, we report on structures of the aptamer domain of the 3',3'-cGAMP riboswitch from Geobacter in the 3',3'-cGAMP and c-di-GMP bound states. The riboswitch adopts a tuning fork-like architecture with a junctional ligand-binding pocket and different orientations of the arms are correlated with the identity of the bound cyclic dinucleotide. Subsequent biochemical experiments revealed that specificity of ligand recognition can be affected by point mutations outside of the binding pocket, which has implications for both the assignment and reengineering of riboswitches in this structural class.

  15. The yjdF riboswitch candidate regulates gene expression by binding diverse azaaromatic compounds.

    Science.gov (United States)

    Li, Sanshu; Hwang, Xue Ying; Stav, Shira; Breaker, Ronald R

    2016-04-01

    The yjdF motif RNA is an orphan riboswitch candidate that almost exclusively associates with the yjdF protein-coding gene in many bacteria. The function of the YjdF protein is unknown, which has made speculation regarding the natural ligand for this putative riboswitch unusually challenging. By using a structure-probing assay for ligand binding, we found that a surprisingly broad diversity of nitrogen-containing aromatic heterocycles, or "azaaromatics," trigger near-identical changes in the structures adopted by representative yjdF motif RNAs. Regions of the RNA that undergo ligand-induced structural modulation reside primarily in portions of the putative aptamer region that are highly conserved in nucleotide sequence, as is typical for riboswitches. Some azaaromatic molecules are bound by the RNA with nanomolar dissociation constants, and a subset of these ligands activate riboswitch-mediated gene expression in cells. Furthermore, genetic elements most commonly adjacent to the yjdF motif RNA or to the yjdF protein-coding region are homologous to protein regulators implicated in mitigating the toxic effects of diverse phenolic acids or polycyclic compounds. Although the precise type of natural ligand sensed by yjdF motif RNAs remains unknown, our findings suggest that this riboswitch class might serve as part of a genetic response system to toxic or signaling compounds with chemical structures similar to azaaromatics.

  16. Structural Basis for Molecular Discrimination by a 3′,3′-cGAMP Sensing Riboswitch

    Directory of Open Access Journals (Sweden)

    Aiming Ren

    2015-04-01

    Full Text Available Cyclic dinucleotides are second messengers that target the adaptor STING and stimulate the innate immune response in mammals. Besides protein receptors, there are bacterial riboswitches that selectively recognize cyclic dinucleotides. We recently discovered a natural riboswitch that targets 3′,3′-cGAMP, which is distinguished from the endogenous mammalian signal 2′,3′-cGAMP by its backbone connectivity. Here, we report on structures of the aptamer domain of the 3′,3′-cGAMP riboswitch from Geobacter in the 3′,3′-cGAMP and c-di-GMP bound states. The riboswitch adopts a tuning fork-like architecture with a junctional ligand-binding pocket and different orientations of the arms are correlated with the identity of the bound cyclic dinucleotide. Subsequent biochemical experiments revealed that specificity of ligand recognition can be affected by point mutations outside of the binding pocket, which has implications for both the assignment and reengineering of riboswitches in this structural class.

  17. The apo riboswitch as a molecular hydra.

    Science.gov (United States)

    Wedekind, Joseph E

    2010-07-14

    Riboswitches "sense" metabolites but knowledge is sparse for structures without bound ligand. Stoddard et al. (2010) determined an apo riboswitch structure "closed" to metabolite binding. Further SAXS, biochemical, and computational analyses support ensemble behavior with interconverting open and closed conformations.

  18. [Novel targets for antibiotics discovery: riboswitches].

    Science.gov (United States)

    Jia, Dong-fang; Jia, Dong-fang; Jia, Dong-fang

    2013-09-01

    Riboswitches are cis-acting domains located in mRNA sequences that could regulate gene expression by sensing small molecules without employing protein. Most known riboswitches in bacteria have naturally evolved to bind essential metabolite ligands and are involved in the regulation of critical genes that are responsible for the biosynthesis or transport of the cognate ligand. The riboswitch-mediated gene expression could be repressed by metabolite analogs, which caused bacterial growth inhibition or even death. A number of leading compounds targeting riboswitches have been discovered. A promising avenue for the development of new class of riboswitch-based antibiotics has been opened. Herein we reviewed the current findings of riboswitches that served as targets for antibacterial drug development and the underlying mechanisms. The development of high-throughput methods and rational drug design for riboswitch-specific drug discovery are relevant challenges are discussed. summarized.

  19. Structural Basis of Ligand Binding by a C-di-GMP Riboswitch

    Energy Technology Data Exchange (ETDEWEB)

    Smith, K.; Lipchock, S; Ames, T; Wang, J; Breaker, R; Strobel, S

    2009-01-01

    The second messenger signaling molecule bis-(3{prime}-5{prime})-cyclic dimeric guanosine monophosphate (c-di-GMP) regulates many processes in bacteria, including motility, pathogenesis and biofilm formation. c-di-GMP-binding riboswitches are important downstream targets in this signaling pathway. Here we report the crystal structure, at 2.7 {angstrom} resolution, of a c-di-GMP riboswitch aptamer from Vibrio cholerae bound to c-di-GMP, showing that the ligand binds within a three-helix junction that involves base-pairing and extensive base-stacking. The symmetric c-di-GMP is recognized asymmetrically with respect to both the bases and the backbone. A mutant aptamer was engineered that preferentially binds the candidate signaling molecule c-di-AMP over c-di-GMP. Kinetic and structural data suggest that genetic regulation by the c-di-GMP riboswitch is kinetically controlled and that gene expression is modulated through the stabilization of a previously unidentified P1 helix, illustrating a direct mechanism for c-di-GMP signaling.

  20. The Quick and the Dead: A Guide to Fast Phasing of Small Ribozyme and Riboswitch Crystal Structures.

    Science.gov (United States)

    Jenkins, Jermaine L; Wedekind, Joseph E

    2016-01-01

    Ribozymes and riboswitches are examples of non-protein-coding (nc)RNA molecules that achieve biological activity by adopting complex three-dimensional folds. Visualization of such molecules at near-atomic resolution can enhance our understanding of how chemical groups are organized spatially, thereby providing novel insight into function. This approach has its challenges, which mainly entail sample crystallization followed by the application of empirical, structure-determination methods that often include experimental "phasing" of X-ray diffraction data. A paucity of high-quality crystals or a low symmetry space group are factors that demand rapid assessment of phasing potential during an ongoing experiment in order to assure a successful outcome. Here we describe the process of evaluating the anomalous signal-to-noise as a prelude to single wavelength or multiwavelength anomalous diffraction (SAD or MAD) phasing. Test cases include an autolytic 62-mer RNA enzyme known as the hairpin ribozyme, and a 33-mer riboswitch that binds the modified guanine metabolite preQ1. The crystals were derivatized with iridium (III) hexammine and osmium (III) pentaammine triflate, respectively. Each data set was then subjected to the XPREP and SHELX programs to assess the anomalous signal-to-noise and to locate the heavy-atom substructure. Subsequent noise filtering was conducted in SHELXE or RESOLVE. The methods described are applicable to the rapid phasing of RNA X-ray diffraction data, and contrast the efficacy of in-house X-rays with those attainable from synchrotron-radiation sources in terms of the potential to plan for and execute an experimental structure determination.

  1. Dissecting electrostatic screening, specific ion binding, and ligand binding in an energetic model for glycine riboswitch folding

    Energy Technology Data Exchange (ETDEWEB)

    Lipfert, Jan; Sim, Adelene Y.L.; Herschlag, Daniel; Doniach, Sebastian (Stanford)

    2010-09-17

    Riboswitches are gene-regulating RNAs that are usually found in the 5{prime}-untranslated regions of messenger RNA. As the sugar-phosphate backbone of RNA is highly negatively charged, the folding and ligand-binding interactions of riboswitches are strongly dependent on the presence of cations. Using small angle X-ray scattering (SAXS) and hydroxyl radical footprinting, we examined the cation dependence of the different folding stages of the glycine-binding riboswitch from Vibrio cholerae. We found that the partial folding of the tandem aptamer of this riboswitch in the absence of glycine is supported by all tested mono- and divalent ions, suggesting that this transition is mediated by nonspecific electrostatic screening. Poisson-Boltzmann calculations using SAXS-derived low-resolution structural models allowed us to perform an energetic dissection of this process. The results showed that a model with a constant favorable contribution to folding that is opposed by an unfavorable electrostatic term that varies with ion concentration and valency provides a reasonable quantitative description of the observed folding behavior. Glycine binding, on the other hand, requires specific divalent ions binding based on the observation that Mg{sup 2+}, Ca{sup 2+}, and Mn{sup 2+} facilitated glycine binding, whereas other divalent cations did not. The results provide a case study of how ion-dependent electrostatic relaxation, specific ion binding, and ligand binding can be coupled to shape the energetic landscape of a riboswitch and can begin to be quantitatively dissected.

  2. Loop-loop interaction in an adenine-sensing riboswitch: a molecular dynamics study.

    Science.gov (United States)

    Allnér, Olof; Nilsson, Lennart; Villa, Alessandra

    2013-07-01

    Riboswitches are mRNA-based molecules capable of controlling the expression of genes. They undergo conformational changes upon ligand binding, and as a result, they inhibit or promote the expression of the associated gene. The close connection between structural rearrangement and function makes a detailed knowledge of the molecular interactions an important step to understand the riboswitch mechanism and efficiency. We have performed all-atom molecular dynamics simulations of the adenine-sensing add A-riboswitch to study the breaking of the kissing loop, one key tertiary element in the aptamer structure. We investigated the aptamer domain of the add A-riboswitch in complex with its cognate ligand and in the absence of the ligand. The opening of the hairpins was simulated using umbrella sampling using the distance between two loops as the reaction coordinate. A two-step process was observed in all the simulated systems. First, a general loss of stacking and hydrogen bond interactions is seen. The last interactions that break are the two base pairs G37-C61 and G38-C60, but the break does not affect the energy profile, indicating their pivotal role in the tertiary structure formation but not in the structure stabilization. The junction area is partially organized before the kissing loop formation and residue A24 anchors together the loop helices. Moreover, when the distance between the loops is increased, one of the hairpins showed more flexibility by changing its orientation in the structure, while the other conserved its coaxial arrangement with the rest of the structure.

  3. Direct observation of multiple tautomers of oxythiamine and their recognition by the thiamine pyrophosphate riboswitch.

    Science.gov (United States)

    Singh, Vipender; Peng, Chunte Sam; Li, Deyu; Mitra, Koyel; Silvestre, Katherine J; Tokmakoff, Andrei; Essigmann, John M

    2014-01-17

    Structural diversification of canonical nucleic acid bases and nucleotide analogues by tautomerism has been proposed to be a powerful on/off switching mechanism allowing regulation of many biological processes mediated by RNA enzymes and aptamers. Despite the suspected biological importance of tautomerism, attempts to observe minor tautomeric forms in nucleic acid or hybrid nucleic acid-ligand complexes have met with challenges due to the lack of sensitive methods. Here, a combination of spectroscopic, biochemical, and computational tools probed tautomerism in the context of an RNA aptamer-ligand complex; studies involved a model ligand, oxythiamine pyrophosphate (OxyTPP), bound to the thiamine pyrophosphate (TPP) riboswitch (an RNA aptamer) as well as its unbound nonphosphorylated form, oxythiamine (OxyT). OxyTPP, similarly to canonical heteroaromatic nucleic acid bases, has a pyrimidine ring that forms hydrogen bonding interactions with the riboswitch. Tautomerism was established using two-dimensional infrared (2D IR) spectroscopy, variable temperature FTIR and NMR spectroscopies, binding isotope effects (BIEs), and computational methods. All three possible tautomers of OxyT, including the minor enol tautomer, were directly identified, and their distributions were quantitated. In the bound form, BIE data suggested that OxyTPP existed as a 4'-keto tautomer that was likely protonated at the N1'-position. These results also provide a mechanistic framework for understanding the activation of riboswitch in response to deamination of the active form of vitamin B1 (or TPP). The combination of methods reported here revealing the fine details of tautomerism can be applied to other systems where the importance of tautomerism is suspected.

  4. Single-molecule conformational dynamics of a biologically functional hydroxocobalamin riboswitch.

    Science.gov (United States)

    Holmstrom, Erik D; Polaski, Jacob T; Batey, Robert T; Nesbitt, David J

    2014-12-03

    Riboswitches represent a family of highly structured regulatory elements found primarily in the leader sequences of bacterial mRNAs. They function as molecular switches capable of altering gene expression; commonly, this occurs via a conformational change in a regulatory element of a riboswitch that results from ligand binding in the aptamer domain. Numerous studies have investigated the ligand binding process, but little is known about the structural changes in the regulatory element. A mechanistic description of both processes is essential for deeply understanding how riboswitches modulate gene expression. This task is greatly facilitated by studying all aspects of riboswitch structure/dynamics/function in the same model system. To this end, single-molecule fluorescence resonance energy transfer (smFRET) techniques have been used to directly observe the conformational dynamics of a hydroxocobalamin (HyCbl) binding riboswitch (env8HyCbl) with a known crystallographic structure.1 The single-molecule RNA construct studied in this work is unique in that it contains all of the structural elements both necessary and sufficient for regulation of gene expression in a biological context. The results of this investigation reveal that the undocking rate constant associated with the disruption of a long-range kissing-loop (KL) interaction is substantially decreased when the ligand is bound to the RNA, resulting in a preferential stabilization of the docked conformation. Notably, the formation of this tertiary KL interaction directly sequesters the Shine-Dalgarno sequence (i.e., the ribosome binding site) via base-pairing, thus preventing translation initiation. These results reveal that the conformational dynamics of this regulatory switch are quantitatively described by a four-state kinetic model, whereby ligand binding promotes formation of the KL interaction. The results of complementary cell-based gene expression experiments conducted in Escherichia coli are highly

  5. The Apo Riboswitch as a Molecular Hydra

    OpenAIRE

    Wedekind, Joseph E.

    2010-01-01

    Riboswitches ‘sense’ metabolites but knowledge is sparse for structures without bound ligand. Stoddard et al. (2010) determined an apo riboswitch structure ‘closed’ to metabolite binding. Further SAXS, biochemical and computational analyses support ensemble behavior with interconverting open and closed conformations.

  6. Riboselector: riboswitch-based synthetic selection device to expedite evolution of metabolite-producing microorganisms.

    Science.gov (United States)

    Jang, Sungho; Yang, Jina; Seo, Sang Woo; Jung, Gyoo Yeol

    2015-01-01

    Many successful metabolic engineering projects have utilized evolutionary approaches, which consist of generating phenotypic diversity and screening for desired phenotype. Since conventional screening methods suffer from low throughput and limited target metabolites, a universal high-throughput screening platform for selection of improved strains should be developed to facilitate evolution of metabolite high producer. Recently, riboswitches have received attention as attractive sensor-actuator hybrids that can control gene expression in response to intracellular metabolite concentration. Our group developed a riboswitch-based selection device called "Riboselector" which can give a growth advantage to metabolite-overproducing strains by modulating expression of a selectable marker gene. We applied the device to expedite evolution of lysine producing Escherichia coli, and the selected strain showed a dramatic improvement of lysine production compared to its parental strain. Moreover, a tryptophan-responsive Riboselector was also developed using synthetic tryptophan aptamer. In this chapter, we provide a step-by-step overview of developing synthetic RNA devices comprising a riboswitch and a selection module that specifically sense inconspicuous metabolites and enrich high producer strains out of library.

  7. Cyclic di-GMP riboswitch-regulated type IV pili contribute to aggregation of Clostridium difficile.

    Science.gov (United States)

    Bordeleau, Eric; Purcell, Erin B; Lafontaine, Daniel A; Fortier, Louis-Charles; Tamayo, Rita; Burrus, Vincent

    2015-03-01

    Clostridium difficile is an anaerobic Gram-positive bacterium that causes intestinal infections with symptoms ranging from mild diarrhea to fulminant colitis. Cyclic diguanosine monophosphate (c-di-GMP) is a bacterial second messenger that typically regulates the switch from motile, free-living to sessile and multicellular behaviors in Gram-negative bacteria. Increased intracellular c-di-GMP concentration in C. difficile was recently shown to reduce flagellar motility and to increase cell aggregation. In this work, we investigated the role of the primary type IV pilus (T4P) locus in c-di-GMP-dependent cell aggregation. Inactivation of two T4P genes, pilA1 (CD3513) and pilB1 (CD3512), abolished pilus formation and significantly reduced cell aggregation under high c-di-GMP conditions. pilA1 is preceded by a putative c-di-GMP riboswitch, predicted to be transcriptionally active upon c-di-GMP binding. Consistent with our prediction, high intracellular c-di-GMP concentration increased transcript levels of T4P genes. In addition, single-round in vitro transcription assays confirmed that transcription downstream of the predicted transcription terminator was dose dependent and specific to c-di-GMP binding to the riboswitch aptamer. These results support a model in which T4P gene transcription is upregulated by c-di-GMP as a result of its binding to an upstream transcriptionally activating riboswitch, promoting cell aggregation in C. difficile.

  8. Selective binding of 2'-F-c-di-GMP to Ct-E88 and Cb-E43, new class I riboswitches from Clostridium tetani and Clostridium botulinum respectively.

    Science.gov (United States)

    Luo, Yiling; Zhou, Jie; Wang, Jingxin; Dayie, T Kwaku; Sintim, Herman O

    2013-06-01

    C-di-GMP is a second messenger in bacteria and partly regulates bacterial physiology by binding to class I and II riboswitches. Four class I c-di-GMP riboswitch aptamer candidates, Ct-E88, Cb-17B, Cb-E43 and Cd-630 RNAs, selected from a GEMM RNA sequence motif in the Rfam database, were expressed and experimentally verified to bind to c-di-GMP. The two newly characterized c-di-GMP riboswitches, Ct-E88 and Cb-E43, bound c-di-GMP with nanomolar Kd whereas the affinities of Cb-17B and Cd-630 for c-di-GMP were at least a 100-fold weaker. Interestingly, whereas the three riboswitches (Vc2, Et-E88 and Cb-E43) bound c-di-GMP with similar Kd values, 2'-modified analogs of c-di-GMP differentially bound to these three class I aptamers. For example, 2'-F-c-di-GMP bound Vc2 with a Kd value of 102 nM whereas the Kd value of 2'-F-c-di-GMP-Ct-E88 is 43 μM (422× higher than that for Vc2 RNA), revealing that there are differences in the binding sites of functional class I c-di-GMP riboswitches.

  9. Structures of riboswitch RNA reaction states by mix-and-inject XFEL serial crystallography

    Science.gov (United States)

    Stagno, J. R.; Liu, Y.; Bhandari, Y. R.; Conrad, C. E.; Panja, S.; Swain, M.; Fan, L.; Nelson, G.; Li, C.; Wendel, D. R.; White, T. A.; Coe, J. D.; Wiedorn, M. O.; Knoska, J.; Oberthuer, D.; Tuckey, R. A.; Yu, P.; Dyba, M.; Tarasov, S. G.; Weierstall, U.; Grant, T. D.; Schwieters, C. D.; Zhang, J.; Ferré-D'Amaré, A. R.; Fromme, P.; Draper, D. E.; Liang, M.; Hunter, M. S.; Boutet, S.; Tan, K.; Zuo, X.; Ji, X.; Barty, A.; Zatsepin, N. A.; Chapman, H. N.; Spence, J. C. H.; Woodson, S. A.; Wang, Y.-X.

    2017-01-01

    Riboswitches are structural RNA elements that are generally located in the 5‧ untranslated region of messenger RNA. During regulation of gene expression, ligand binding to the aptamer domain of a riboswitch triggers a signal to the downstream expression platform. A complete understanding of the structural basis of this mechanism requires the ability to study structural changes over time. Here we use femtosecond X-ray free electron laser (XFEL) pulses to obtain structural measurements from crystals so small that diffusion of a ligand can be timed to initiate a reaction before diffraction. We demonstrate this approach by determining four structures of the adenine riboswitch aptamer domain during the course of a reaction, involving two unbound apo structures, one ligand-bound intermediate, and the final ligand-bound conformation. These structures support a reaction mechanism model with at least four states and illustrate the structural basis of signal transmission. The three-way junction and the P1 switch helix of the two apo conformers are notably different from those in the ligand-bound conformation. Our time-resolved crystallographic measurements with a 10-second delay captured the structure of an intermediate with changes in the binding pocket that accommodate the ligand. With at least a 10-minute delay, the RNA molecules were fully converted to the ligand-bound state, in which the substantial conformational changes resulted in conversion of the space group. Such notable changes in crystallo highlight the important opportunities that micro- and nanocrystals may offer in these and similar time-resolved diffraction studies. Together, these results demonstrate the potential of ‘mix-and-inject’ time-resolved serial crystallography to study biochemically important interactions between biomacromolecules and ligands, including those that involve large conformational changes.

  10. Structures of riboswitch RNA reaction states by mix-and-inject XFEL serial crystallography

    Science.gov (United States)

    Stagno, J. R.; Liu, Y.; Bhandari, Y. R.; Conrad, C. E.; Panja, S.; Swain, M.; Fan, L.; Nelson, G.; Li, C.; Wendel, D. R.; White, T. A.; Coe, J. D.; Wiedorn, M. O.; Knoska, J.; Oberthuer, D.; Tuckey, R. A.; Yu, P.; Dyba, M.; Tarasov, S. G.; Weierstall, U.; Grant, T. D.; Schwieters, C. D.; Zhang, J.; Ferré-D'Amaré, A. R.; Fromme, P.; Draper, D. E.; Liang, M.; Hunter, M. S.; Boutet, S.; Tan, K.; Zuo, X.; Ji, X.; Barty, A.; Zatsepin, N. A.; Chapman, H. N.; Spence, J. C. H.; Woodson, S. A.; Wang, Y.-X.

    2016-11-01

    Riboswitches are structural RNA elements that are generally located in the 5‧ untranslated region of messenger RNA. During regulation of gene expression, ligand binding to the aptamer domain of a riboswitch triggers a signal to the downstream expression platform. A complete understanding of the structural basis of this mechanism requires the ability to study structural changes over time. Here we use femtosecond X-ray free electron laser (XFEL) pulses to obtain structural measurements from crystals so small that diffusion of a ligand can be timed to initiate a reaction before diffraction. We demonstrate this approach by determining four structures of the adenine riboswitch aptamer domain during the course of a reaction, involving two unbound apo structures, one ligand-bound intermediate, and the final ligand-bound conformation. These structures support a reaction mechanism model with at least four states and illustrate the structural basis of signal transmission. The three-way junction and the P1 switch helix of the two apo conformers are notably different from those in the ligand-bound conformation. Our time-resolved crystallographic measurements with a 10-second delay captured the structure of an intermediate with changes in the binding pocket that accommodate the ligand. With at least a 10-minute delay, the RNA molecules were fully converted to the ligand-bound state, in which the substantial conformational changes resulted in conversion of the space group. Such notable changes in crystallo highlight the important opportunities that micro- and nanocrystals may offer in these and similar time-resolved diffraction studies. Together, these results demonstrate the potential of ‘mix-and-inject’ time-resolved serial crystallography to study biochemically important interactions between biomacromolecules and ligands, including those that involve large conformational changes.

  11. Intracellular light-activation of riboswitch activity.

    Science.gov (United States)

    Walsh, Steven; Gardner, Laura; Deiters, Alexander; Williams, Gavin J

    2014-06-16

    By combining a riboswitch with a cell-permeable photocaged small-molecule ligand, an optochemical gene control element was constructed that enabled spatial and temporal control of gene expression in bacterial cells. The simplicity of this strategy, coupled with the ability to create synthetic riboswitches with tailored ligand specificities and output in a variety of microorganisms, plants, and fungi might afford a general strategy to photocontrol gene expression in vivo. The ability to activate riboswitches by using light enables the interrogation and manipulation of a wide range of biological processes with high precision, and will have broad utility in the regulation of artificial genetic circuits.

  12. Folding Kinetics of Riboswitch Transcriptional Terminators

    Science.gov (United States)

    Sauerwine, Benjamin; Widom, Michael

    2009-03-01

    Riboswitches control the expression of genes in bacteria by halting gene transcription or allowing it to proceed based on the presence of ligands in solution. A key feature of every riboswitch is a transcriptional terminator in which the messenger RNA folds into a secondary structure with the stem-loop structure of a hairpin. Through kinetic Monte Carlo simulation we show that terminators have been naturally selected to fold with high reliability on the time-scale of gene transcription. This efficient folding behavior is preserved among two classes of riboswitch and among two species of bacteria.

  13. Structural Basis of Differential Ligand Recognition by Two Classes of bis-(3-5)-cyclic Dimeric Guanosine Monophosphate-binding Riboswitches

    Energy Technology Data Exchange (ETDEWEB)

    K Smith; C Shanahan; E Moore; A Simon; S Strobel

    2011-12-31

    The bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) signaling pathway regulates biofilm formation, virulence, and other processes in many bacterial species and is critical for their survival. Two classes of c-di-GMP-binding riboswitches have been discovered that bind this second messenger with high affinity and regulate diverse downstream genes, underscoring the importance of RNA receptors in this pathway. We have solved the structure of a c-di-GMP-II riboswitch, which reveals that the ligand is bound as part of a triplex formed with a pseudoknot. The structure also shows that the guanine bases of c-di-GMP are recognized through noncanonical pairings and that the phosphodiester backbone is not contacted by the RNA. Recognition is quite different from that observed in the c-di-GMP-I riboswitch, demonstrating that at least two independent solutions for RNA second messenger binding have evolved. We exploited these differences to design a c-di-GMP analog that selectively binds the c-di-GMP-II aptamer over the c-di-GMP-I RNA. There are several bacterial species that contain both types of riboswitches, and this approach holds promise as an important tool for targeting one riboswitch, and thus one gene, over another in a selective fashion.

  14. Amplifying Riboswitch Signal Output using Cellular Wiring

    Science.gov (United States)

    2017-01-30

    replication and chloramphenicol resistance gene. The 2AP riboswitch amplification circuit was compared to the plasmid containing the 2AP riboswitch directly... antibiotic with varying concentrations of 2AP and incubated at 37oC with shaking for 24 h. Fluorescence and OD were measured spectrophotometrically...circuit in JM109 cells. Cells were inoculated into 3 mL LB- antibiotic with 500 M 2AP and incubated at 37oC with shaking. Fluorescence and OD was measured

  15. Microfluidic Screening of Electrophoretic Mobility Shifts Elucidates Riboswitch Binding Function

    OpenAIRE

    Karns, Kelly; Vogan, Jacob M.; Qin, Qian; Hickey, Scott F.; Wilson, Stephen C.; Hammond, Ming C.; Herr, Amy E.

    2013-01-01

    Riboswitches are RNA sensors that change conformation upon binding small molecule metabolites, in turn modulating gene expression. Our understanding of riboswitch regulatory function would be accelerated by a high throughput, quantitative screening tool capable of measuring riboswitch-ligand binding. We introduce a microfluidic mobility shift assay that enables precise and rapid quantitation of ligand binding and subsequent riboswitch conformational change. In 0.3% of the time required for be...

  16. Riboswitch function: Flipping the switch or tuning the dimmer?

    Energy Technology Data Exchange (ETDEWEB)

    Baird, Nathan J.; Kulshina, Nadia; Ferré D' Amaré, Adrian R. (FHCRC)

    2010-10-08

    Riboswitches are structured mRNA elements involved in gene regulation that respond to the intracellular concentration of specific small molecules. Binding of their cognate ligand is thought to elicit a global conformational change of the riboswitch, in addition to modulating the fine structure of the binding site. X-ray crystallography has produced detailed descriptions of the three-dimensional structures of the ligand-bound conformations of several riboswitches. We have employed small-angle X-ray scattering (SAXS) to generate low-resolution reconstructions of the ligand-free states of the ligand-binding domains of riboswitches that respond to thiamine pyrophosphate (TPP), and cyclic diguanylate (c-di-GMP), a bacterial second messenger. Comparison of the SAXS reconstructions with the crystal structures of these two riboswitches demonstrates that the RNAs undergo dramatic ligand-induced global conformational changes. However, this is not an universal feature of riboswitches. SAXS analysis of the solution behavior of several other riboswitch ligand-binding domains demonstrates a broad spectrum of conformational switching behaviors, ranging from the unambiguous switching of the TPP and c-di-GMP riboswitches to complete lack of switching for the flavin mononucleotide (FMN) riboswitch. Moreover, the switching behavior varies between examples of the same riboswitch from different organisms. The range of observed behaviors suggests that in response to the evolutionary need for precise genetic regulation, riboswitches may be tuned to function more as dimmers or rheostats than binary on/off switches.

  17. Rational design of a synthetic mammalian riboswitch as a ligand-responsive -1 ribosomal frame-shifting stimulator

    Science.gov (United States)

    Lin, Ya-Hui; Chang, Kung-Yao

    2016-01-01

    Metabolite-responsive RNA pseudoknots derived from prokaryotic riboswitches have been shown to stimulate −1 programmed ribosomal frameshifting (PRF), suggesting −1 PRF as a promising gene expression platform to extend riboswitch applications in higher eukaryotes. However, its general application has been hampered by difficulty in identifying a specific ligand-responsive pseudoknot that also functions as a ligand-dependent -1 PRF stimulator. We addressed this problem by using the −1 PRF stimulation pseudoknot of SARS-CoV (SARS-PK) to build a ligand-dependent −1 PRF stimulator. In particular, the extra stem of SARS-PK was replaced by an RNA aptamer of theophylline and designed to couple theophylline binding with the stimulation of −1 PRF. Conformational and functional analyses indicate that the engineered theophylline-responsive RNA functions as a mammalian riboswitch with robust theophylline-dependent −1 PRF stimulation activity in a stable human 293T cell-line. Thus, RNA–ligand interaction repertoire provided by in vitro selection becomes accessible to ligand-specific −1 PRF stimulator engineering using SARS-PK as the scaffold for synthetic biology application. PMID:27521370

  18. Aptamers for biosensors

    OpenAIRE

    Bini, A

    2009-01-01

    Aptamers are single-stranded DNA or RNA molecules isolated in vitro by a selection and amplification method. Aptamers bind with high specificity and affinity to a wide range of target molecules, with dissociation constant comparable to antibodies. In this work aptamers were employed as a new kind of bio-recognition element in affinity biosensors for the detection of clinically relevant proteins in heterogeneous assay, using Piezoelectric Quartz Crystal Microbalance and Surface ...

  19. Evolutionary Origin and Conserved Structural Building Blocks of Riboswitches and Ribosomal RNAs: Riboswitches as Probable Target Sites for Aminoglycosides Interaction

    Directory of Open Access Journals (Sweden)

    Elnaz Mehdizadeh Aghdam

    2014-05-01

    Full Text Available Purpose: Riboswitches, as noncoding RNA sequences, control gene expression through direct ligand binding. Sporadic reports on the structural relation of riboswitches with ribosomal RNAs (rRNA, raises an interest in possible similarity between riboswitches and rRNAs evolutionary origins. Since aminoglycoside antibiotics affect microbial cells through binding to functional sites of the bacterial rRNA, finding any conformational and functional relation between riboswitches/rRNAs is utmost important in both of medicinal and basic research. Methods: Analysis of the riboswitches structures were carried out using bioinformatics and computational tools. The possible functional similarity of riboswitches with rRNAs was evaluated based on the affinity of paromomycin antibiotic (targeting “A site” of 16S rRNA to riboswitches via docking method. Results: There was high structural similarity between riboswitches and rRNAs, but not any particular sequence based similarity between them was found. The building blocks including "hairpin loop containing UUU", "peptidyl transferase center conserved hairpin A loop"," helix 45" and "S2 (G8 hairpin" as high identical rRNA motifs were detected in all kinds of riboswitches. Surprisingly, binding energies of paromomycin with different riboswitches are considerably better than the binding energy of paromomycin with “16S rRNA A site”. Therefore the high affinity of paromomycin to bind riboswitches in comparison with rRNA “A site” suggests a new insight about riboswitches as possible targets for aminoglycoside antibiotics. Conclusion: These findings are considered as a possible supporting evidence for evolutionary origin of riboswitches/rRNAs and also their role in the exertion of antibiotics effects to design new drugs based on the concomitant effects via rRNA/riboswitches.

  20. Structural and Biochemical Determinants of Ligand Binding by the c-di-GMP Riboswitch

    Energy Technology Data Exchange (ETDEWEB)

    Smith, K.; Lipchock, S; Livingston,; Shanahan, C; Strobel, S

    2010-01-01

    The bacterial second messenger c-di-GMP is used in many species to control essential processes that allow the organism to adapt to its environment. The c-di-GMP riboswitch (GEMM) is an important downstream target in this signaling pathway and alters gene expression in response to changing concentrations of c-di-GMP. The riboswitch selectively recognizes its second messenger ligand primarily through contacts with two critical nucleotides. However, these two nucleotides are not the most highly conserved residues within the riboswitch sequence. Instead, nucleotides that stack with c-di-GMP and that form tertiary RNA contacts are the most invariant. Biochemical and structural evidence reveals that the most common natural variants are able to make alternative pairing interactions with both guanine bases of the ligand. Additionally, a high-resolution (2.3 {angstrom}) crystal structure of the native complex reveals that a single metal coordinates the c-di-GMP backbone. Evidence is also provided that after transcription of the first nucleotide on the 3{prime}-side of the P1 helix, which is predicted to be the molecular switch, the aptamer is functional for ligand binding. Although large energetic effects occur when several residues in the RNA are altered, mutations at the most conserved positions, rather than at positions that base pair with c-di-GMP, have the most detrimental effects on binding. Many mutants retain sufficient c-di-GMP affinity for the RNA to remain biologically relevant, which suggests that this motif is quite resilient to mutation.

  1. Abundance and functional diversity of riboswitches in microbial communities

    Directory of Open Access Journals (Sweden)

    Gelfand Mikhail S

    2007-10-01

    Full Text Available Abstract Background Several recently completed large-scale enviromental sequencing projects produced a large amount of genetic information about microbial communities ('metagenomes' which is not biased towards cultured organisms. It is a good source for estimation of the abundance of genes and regulatory structures in both known and unknown members of microbial communities. In this study we consider the distribution of RNA regulatory structures, riboswitches, in the Sargasso Sea, Minnesota Soil and Whale Falls metagenomes. Results Over three hundred riboswitches were found in about 2 Gbp metagenome DNA sequences. The abundabce of riboswitches in metagenomes was highest for the TPP, B12 and GCVT riboswitches; the S-box, RFN, YKKC/YXKD, YYBP/YKOY regulatory elements showed lower but significant abundance, while the LYS, G-box, GLMS and YKOK riboswitches were rare. Regions downstream of identified riboswitches were scanned for open reading frames. Comparative analysis of identified ORFs revealed new riboswitch-regulated functions for several classes of riboswitches. In particular, we have observed phosphoserine aminotransferase serC (COG1932 and malate synthase glcB (COG2225 to be regulated by the glycine (GCVT riboswitch; fatty acid desaturase ole1 (COG1398, by the cobalamin (B12 riboswitch; 5-methylthioribose-1-phosphate isomerase ykrS (COG0182, by the SAM-riboswitch. We also identified conserved riboswitches upstream of genes of unknown function: thiamine (TPP, cobalamine (B12, and glycine (GCVT, upstream of genes from COG4198. Conclusion This study demonstrates applicability of bioinformatics to the analysis of RNA regulatory structures in metagenomes.

  2. Cyclic dinucleotide detection with riboswitch-G-quadruplex hybrid.

    Science.gov (United States)

    Tsuji, Genichiro; Sintim, Herman O

    2016-03-01

    A cyclic dinucleotide riboswitch has been fused with a G-quadruplex motif to produce a conditional riboswitch-peroxidase-mimicking sensor that oxidizes both colorimetric and fluorogenic substrates in the presence of c-di-GMP. We find that signal-to-noise ratio could be improved by using a two-, not three-, floor split G-quadruplex for this conditional peroxidase-mimicking riboswitch.

  3. Riboswitch control of Rho-dependent transcription termination.

    Science.gov (United States)

    Hollands, Kerry; Proshkin, Sergey; Sklyarova, Svetlana; Epshtein, Vitaly; Mironov, Alexander; Nudler, Evgeny; Groisman, Eduardo A

    2012-04-03

    Riboswitches are RNA sensors that regulate gene expression upon binding specific metabolites or ions. Bacterial riboswitches control gene expression primarily by promoting intrinsic transcription termination or by inhibiting translation initiation. We now report a third general mechanism of riboswitch action: governing the ability of the RNA-dependent helicase Rho to terminate transcription. We establish that Rho promotes transcription termination in the Mg(2+)-sensing mgtA riboswitch from Salmonella enterica serovar Typhimurium and the flavin mononucleotide-sensing ribB riboswitch from Escherichia coli when the corresponding riboswitch ligands are present. The Rho-specific inhibitor bicyclomycin enabled transcription of the coding regions at these two loci in bacteria experiencing repressing concentrations of the riboswitch ligands in vivo. A mutation in the mgtA leader that favors the "high Mg(2+)" conformation of the riboswitch promoted Rho-dependent transcription termination in vivo and in vitro and enhanced the ability of the RNA to stimulate Rho's ATPase activity in vitro. These effects were overcome by mutations in a C-rich region of the mRNA that is alternately folded at high and low Mg(2+), suggesting a role for this region in regulating the activity of Rho. Our results reveal a potentially widespread mode of gene regulation whereby riboswitches dictate whether a protein effector can interact with the transcription machinery to prematurely terminate transcription.

  4. Aptamer-based nanobiosensors.

    Science.gov (United States)

    Kim, Yeon Seok; Raston, Nurul Hanun Ahmad; Gu, Man Bock

    2016-02-15

    It has been more than two decades since aptamer and the systematic evolution of ligands by exponential enrichment (SELEX) method were discovered by Larry Gold and Andrew Ellington in 1990, respectively. Based on the various advantages of aptamers, they have become a potent rival of antibodies in therapeutics and bio-analysis. Especially, the recent advances in aptamer biosensor application are remarkable due to its intrinsic properties of aptamers as nucleic acids and target induced conformational changes, in addition to the introduction of graphene oxide-based easy and simple immobilization-free screening method even for dual aptamers. In addition, the incorporation of various nanomaterials such as metallic nanoparticles, carbon materials, and functional nanospheres in aptasensors has facilitated the improvement of analytical performance and commercial application of aptasensors. In this review, recent prominent reports on aptasensors utilizing nanomaterials were introduced to understand the principle of aptamer-based biosensors and provide an insight for new strategies of aptasensors and the application of various nanomaterials. The perspective on aptamer-based biosensors and diagnostics was also discussed in view of technology and market.

  5. Dynamic behaviour of the B12 riboswitch

    Science.gov (United States)

    Santillán, Moisés; Mackey, Michael C.

    2005-03-01

    Riboswitches are RNA segments that serve as ligand-responsive genetic control elements. They modulate the expression of certain genes in response to changing concentrations of metabolites. In this paper, we study the dynamic behaviour of the B12 riboswitch in E. coli—perhaps the most widely studied and best known of all riboswitches—through a mathematical model of its regulatory pathway. To carry this out, we simulate dynamic experiments in which the bacterial B12 uptake capacity is measured after being depleted of this vitamin for a long time. The results of these simulations compare favourably with reported experimental data. The model also predicts that an overshoot of intracellular B12 should be observed if the replenishment experiments were to be carried out for longer times. This behaviour is discussed in terms of a possible evolutionary advantage for E. coli, together with the fact that regulation at the transcriptional and translational levels is almost equivalent dynamically.

  6. Carba-sugars Activate the glmS-Riboswitch of Staphylococcus aureus

    OpenAIRE

    Lünse, Christina E.; Schmidt, Magnus S.; Wittmann, Valentin; Mayer, Günter

    2011-01-01

    The glmS-riboswitch is unique among riboswitch families as it represents a metabolite-dependent ribozyme that undergoes self-cleavage upon recognition of glucosamin-6-phosphate. The glmS-riboswitch is located in the 5′-untranslated region of bacterial genes involved in cell wall biosynthesis. Therefore, this riboswitch represents a promising target for developing new antibiotics. We describe the metabolite-dependent glmS-riboswitch of pathologically relevant and vancomycin-resistant Staphyloc...

  7. Fluorescence monitoring of riboswitch transcription regulation using a dual molecular beacon assay

    OpenAIRE

    Chinnappan, Raja; Dubé, Audrey; Lemay, Jean-François; Daniel A Lafontaine

    2013-01-01

    Riboswitches are mRNA elements that specifically bind cellular metabolites and control gene expression by modifying their structure. As riboswitches often control essential genes in pathogenic bacteria, riboswitches have been proposed as new targets for antibiotics. High-throughput screening provides a powerful approach to identify riboswitch ligand analogs that could act as powerful antibacterial drugs. Biochemical assays have already been used to find riboswitch-binding analogs, but those m...

  8. Targeting of Antibodies using Aptamers

    OpenAIRE

    2003-01-01

    The chapter presents a methodology for the rapid selection of aptamers against antibody targets. It is a detailed account of the various methodological steps that describe the selection of aptamers, including PCR steps, buffers to be used, target immobilisation, partitioning and amplification of aptamers, clonning and sequencing, to results in high affinity and specificity ligands for the chosen target antibody.

  9. Novel TPP-riboswitch activators bypass metabolic enzyme dependency

    Directory of Open Access Journals (Sweden)

    Günter eMayer

    2014-07-01

    Full Text Available Riboswitches are conserved regions within mRNA molecules that bind specific metabolites and regulate gene expression. TPP-riboswitches, which respond to thiamine pyrophosphate (TPP, are involved in the regulation of thiamine metabolism in numerous bacteria. As these regulatory RNAs are often modulating essential biosynthesis pathways they have become increasingly interesting as promising antibacterial targets. Here, we describe thiamine analogs containing a central 1,2,3-triazole group to induce repression of thiM-riboswitch dependent gene expression in different E. coli strains. Additionally, we show that compound activation is dependent on proteins involved in the metabolic pathways of thiamine uptake and synthesis. The most promising molecule, triazolethiamine (TT, shows concentration dependent reporter gene repression that is dependent on the presence of thiamine kinase ThiK, whereas the effect of pyrithiamine (PT, a known TPP-riboswitch modulator, is ThiK independent. We further show that this dependence can be bypassed by triazolethiamine-derivatives that bear phosphate-mimicking moieties. As triazolethiamine reveals superior activity compared to pyrithiamine, it represents a very promising starting point for developing novel antibacterial compounds that target TPP-riboswitches. Riboswitch-targeting compounds engage diverse endogenous mechanisms to attain in vivo activity. These findings are of importance for the understanding of compounds that require metabolic activation to achieve effective riboswitch modulation and they enable the design of novel compound generations that are independent of endogenous activation mechanisms.

  10. Novel TPP-riboswitch activators bypass metabolic enzyme dependency

    Science.gov (United States)

    Mayer, Günter; Lünse, Christina; Suckling, Colin; Scott, Fraser

    2014-07-01

    Riboswitches are conserved regions within mRNA molecules that bind specific metabolites and regulate gene expression. TPP-riboswitches, which respond to thiamine pyrophosphate (TPP), are involved in the regulation of thiamine metabolism in numerous bacteria. As these regulatory RNAs are often modulating essential biosynthesis pathways they have become increasingly interesting as promising antibacterial targets. Here, we describe thiamine analogs containing a central 1,2,3-triazole group to induce repression of thiM-riboswitch dependent gene expression in different E. coli strains. Additionally, we show that compound activation is dependent on proteins involved in the metabolic pathways of thiamine uptake and synthesis. The most promising molecule, triazolethiamine (TT), shows concentration dependent reporter gene repression that is dependent on the presence of thiamine kinase ThiK, whereas the effect of pyrithiamine (PT), a known TPP-riboswitch modulator, is ThiK independent. We further show that this dependence can be bypassed by triazolethiamine-derivatives that bear phosphate-mimicking moieties. As triazolethiamine reveals superior activity compared to pyrithiamine, it represents a very promising starting point for developing novel antibacterial compounds that target TPP-riboswitches. Riboswitch-targeting compounds engage diverse endogenous mechanisms to attain in vivo activity. These findings are of importance for the understanding of compounds that require metabolic activation to achieve effective riboswitch modulation and they enable the design of novel compound generations that are independent of endogenous activation mechanisms.

  11. Structural principles of nucleoside selectivity in a 2'-deoxyguanosine riboswitch.

    Science.gov (United States)

    Pikovskaya, Olga; Polonskaia, Anna; Patel, Dinshaw J; Serganov, Alexander

    2011-08-14

    Purine riboswitches have an essential role in genetic regulation of bacterial metabolism. This family includes the 2'-deoxyguanosine (dG) riboswitch, which is involved in feedback control of deoxyguanosine biosynthesis. To understand the principles that define dG selectivity, we determined crystal structures of the natural Mesoplasma florum riboswitch bound to cognate dG as well as to noncognate guanosine, deoxyguanosine monophosphate and guanosine monophosphate. Comparison with related purine riboswitch structures reveals that the dG riboswitch achieves its specificity through modification of key interactions involving the nucleobase and rearrangement of the ligand-binding pocket to accommodate the additional sugar moiety. In addition, we observe new conformational changes beyond the junctional binding pocket extending as far as peripheral loop-loop interactions. It appears that re-engineering riboswitch scaffolds will require consideration of selectivity features dispersed throughout the riboswitch tertiary fold, and structure-guided drug design efforts targeted to junctional RNA scaffolds need to be addressed within such an expanded framework.

  12. RNA-Puzzles Round III: 3D RNA structure prediction of five riboswitches and one ribozyme.

    Science.gov (United States)

    Miao, Zhichao; Adamiak, Ryszard W; Antczak, Maciej; Batey, Robert T; Becka, Alexander J; Biesiada, Marcin; Boniecki, Michał J; Bujnicki, Janusz; Chen, Shi-Jie; Cheng, Clarence Yu; Chou, Fang-Chieh; Ferré-D'Amaré, Adrian R; Das, Rhiju; Dawson, Wayne K; Feng, Ding; Dokholyan, Nikolay V; Dunin-Horkawicz, Stanisław; Geniesse, Caleb; Kappel, Kalli; Kladwang, Wipapat; Krokhotin, Andrey; Łach, Grzegorz E; Major, François; Mann, Thomas H; Magnus, Marcin; Pachulska-Wieczorek, Katarzyna; Patel, Dinshaw J; Piccirilli, Joseph A; Popenda, Mariusz; Purzycka, Katarzyna J; Ren, Aiming; Rice, Greggory M; Santalucia, John; Sarzynska, Joanna; Szachniuk, Marta; Tandon, Arpit; Trausch, Jeremiah J; Tian, Siqi; Wang, Jian; Weeks, Kevin M; Williams, Benfeard; Xiao, Yi; Xu, Xiaojun; Zhang, Dong; Zok, Tomasz; Westhof, Eric

    2017-01-30

    RNA-Puzzles is a collective experiment in blind 3D RNA structure prediction. We report here a third round of RNA-Puzzles. Five puzzles, 4, 8, 12, 13, 14, all structures of riboswitch aptamers and puzzle 7, a ribozyme structure, are included in this round of the experiment. The riboswitch structures include biological binding sites for small molecules (S-adenosyl methionine, cyclic diadenosine monophosphate, 5-amino 4-imidazole carboxamide riboside 5'-triphosphate, glutamine) and proteins (YbxF) and one set describes large conformational changes between ligand-free and ligand-bound states; the Varkud satellite ribozyme is the most recently solved structure of a known large ribozyme. All the puzzles have established biological functions and require structural understanding to appreciate their molecular mechanisms. Through the use of fast-track experimental data, including multidimensional chemical mapping, and accurate prediction of RNA secondary structure, a large portion of the contacts in 3D have been predicted correctly leading to similar topologies for the top ranking predictions. Template-based and homology-derived predictions could predict structures to particularly high accuracies. However, achieving biological insights from de novo prediction of RNA 3D structures still depends on the size and complexity of the RNA. Blind computational predictions of RNA structures already appear to provide useful structural information in many cases. Similar to the previous RNA-Puzzles Round II experiment, the prediction of non-Watson-Crick interactions and the observed high atomic clash scores reveal notable need for algorithm of improvement. All prediction models and assessment results are available at http://ahsoka.u-strasbg.fr/rnapuzzles/.

  13. Structure-guided mutational analysis of gene regulation by the Bacillus subtilis pbuE adenine-responsive riboswitch in a cellular context.

    Science.gov (United States)

    Marcano-Velázquez, Joan G; Batey, Robert T

    2015-02-13

    Riboswitches are a broadly distributed form of RNA-based gene regulation in Bacteria and, more rarely, Archaea and Eukarya. Most often found in the 5'-leader sequence of bacterial mRNAs, they are generally composed of two functional domains: a receptor (aptamer) domain that binds an effector molecule and a regulatory domain (or expression platform) that instructs the expression machinery. One of the most studied riboswitches is the Bacillus subtilis adenine-responsive pbuE riboswitch, which regulates gene expression at the transcriptional level, up-regulating expression in response to increased intracellular effector concentrations. In this work, we analyzed sequence and structural elements that contribute to efficient ligand-dependent regulatory activity in a co-transcriptional and cellular context. Unexpectedly, we found that the P1 helix, which acts as the antitermination element of the switch in this RNA, supported ligand-dependent activation of a reporter gene over a broad spectrum of lengths from 3 to 10 bp. This same trend was also observed using a minimal in vitro single-turnover transcription assay, revealing that this behavior is intrinsic to the RNA sequence. We also found that the sequences at the distal tip of the terminator not directly involved in alternative secondary structure formation are highly important for efficient regulation. These data strongly support a model in which the switch is highly localized to the P1 helix adjacent to the ligand-binding pocket that likely presents a local kinetic block to invasion of the aptamer by the terminator.

  14. Beyond Crystallography: Investigating the Conformational Dynamics of the Purine Riboswitch

    Science.gov (United States)

    Stoddard, Colby D.; Batey, Robert T.

    Riboswitches are structured elements located in the 5'-untranslated regions of numerous bacterial mRNAs that serve to regulate gene expression via their ability to specifically bind metabolites. The purine riboswitch ligand-binding domain has emerged as an important model system for investigating the relationship between RNA structure and function. Directed by NMR and crystallographically generated structures of this RNA, a variety of biophysical and biochemical techniques have been utilized to understand its dynamic nature. In this review, we describe these various approaches and what they reveal about the purine riboswitch.

  15. Imaging metabolite dynamics in living cells using a Spinach-based riboswitch.

    Science.gov (United States)

    You, Mingxu; Litke, Jacob L; Jaffrey, Samie R

    2015-05-26

    Riboswitches are natural ligand-sensing RNAs typically that are found in the 5' UTRs of mRNA. Numerous classes of riboswitches have been discovered, enabling mRNA to be regulated by diverse and physiologically important cellular metabolites and small molecules. Here we describe Spinach riboswitches, a new class of genetically encoded metabolite sensor derived from naturally occurring riboswitches. Drawing upon the structural switching mechanism of natural riboswitches, we show that Spinach can be swapped for the expression platform of various riboswitches, allowing metabolite binding to induce Spinach fluorescence directly. In the case of the thiamine 5'-pyrophosphate (TPP) riboswitch from the Escherichia coli thiM gene encoding hydroxyethylthiazole kinase, we show that insertion of Spinach results in an RNA sensor that exhibits fluorescence upon binding TPP. This TPP Spinach riboswitch binds TPP with affinity and selectivity similar to that of the endogenous riboswitch and enables the discovery of agonists and antagonists of the TPP riboswitch using simple fluorescence readouts. Furthermore, expression of the TPP Spinach riboswitch in Escherichia coli enables live imaging of dynamic changes in intracellular TPP concentrations in individual cells. Additionally, we show that other riboswitches that use a structural mechanism similar to that of the TPP riboswitch, including the guanine and adenine riboswitches from the Bacillus subtilis xpt gene encoding xanthine phosphoribosyltransferase, and the S-adenosyl-methionine-I riboswitch from the B. subtilis yitJ gene encoding methionine synthase, can be converted into Spinach riboswitches. Thus, Spinach riboswitches constitute a novel class of RNA-based fluorescent metabolite sensors that exploit the diversity of naturally occurring ligand-binding riboswitches.

  16. Comparative study between transcriptionally- and translationally-acting adenine riboswitches reveals key differences in riboswitch regulatory mechanisms.

    Directory of Open Access Journals (Sweden)

    Jean-François Lemay

    2011-01-01

    Full Text Available Many bacterial mRNAs are regulated at the transcriptional or translational level by ligand-binding elements called riboswitches. Although they both bind adenine, the adenine riboswitches of Bacillus subtilis and Vibrio vulnificus differ by controlling transcription and translation, respectively. Here, we demonstrate that, beyond the obvious difference in transcriptional and translational modulation, both adenine riboswitches exhibit different ligand binding properties and appear to operate under different regulation regimes (kinetic versus thermodynamic. While the B. subtilis pbuE riboswitch fully depends on co-transcriptional binding of adenine to function, the V. vulnificus add riboswitch can bind to adenine after transcription is completed and still perform translation regulation. Further investigation demonstrates that the rate of transcription is critical for the B. subtilis pbuE riboswitch to perform efficiently, which is in agreement with a co-transcriptional regulation. Our results suggest that the nature of gene regulation control, that is transcription or translation, may have a high importance in riboswitch regulatory mechanisms.

  17. Folding Kinetics of Riboswitch Transcriptional Terminators and Sequesterers

    Directory of Open Access Journals (Sweden)

    Ben Sauerwine

    2013-07-01

    Full Text Available To function as gene regulatory elements in response to environmental signals, riboswitches must adopt specific secondary structures on appropriate time scales. We employ kinetic Monte Carlo simulation to model the time-dependent folding during transcription of thiamine pyrophosphate (TPP riboswitch expression platforms. According to our simulations, riboswitch transcriptional terminators, which must adopt a specific hairpin configuration by the time they have been transcribed, fold with higher efficiency than Shine-Dalgarno sequesterers, whose proper structure is required only at the time of ribosomal binding. Our findings suggest both that riboswitch transcriptional terminator sequences have been naturally selected for high folding efficiency, and that sequesterers can maintain their function even in the presence of significant misfolding.

  18. A riboswitch-based inducible gene expression system for mycobacteria.

    Directory of Open Access Journals (Sweden)

    Jessica C Seeliger

    Full Text Available Research on the human pathogen Mycobacterium tuberculosis (Mtb would benefit from novel tools for regulated gene expression. Here we describe the characterization and application of a synthetic riboswitch-based system, which comprises a mycobacterial promoter for transcriptional control and a riboswitch for translational control. The system was used to induce and repress heterologous protein overexpression reversibly, to create a conditional gene knockdown, and to control gene expression in a macrophage infection model. Unlike existing systems for controlling gene expression in Mtb, the riboswitch does not require the co-expression of any accessory proteins: all of the regulatory machinery is encoded by a short DNA segment directly upstream of the target gene. The inducible riboswitch platform has the potential to be a powerful general strategy for creating customized gene regulation systems in Mtb.

  19. Folding Kinetics of Riboswitch Transcriptional Terminators and Sequesterers

    Science.gov (United States)

    Sauerwine, Ben; Widom, Michael

    2013-07-01

    To function as gene regulatory elements in response to environmental signals, riboswitches must adopt specific secondary structures on appropriate time scales. We employ kinetic Monte Carlo simulation to model the time-dependent folding during transcription of TPP riboswitch expression platforms. According to our simulations, riboswitch transcriptional terminators, which must adopt a specific hairpin configuration by the time they have been transcribed, fold with higher efficiency than Shine-Dalgarno sequesterers, whose proper structure is required only at the time of ribosomal binding. Our findings suggest both that riboswitch transcriptional terminator sequences have been naturally selected for high folding efficiency, and that sequesterers can maintain their function even in the presence of significant misfolding.

  20. Recent advances of aptamer sensors

    Institute of Scientific and Technical Information of China (English)

    LI YiLin; GUO Lei; ZHANG ZhaoYang; TANG JiJun; XIE JianWei

    2008-01-01

    Aptamers are a series of high-affinity and high-specificity oligoneucleotides (single-stranded DNA or RNA) to the target, usually selected by the combinatorial chemistry SELEX technique (systematic evolution of ligands by exponential enrichment). Aptamers have proved to be one kind of novel func-tional molecules in life science and chemistry. After being labeled by signaling groups, the aptamer probe can conveniently transfer the characteristics of aptamer-target recognition to a form of high-sensitive signal, and the high-affinity, high-specificity measurements of metal ion, organic mole-cules, nucleic acid, proteins, or cells become possible. This article summarizes the recent advances of aptamer probes in different sensing fields, with special emphasis on aptamer probes as fluorescent sensors.

  1. Recent advances of aptamer sensors

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Aptamers are a series of high-affinity and high-specificity oligoneucleotides (single-stranded DNA or RNA) to the target, usually selected by the combinatorial chemistry SELEX technique (systematic evolution of ligands by exponential enrichment). Aptamers have proved to be one kind of novel functional molecules in life science and chemistry. After being labeled by signaling groups, the aptamer probe can conveniently transfer the characteristics of aptamer-target recognition to a form of high-sensitive signal, and the high-affinity, high-specificity measurements of metal ion, organic molecules, nucleic acid, proteins, or cells become possible. This article summarizes the recent advances of aptamer probes in different sensing fields, with special emphasis on aptamer probes as fluorescent sensors.

  2. The Toolbox for Modified Aptamers.

    Science.gov (United States)

    Lapa, Sergey A; Chudinov, Alexander V; Timofeev, Edward N

    2016-02-01

    Aptamers are nucleic acid-based scaffolds that can bind with high affinity to a variety of biological targets. Aptamers are identified from large DNA or RNA libraries through a process of directed molecular evolution (SELEX). Chemical modification of nucleic acids considerably increases the functional and structural diversity of aptamer libraries and substantially increases the affinity of the aptamers. Additionally, modified aptamers exhibit much greater resistance to biodegradation. The evolutionary selection of modified aptamers is conditioned by the possibility of the enzymatic synthesis and replication of non-natural nucleic acids. Wild-type or mutant polymerases and their non-natural nucleotide substrates that can support SELEX are highlighted in the present review. A focus is made on the efforts to find the most suitable type of nucleotide modifications and the engineering of new polymerases. Post-SELEX modification as a complementary method will be briefly considered as well.

  3. Aptamers: The "evolution" of SELEX.

    Science.gov (United States)

    Wu, Yi Xi; Kwon, Young Jik

    2016-08-15

    It has been more than two decades since the first aptamer molecule was discovered. Since then, aptamer molecules have gain much attention in the scientific field. This increasing traction can be attributed to their many desirable traits, such as 1) their potentials to bind a wide range of molecules, 2) their malleability, and 3) their low cost of production. These traits have made aptamer molecules an ideal platform to pursue in the realm of pharmaceuticals and bio-sensors. Despite the broad applications of aptamers, tedious procedure, high resource consumption, and limited nucleobase repertoire have hindered aptamer in application usage. To address these issues, new innovative methodologies, such as automation and single round SELEX, are being developed to improve the outcomes and rates in which aptamers are discovered. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Aptamers and Their Biological Applications

    Directory of Open Access Journals (Sweden)

    Changill Ban

    2012-01-01

    Full Text Available Recently, aptamers have attracted the attention of many scientists, because they not only have all of the advantages of antibodies, but also have unique merits, such as thermal stability, low cost, and unlimited applications. In this review, we present the reasons why aptamers are known as alternatives to antibodies. Furthermore, several types of in vitro selection processes, including nitrocellulose membrane filtration, affinity chromatography, magnetic bead, and capillary electrophoresis-based selection methods, are explained in detail. We also introduce various applications of aptamers for the diagnosis of diseases and detection of small molecules. Numerous analytical techniques, such as electrochemical, colorimetric, optical, and mass-sensitive methods, can be utilized to detect targets, due to convenient modifications and the stability of aptamers. Finally, several medical and analytical applications of aptamers are presented. In summary, aptamers are promising materials for diverse areas, not just as alternatives to antibodies, but as the core components of medical and analytical equipment.

  5. Use of aptamers in immunoassays.

    Science.gov (United States)

    Nezlin, Roald

    2016-02-01

    Aptamers, short single-chain DNA or RNA oligonucleotides, react specifically with small molecules, as well as with proteins. Unlike antibodies, they may be obtained relatively easily. Aptamers are now widely employed in immunological studies and could replace antibodies in immunoassays. In this short review, methods for immobilizing aptamers on various insoluble materials (so-called apta-sorbents) are described. Recent findings on their use in the detection and isolation of immunoglobulins and their application in various immunoassays are also discussed.

  6. Nucleic Acid Aptamers Against Proteases

    DEFF Research Database (Denmark)

    Dupont, D M; Andersen, L M; Bøtkjær, Kenneth Alrø

    2011-01-01

    Proteases are potential or realized therapeutic targets in a wide variety of pathological conditions. Moreover, proteases are classical subjects for studies of enzymatic and regulatory mechanisms. We here review the literature on nucleic acid aptamers selected with proteases as targets. Designing...... strategies and of new principles for regulating the activity of the inhibitory action of aptamers of general interest to researchers working with nucleic acid aptamers...

  7. Nucleic Acid Aptamers Against Proteases

    DEFF Research Database (Denmark)

    Dupont, D M; Andersen, L M; Bøtkjær, Kenneth Alrø

    2011-01-01

    -specifically, for instance with vastly different affinities to zymogen and active enzyme forms. Furthermore, aptamers can be selected to inhibit the enzyme activity of the target proteases, but also to inhibit functionally important exosite interactions, for instance cofactor binding. Several protease-inhibiting aptamers......, directed against blood coagulation factors, are in clinical trials as anticoagulant drugs. Several of the studies on protease-binding aptamers have been pioneering and trend-setting in the field. The work with protease-binding aptamers also demonstrates many interesting examples of non-standard selection...

  8. Fluorescence monitoring of riboswitch transcription regulation using a dual molecular beacon assay.

    Science.gov (United States)

    Chinnappan, Raja; Dubé, Audrey; Lemay, Jean-François; Lafontaine, Daniel A

    2013-05-01

    Riboswitches are mRNA elements that specifically bind cellular metabolites and control gene expression by modifying their structure. As riboswitches often control essential genes in pathogenic bacteria, riboswitches have been proposed as new targets for antibiotics. High-throughput screening provides a powerful approach to identify riboswitch ligand analogs that could act as powerful antibacterial drugs. Biochemical assays have already been used to find riboswitch-binding analogs, but those methods do take into account the transcriptional context for riboswitch regulation. As the importance of co-transcriptional ligand binding has been shown for several riboswitches, it is vital to develop an assay that screens riboswitch-binding analogs during the transcriptional process. Here, we describe the development of a dual molecular beacon system monitoring the transcriptional regulation activity of the Bacillus subtilis pbuE adenine riboswitch. This system relies on two molecular beacons that enable the monitoring of transcription efficiency, as well as the regulatory activity of the riboswitch. Different analogs were tested using our system, and a good correlation was observed between riboswitch activity and reported metabolite affinities. This method is specific, reliable and could be applied at the high-throughput level for the identification of new potential antibiotics targeting any riboswitch-regulating gene expression at the mRNA level.

  9. Aptamer-Drug Conjugates.

    Science.gov (United States)

    Zhu, Guizhi; Niu, Gang; Chen, Xiaoyuan

    2015-11-18

    Western medicine often aims to specifically treat diseased tissues or organs. However, the majority of current therapeutics failed to do so owing to their limited selectivity and the consequent "off-target" side effects. Targeted therapy aims to enhance the selectivity of therapeutic effects and reduce adverse side effects. One approach toward this goal is to utilize disease-specific ligands to guide the delivery of less-specific therapeutics, such that the therapeutic effects can be guided specifically to diseased tissues or organs. Among these ligands, aptamers, also known as chemical antibodies, have emerged over the past decades as a novel class of targeting ligands that are capable of specific binding to disease biomarkers. Compared with other types of targeting ligands, aptamers have an array of unique advantageous features, which make them promising for developing aptamer-drug conjugates (ApDCs) for targeted therapy. In this Review, we will discuss ApDCs for targeted drug delivery in chemotherapy, gene therapy, immunotherapy, photodynamic therapy, and photothermal therapy, primarily of cancer.

  10. Protein Detection with Aptamer Biosensors

    Directory of Open Access Journals (Sweden)

    Regina Stoltenburg

    2008-07-01

    Full Text Available Aptamers have been developed for different applications. Their use as new biological recognition elements in biosensors promises progress for fast and easy detection of proteins. This new generation of biosensor (aptasensors will be more stable and well adapted to the conditions of real samples because of the specific properties of aptamers.

  11. Coenzyme Recognition and Gene Regulation by a Flavin Mononucleotide Riboswitch

    Energy Technology Data Exchange (ETDEWEB)

    Serganov, A.; Huang, L; Patel, D

    2009-01-01

    The biosynthesis of several protein cofactors is subject to feedback regulation by riboswitches. Flavin mononucleotide (FMN)-specific riboswitches also known as RFN elements, direct expression of bacterial genes involved in the biosynthesis and transport of riboflavin (vitamin B2) and related compounds. Here we present the crystal structures of the Fusobacterium nucleatum riboswitch bound to FMN, riboflavin and antibiotic roseoflavin. The FMN riboswitch structure, centred on an FMN-bound six-stem junction, does not fold by collinear stacking of adjacent helices, typical for folding of large RNAs. Rather, it adopts a butterfly-like scaffold, stapled together by opposingly directed but nearly identically folded peripheral domains. FMN is positioned asymmetrically within the junctional site and is specifically bound to RNA through interactions with the isoalloxazine ring chromophore and direct and Mg{sup 2+}-mediated contacts with the phosphate moiety. Our structural data, complemented by binding and footprinting experiments, imply a largely pre-folded tertiary RNA architecture and FMN recognition mediated by conformational transitions within the junctional binding pocket. The inherent plasticity of the FMN-binding pocket and the availability of large openings make the riboswitch an attractive target for structure-based design of FMN-like antimicrobial compounds. Our studies also explain the effects of spontaneous and antibiotic-induced deregulatory mutations and provided molecular insights into FMN-based control of gene expression in normal and riboflavin-overproducing bacterial strains.

  12. Role of riboswitches in gene regulation and their potential for algal biotechnology.

    Science.gov (United States)

    Nguyen, Ginnie T D T; Scaife, Mark A; Helliwell, Katherine E; Smith, Alison G

    2016-06-01

    Riboswitches are regulatory elements in messenger RNA to which specific ligands can bind directly in the absence of proteins. Ligand binding alters the mRNA secondary structure, thereby affecting expression of the encoded protein. Riboswitches are widespread in prokaryotes, with over 20 different effector ligands known, including amino acids, cofactors, and Mg(2+) ions, and gene expression is generally regulated by affecting translation or termination of transcription. In plants, fungi, and microalgae, riboswitches have been found, but only those that bind thiamine pyrophosphate. These eukaryotic riboswitches operate by causing alternative splicing of the transcript. Here, we review the current status of riboswitch research with specific emphasis on microalgae. We discuss new riboswitch discoveries and insights into the underlying mechanism of action, and how next generation sequencing technology provides the motivation and opportunity to improve our understanding of these rare but important regulatory elements. We also highlight the potential of microalgal riboswitches as a tool for synthetic biology and industrial biotechnology.

  13. Kinetic regulation mechanism of pbuE riboswitch

    Science.gov (United States)

    Gong, Sha; Wang, Yujie; Zhang, Wenbing

    2015-01-01

    Riboswitches are RNA residue segments located in untranslated regions of messenger RNAs. These folded segments directly bind ligands through shape complementarity and specific interactions in cells and alter the expression of genes at the transcriptional or translational level through conformation change. Using the recently developed systematic helix-based computational method to predict the cotranscription folding kinetics, we theoretically studied the cotranscription folding behavior of the Bacillus subtilis pbuE riboswitch in the absence and presence of the ligand. The ligand concentration, the transcription speed, and the transcription pausing are incorporated into the method. The results are in good agreement with the experimental results. We find that there are no obvious misfolded structures formed during the transcription and the formation of the ligand bound state is rate-limited by the association of the ligand and the RNA. For this kinetically driven riboswitch, the ligand concentration, the transcription speed, and the transcription pausing are coupled to perform regulatory activity.

  14. A riboswitch-regulated antisense RNA in Listeria monocytogenes.

    Science.gov (United States)

    Mellin, J R; Tiensuu, Teresa; Bécavin, Christophe; Gouin, Edith; Johansson, Jörgen; Cossart, Pascale

    2013-08-06

    Riboswitches are ligand-binding elements located in 5' untranslated regions of messenger RNAs, which regulate expression of downstream genes. In Listeria monocytogenes, a vitamin B12-binding (B12) riboswitch was identified, not upstream of a gene but downstream, and antisense to the adjacent gene, pocR, suggesting it might regulate pocR in a nonclassical manner. In Salmonella enterica, PocR is a transcription factor that is activated by 1,2-propanediol, and subsequently activates expression of the pdu genes. The pdu genes mediate propanediol catabolism and are implicated in pathogenesis. As enzymes involved in propanediol catabolism require B12 as a cofactor, we hypothesized that the Listeria B12 riboswitch might be involved in pocR regulation. Here we demonstrate that the B12 riboswitch is transcribed as part of a noncoding antisense RNA, herein named AspocR. In the presence of B12, the riboswitch induces transcriptional termination, causing aspocR to be transcribed as a short transcript. In contrast, in the absence of B12, aspocR is transcribed as a long antisense RNA, which inhibits pocR expression. Regulation by AspocR ensures that pocR, and consequently the pdu genes, are maximally expressed only when both propanediol and B12 are present. Strikingly, AspocR can inhibit pocR expression in trans, suggesting it acts through a direct interaction with pocR mRNA. Together, this study demonstrates how pocR and the pdu genes can be regulated by B12 in bacteria and extends the classical definition of riboswitches from elements governing solely the expression of mRNAs to a wider role in controlling transcription of noncoding RNAs.

  15. Molecular Buffers Permit Sensitivity Tuning and Inversion of Riboswitch Signals

    DEFF Research Database (Denmark)

    Rugbjerg, Peter; Genee, Hans Jasper; Jensen, Kristian;

    2016-01-01

    Predictable integration of foreign biological signals and parts remains a key challenge in the systematic engineering of synthetic cellular actuations, and general methods to improve signal transduction and sensitivity are needed. To address this problem we modeled and built a molecular signal...... buffer network in Saccharomyces cerevisiae inspired by chemical pH buffer systems. The molecular buffer system context-insulates a riboswitch enabling synthetic control of colony formation and modular signal manipulations. The riboswitch signal is relayed to a transcriptional activation domain of a split...

  16. Riboswitches. A riboswitch-containing sRNA controls gene expression by sequestration of a response regulator.

    Science.gov (United States)

    DebRoy, Sruti; Gebbie, Margo; Ramesh, Arati; Goodson, Jonathan R; Cruz, Melissa R; van Hoof, Ambro; Winkler, Wade C; Garsin, Danielle A

    2014-08-22

    The ethanolamine utilization (eut) locus of Enterococcus faecalis, containing at least 19 genes distributed over four polycistronic messenger RNAs, appears to be regulated by a single adenosyl cobalamine (AdoCbl)-responsive riboswitch. We report that the AdoCbl-binding riboswitch is part of a small, trans-acting RNA, EutX, which additionally contains a dual-hairpin substrate for the RNA binding-response regulator, EutV. In the absence of AdoCbl, EutX uses this structure to sequester EutV. EutV is known to regulate the eut messenger RNAs by binding dual-hairpin structures that overlap terminators and thus prevent transcription termination. In the presence of AdoCbl, EutV cannot bind to EutX and, instead, causes transcriptional read through of multiple eut genes. This work introduces riboswitch-mediated control of protein sequestration as a posttranscriptional mechanism to coordinately regulate gene expression.

  17. Glutamate Receptor Aptamers and ALS

    Science.gov (United States)

    2009-01-01

    proposed, including oxidative stress, excitotoxicity, mitochondrial dysfunction, etc., the cause(s) of the disease, including the pathogenesis of the...GluR6-Selective Aptamers for Potential Autism Therapy This project is to develop RNA aptamers against a GluR6 kainate receptor mutant thought to be...involved in autism . Role: PI Department of Defense (PI: Niu) 4/1/09-3/30/14 Advanced Tech./Therapeutic Develop. Grant Developing Biostable

  18. Aptamers for Targeted Drug Delivery

    Directory of Open Access Journals (Sweden)

    Partha Ray

    2010-05-01

    Full Text Available Aptamers are a class of therapeutic oligonucleotides that form specific three-dimensional structures that are dictated by their sequences. They are typically generated by an iterative screening process of complex nucleic acid libraries employing a process termed Systemic Evolution of Ligands by Exponential Enrichment (SELEX. SELEX has traditionally been performed using purified proteins, and cell surface receptors may be challenging to purify in their properly folded and modified conformations. Therefore, relatively few aptamers have been generated that bind cell surface receptors. However, improvements in recombinant fusion protein technology have increased the availability of receptor extracellular domains as purified protein targets, and the development of cell-based selection techniques has allowed selection against surface proteins in their native configuration on the cell surface. With cell-based selection, a specific protein target is not always chosen, but selection is performed against a target cell type with the goal of letting the aptamer choose the target. Several studies have demonstrated that aptamers that bind cell surface receptors may have functions other than just blocking receptor-ligand interactions. All cell surface proteins cycle intracellularly to some extent, and many surface receptors are actively internalized in response to ligand binding. Therefore, aptamers that bind cell surface receptors have been exploited for the delivery of a variety of cargoes into cells. This review focuses on recent progress and current challenges in the field of aptamer-mediated delivery.

  19. Riboswitch-based sensor in low optical background

    Science.gov (United States)

    Harbaugh, Svetlana V.; Davidson, Molly E.; Chushak, Yaroslav G.; Kelley-Loughnane, Nancy; Stone, Morley O.

    2008-08-01

    Riboswitches are a type of natural genetic control element that use untranslated sequence in the RNA to recognize and bind to small molecules that regulate expression of that gene. Creation of synthetic riboswitches to novel ligands depends on the ability to screen for analyte binding sensitivity and specificity. In our work, we have coupled a synthetic riboswitch to an optical reporter assay based on fluorescence resonance energy transfer (FRET) between two genetically-coded fluorescent proteins. Specifically, a theophylline-sensitive riboswitch was placed upstream of the Tobacco Etch Virus (TEV) protease coding sequence, and a FRET-based construct, BFP-eGFP or eGFP-REACh, was linked by a peptide encoding the recognition sequence for TEV protease. Cells expressing the riboswitch showed a marked optical difference in fluorescence emission in the presence of theophylline. However, the BFP-eGFP FRET pair posses significant optical background that reduces the sensitivity of a FRET-based assay. To improve the optical assay, we designed a nonfluorescent yellow fluorescent protein (YFP) mutant called REACh (for Resonance Energy-Accepting Chromoprotein) as the FRET acceptor for eGFP. The advantage of using an eGFP-REACh pair is the elimination of acceptor fluorescence which leads to an improved detection of FRET via better signal-to-noise ratio. The EGFP-REACh fusion protein was constructed with the TEV protease cleavage site; thus upon TEV translation, cleavage occurs diminishing REACh quenching and increasing eGFP emission resulting in a 4.5-fold improvement in assay sensitivity.

  20. Carba-sugars activate the glmS-riboswitch of Staphylococcus aureus.

    Science.gov (United States)

    Lünse, Christina E; Schmidt, Magnus S; Wittmann, Valentin; Mayer, Günter

    2011-07-15

    The glmS-riboswitch is unique among riboswitch families as it represents a metabolite-dependent ribozyme that undergoes self-cleavage upon recognition of glucosamin-6-phosphate. The glmS-riboswitch is located in the 5'-untranslated region of bacterial genes involved in cell wall biosynthesis. Therefore, this riboswitch represents a promising target for developing new antibiotics. We describe the metabolite-dependent glmS-riboswitch of pathologically relevant and vancomycin-resistant Staphylococcus aureus and the discovery and synthesis of a carba-sugar with potency similar to that of the native metabolite glucosamine-6-phosphate in modulating riboswitch activity. This compound represents a valuable lead structure for the development of antibiotics with a novel mode of action.

  1. Aptamers in Virology: Recent Advances and Challenges

    OpenAIRE

    Binning, Jennifer M.; Leung, Daisy W.; Amarasinghe, Gaya K.

    2012-01-01

    Aptamers generated from randomized libraries of nucleic acids have found utility in a wide variety of fields and in the clinic. Aptamers can be used to target both intracellular and extracellular components, including small molecules, proteins, cells, and viruses. With recent technological developments in stringent selection and rapid isolation strategies, it is likely that aptamers will continue to make an impact as useful tools and reagents. Although many recently developed aptamers are int...

  2. Structural Insights Into Amino Acid Binding and Gene Control by a Lysine Riboswitch

    Energy Technology Data Exchange (ETDEWEB)

    Serganov, A.; Huang, L; Patel, D

    2008-01-01

    In bacteria, the intracellular concentration of several amino acids is controlled by riboswitches1, 2, 3, 4. One of the important regulatory circuits involves lysine-specific riboswitches, which direct the biosynthesis and transport of lysine and precursors common for lysine and other amino acids. To understand the molecular basis of amino acid recognition by riboswitches, here we present the crystal structure of the 174-nucleotide sensing domain of the Thermotoga maritima lysine riboswitch in the lysine-bound (1.9 A) and free (3.1 A) states. The riboswitch features an unusual and intricate architecture, involving three-helical and two-helical bundles connected by a compact five-helical junction and stabilized by various long-range tertiary interactions. Lysine interacts with the junctional core of the riboswitch and is specifically recognized through shape-complementarity within the elongated binding pocket and through several direct and K+-mediated hydrogen bonds to its charged ends. Our structural and biochemical studies indicate preformation of the riboswitch scaffold and identify conformational changes associated with the formation of a stable lysine-bound state, which prevents alternative folding of the riboswitch and facilitates formation of downstream regulatory elements. We have also determined several structures of the riboswitch bound to different lysine analogues5, including antibiotics, in an effort to understand the ligand-binding capabilities of the lysine riboswitch and understand the nature of antibiotic resistance. Our results provide insights into a mechanism of lysine-riboswitch-dependent gene control at the molecular level, thereby contributing to continuing efforts at exploration of the pharmaceutical and biotechnological potential of riboswitches.

  3. Novel riboswitch ligand analogs as selective inhibitors of guanine-related metabolic pathways.

    Directory of Open Access Journals (Sweden)

    Jérôme Mulhbacher

    2010-04-01

    Full Text Available Riboswitches are regulatory elements modulating gene expression in response to specific metabolite binding. It has been recently reported that riboswitch agonists may exhibit antimicrobial properties by binding to the riboswitch domain. Guanine riboswitches are involved in the regulation of transport and biosynthesis of purine metabolites, which are critical for the nucleotides cellular pool. Upon guanine binding, these riboswitches stabilize a 5'-untranslated mRNA structure that causes transcription attenuation of the downstream open reading frame. In principle, any agonistic compound targeting a guanine riboswitch could cause gene repression even when the cell is starved for guanine. Antibiotics binding to riboswitches provide novel antimicrobial compounds that can be rationally designed from riboswitch crystal structures. Using this, we have identified a pyrimidine compound (PC1 binding guanine riboswitches that shows bactericidal activity against a subgroup of bacterial species including well-known nosocomial pathogens. This selective bacterial killing is only achieved when guaA, a gene coding for a GMP synthetase, is under the control of the riboswitch. Among the bacterial strains tested, several clinical strains exhibiting multiple drug resistance were inhibited suggesting that PC1 targets a different metabolic pathway. As a proof of principle, we have used a mouse model to show a direct correlation between the administration of PC1 and the reduction of Staphylococcus aureus infection in mammary glands. This work establishes the possibility of using existing structural knowledge to design novel guanine riboswitch-targeting antibiotics as powerful and selective antimicrobial compounds. Particularly, the finding of this new guanine riboswitch target is crucial as community-acquired bacterial infections have recently started to emerge.

  4. Long-range pseudoknot interactions dictate the regulatory response in the tetrahydrofolate riboswitch

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Lili; Ishibe-Murakami, Satoko; Patel, Dinshaw J.; Serganov, Alexander (MSKCC)

    2011-09-15

    Tetrahydrofolate (THF), a biologically active form of the vitamin folate (B{sub 9}), is an essential cofactor in one-carbon transfer reactions. In bacteria, expression of folate-related genes is controlled by feedback modulation in response to specific binding of THF and related compounds to a riboswitch. Here, we present the X-ray structures of the THF-sensing domain from the Eubacterium siraeum riboswitch in the ligand-bound and unbound states. The structure reveals an 'inverted' three-way junctional architecture, most unusual for riboswitches, with the junction located far from the regulatory helix P1 and not directly participating in helix P1 formation. Instead, the three-way junction, stabilized by binding to the ligand, aligns the riboswitch stems for long-range tertiary pseudoknot interactions that contribute to the organization of helix P1 and therefore stipulate the regulatory response of the riboswitch. The pterin moiety of the ligand docks in a semiopen pocket adjacent to the junction, where it forms specific hydrogen bonds with two moderately conserved pyrimidines. The aminobenzoate moiety stacks on a guanine base, whereas the glutamate moiety does not appear to make strong interactions with the RNA. In contrast to other riboswitches, these findings demonstrate that the THF riboswitch uses a limited number of available determinants for ligand recognition. Given that modern antibiotics target folate metabolism, the THF riboswitch structure provides insights on mechanistic aspects of riboswitch function and may help in manipulating THF levels in pathogenic bacteria

  5. Long-range pseudoknot interactions dictate the regulatory response in the tetrahydrofolate riboswitch.

    Science.gov (United States)

    Huang, Lili; Ishibe-Murakami, Satoko; Patel, Dinshaw J; Serganov, Alexander

    2011-09-06

    Tetrahydrofolate (THF), a biologically active form of the vitamin folate (B(9)), is an essential cofactor in one-carbon transfer reactions. In bacteria, expression of folate-related genes is controlled by feedback modulation in response to specific binding of THF and related compounds to a riboswitch. Here, we present the X-ray structures of the THF-sensing domain from the Eubacterium siraeum riboswitch in the ligand-bound and unbound states. The structure reveals an "inverted" three-way junctional architecture, most unusual for riboswitches, with the junction located far from the regulatory helix P1 and not directly participating in helix P1 formation. Instead, the three-way junction, stabilized by binding to the ligand, aligns the riboswitch stems for long-range tertiary pseudoknot interactions that contribute to the organization of helix P1 and therefore stipulate the regulatory response of the riboswitch. The pterin moiety of the ligand docks in a semiopen pocket adjacent to the junction, where it forms specific hydrogen bonds with two moderately conserved pyrimidines. The aminobenzoate moiety stacks on a guanine base, whereas the glutamate moiety does not appear to make strong interactions with the RNA. In contrast to other riboswitches, these findings demonstrate that the THF riboswitch uses a limited number of available determinants for ligand recognition. Given that modern antibiotics target folate metabolism, the THF riboswitch structure provides insights on mechanistic aspects of riboswitch function and may help in manipulating THF levels in pathogenic bacteria.

  6. Aptamers: Biomedical Interest and Applications

    Directory of Open Access Journals (Sweden)

    Cristina Romero-López

    2017-03-01

    Full Text Available Aptamers are short DNA or RNA oligonucleotides specialized in the specific and efficient binding to a target molecule. They are obtained by in vitro selection or evolution processes. It was in 1990 that two independent research groups described the bases of a new in vitro technology for the identification of RNA molecules able to specifically bind to a target [1,2]. Tuerk and Gold established the principals of the in vitro selection process that was named SELEX (Systematic Evolution of Ligands by Exponential enrichment, which is based on iterative cycles of binding, partitioning, and amplification of oligonucleotides from a pool of variant sequences [2]. Ellington and Szostak coined the term aptamer to define the selected molecules by the application of this method [1]. To date, numerous reports have described the isolation of aptamers directed against a great variety of targets covering a wide diversity of molecules varying in nature, size, and complexity ranging from ions to whole cells, including small molecules (e.g., aminoacids, nucleotides, antibiotics, peptides, proteins, nucleic acids, and viruses, among others (for example, see [3–6]. Modifications and optimization of the SELEX procedure aimed to get newly modified aptamers has also attracted much interest (examples can be found in [7,8]. These advances along with the parallel progresses in the nucleic acids chemistry and cellular delivery fields have allowed for the rise of a new hope in developing aptamers as efficient molecular tools for diagnostics and therapeutics (for recent comprehensive reviews, see [9–11].

  7. Developing aptamers into tumor diagnostics and therapeutics

    Institute of Scientific and Technical Information of China (English)

    Jing Mi; Bryan M. Clary; Bruce A. Sullenger

    2008-01-01

    Aptamers are small single-stranded nucleic acid molecules that bind a target protein with high affinity and specificity. Due to their stability, low toxicity and immunogenicity, as well as improved safety, aptamers are attractive alternatives to antibody and are therefore suitable for in vivo applications. Aptamers are typically isolated, through a process termed SELEX (systematic evolution of ligands by exponential enrichment), from combinatorial libraries with desired proteins. In the present review, the recent non-conventional aptamer selection process will be discussed together with an overview on the aptamer application in cancer diagnosis and therapy.

  8. Characterization of a natural triple-tandem c-di-GMP riboswitch and application of the riboswitch-based dual-fluorescence reporter.

    Science.gov (United States)

    Zhou, Hang; Zheng, Cao; Su, Jianmei; Chen, Bo; Fu, Yang; Xie, Yuqun; Tang, Qing; Chou, Shan-Ho; He, Jin

    2016-02-19

    c-di-GMP riboswitches are structured RNAs located in the 5'-untranslated regions (5'-UTRs) of mRNAs that regulate expression of downstream genes in response to changing concentrations of the second messenger c-di-GMP. We discovered three complete c-di-GMP riboswitches (Bc3, Bc4 and Bc5 RNA) with similar structures, which are arranged in tandem to constitute a triple-tandem (Bc3-5 RNA) riboswitch in the 5'-UTR of the cspABCDE mRNA in Bacillus thuringiensis subsp. chinensis CT-43. Our results showed that this natural triple-tandem riboswitch controlled the expression of the reporter gene more stringently and digitally than the double-tandem or single riboswitch. A sandwich-like dual-fluorescence reporter was further constructed by fusing the Bc3-5 RNA gene between the two fluorescence protein genes amcyan and turborfp. This reporter strain was found to exhibit detectable fluorescence color changes under bright field in response to intracellular c-di-GMP level altered by induced expression of diguanylate cyclase (DGC) PleD. Using this system, two putative membrane-bound DGCs from B. thuringiensis and Xanthomonas oryzae were verified to be functional by replacing pleD with the corresponding DGC genes. This report represented the first native triple-tandem riboswitch that was applied to serve as a riboswitch-based dual-fluorescence reporter for the efficient and convenient verification of putative DGC activity in vivo.

  9. G4 Aptamers: Trends in Structural Design.

    Science.gov (United States)

    Varizhuk, Anna; Ilyinsky, Nikolay; Smirnov, Igor; Pozmogova, Galina

    2016-01-01

    Many potent DNA aptamers are known to contain a G-quadruplex (G4) core. Structures and applications of the majority of such aptamers have been reviewed previously. The present review focuses on the design and optimization of G4 aptamers. General features of bioactive G4s are analyzed, and the main strategies for construction of aptamers with desired properties and topologies, including modular assembly, control of an aptamer folding and some others, are outlined. Chemical modification as a method for post-SELEX G4 aptamer optimization is also discussed, and the effects of loop and core modifications are compared. Particular attention is paid to the emerging trends, such as the development of genomic G4- inspired aptamers and the combinatorial approaches which aim to find a balance between rational design and selection.

  10. Preparation of modified long-mer RNAs and analysis of FMN binding to the ypaA aptamer from B. subtilis.

    Science.gov (United States)

    Frommer, Jennifer; Hieronymus, Robert; Selvi Arunachalam, Tamil; Heeren, Sabine; Jenckel, Maria; Strahl, Anne; Appel, Bettina; Müller, Sabine

    2014-01-01

    In recent years, RNA has been shown to fulfil a number of cellular functions. This has led to much interest in elucidation of the structure of functional RNA molecules, and thus, in the preparation of suitably functionalized RNAs. The chemical synthesis of RNAs allows for the site-specific modification; however, is limited to sequences of about 60-70 nucleotides in length. At the example of the flavine mononucleotide (FMN) responsive aptamer of the ypaA riboswitch from B. subtilis, we demonstrate the highly efficient preparation of site-specifically modified long-mer RNAs. Our strategy consists of the chemical synthesis of fragments followed by enzymatic or chemical ligation. Splint ligation with T4 RNA ligase turned out to be most successful among the enyzymatic protocols. Highly efficient chemical ligation was performed by azide-alkyne cycloaddition of suitably modified RNA fragments. Wild-type and 2-aminopurine (2-AP)-modified variants of the ypaA aptamer were prepared. FMN binding to all synthesized ypaA aptamer variants is demonstrated. However, dissociation of FMN from its binding site by reduction of the isoalloxazin unit as demonstrated before for a small-hairpin-derived aptazyme could not be shown. This implies that either FMN is less accessible to reduction when it is bound to its natural aptamer; that reduced FMN remains bound to the aptamer; or that FMN upon reduction indeed is released from its binding site, without the aptamer folding back in the natural ligand-free state. The results of this study are of general interest to the preparation of site-specifically modified RNAs for investigation into structure and function.

  11. Characterization of a natural triple-tandem c-di-GMP riboswitch and application of the riboswitch-based dual-fluorescence reporter

    OpenAIRE

    Hang Zhou; Cao Zheng; Jianmei Su; Bo Chen; Yang Fu; Yuqun Xie; Qing Tang; Shan-Ho Chou; Jin He

    2016-01-01

    c-di-GMP riboswitches are structured RNAs located in the 5′-untranslated regions (5′-UTRs) of mRNAs that regulate expression of downstream genes in response to changing concentrations of the second messenger c-di-GMP. We discovered three complete c-di-GMP riboswitches (Bc3, Bc4 and Bc5 RNA) with similar structures, which are arranged in tandem to constitute a triple-tandem (Bc3-5 RNA) riboswitch in the 5′-UTR of the cspABCDE mRNA in Bacillus thuringiensis subsp. chinensis CT-43. Our results s...

  12. Thiamine pyrophosphate riboswitch in some representative plant species: a bioinformatics study.

    Science.gov (United States)

    Yadav, Sunita; Swati, D; Chandrasekharan, Hariharan

    2015-01-01

    Metabolites regulate their own production by directly interacting with highly conserved regions of mRNA that are capable of forming discrete tertiary structures. Such regions of mRNA are called riboswitches. The thiamine pyrophosphate (TPP) riboswitch is the most common riboswitch in different organisms. The TPP is an essential coenzyme that is synthesized by the coupling of pyrimidine (hydroxymethyl pyrimidine) and thiazole (hydroxyethyl thiazole). The TPP riboswitch was searched across all possible phyla of plant kingdom by using Arabidopsis thaliana, a model organism in which TPP riboswitch is already found. The aptameric domain of the TPP riboswitch is conserved at the sequence as well as structure levels in all chosen plant species. The sequence of the noncoding RNA that acts as a riboswitch and is folded into an appropriate stem-loop hairpin secondary structure with minimum free energy is predicted by several computational tools. Most of the secondary structures are similar but not the same, because of variation in sequence size. The TPP ligand can bind to the 3' untranslated region of the aptameric sequence, between the loops P2, P4, and P5 and the region between J2/3 and J4/5. The sequence of these loop regions in all predicted tertiary structure of the riboswitch in representative plant species--green algae to flowering plants--is the same, and the residues situated in these junctions are directly involved in binding thymine pyrophosphate and are conserved in all the representative species.

  13. Single-Molecule Approaches for the Characterization of Riboswitch Folding Mechanisms.

    Science.gov (United States)

    Boudreault, Julien; Perez-Gonzalez, D Cibran; Penedo, J Carlos; Lafontaine, Daniel A

    2015-01-01

    Riboswitches are highly structured RNA molecules that control genetic expression by altering their structure as a function of metabolite binding. Accumulating evidence suggests that riboswitch structures are highly dynamic and perform conformational exchange between structural states that are important for the outcome of genetic regulation. To understand how ligand binding influences the folding of riboswitches, it is important to monitor in real time the riboswitch folding pathway as a function of experimental conditions. Single-molecule FRET (sm-FRET) is unique among biophysical techniques to study riboswitch conformational changes as it allows to both monitor steady-state populations of riboswitch conformers and associated interconversion dynamics. Since FRET fluorophores can be attached to virtually any nucleotide position, FRET assays can be adapted to monitor specific conformational changes, thus enabling to deduce complex riboswitch folding pathways. Herein, we show how to employ sm-FRET to study the folding pathway of the S-adenosylmethionine (SAM) and how this can be used to understand very specific conformational changes that are at the heart of riboswitch regulation mechanism.

  14. Folding of SAM-II riboswitch explored by replica-exchange molecular dynamics simulation.

    Science.gov (United States)

    Xue, Xu; Yongjun, Wang; Zhihong, Li

    2015-01-21

    Riboswitches are cis-acting RNA fragments that function via a conformational transition mechanism when a specific target molecule binds to its binding pocket, representing an inviting new class of biomolecular target for the development of antibiotics. To understand the folding mechanism of SAM-II riboswitch, occurring predominantly in proteobacteria, a 100ns replica-exchange molecular dynamics simulation in explicit solvent is performed. Our results show that this RNA pseudoknot has multiple folding pathways, and various intermediate structures. The resultant riboswitch conformational transition map is well consistent with the recent fluorescence measurement, which confirms the dynamical properties of this pseudoknot. Moreover, a novel transition pathway is predicted. The global folding dynamics is mainly coupled with the helix rather than the loop region. The potential folding pathways of the riboswitch presented here should lead to a deeper understanding of the folding mechanism of the riboswitch, as well as the conformational change of RNA pseudoknot.

  15. Strategies for the discovery of therapeutic Aptamers

    Science.gov (United States)

    Yang, Xianbin; Li, Na; Gorenstein, David G.

    2010-01-01

    Importance of the field Therapeutic aptamers are synthetic, structured oligonucleotides that bind to a very broad range of targets with high affinity and specificity. They are an emerging class of targeting ligand that show great promise for treating a number of diseases. A series of aptamers currently in various stages of clinical development highlights the potential of aptamers for therapeutic applications. Area covered in this review This review will cover in vitro selection of oligonucleotide ligands, called aptamers, from a combinatorial library using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process as well as the other known strategies for finding aptamers against various targets. What the reader will gain Readers will gain an understanding of the highly useful strategies for successful aptamer discovery. They may also be able combine two or more of the presented strategies for their aptamer discovery projects. Take home message Although many processes are available for discovering aptamers, it is not trivial to discover an aptamer candidate that is ready to move toward pharmaceutical drug development. It is also apparent that there have been relatively few therapeutic advances and clinical trials undertaken due to the small number of companies that participate in aptamer development. PMID:21359096

  16. Engineering a Lysine-ON Riboswitch for Metabolic Control of Lysine Production in Corynebacterium glutamicum.

    Science.gov (United States)

    Zhou, Li-Bang; Zeng, An-Ping

    2015-12-18

    Riboswitches are natural RNA elements that regulate gene expression by binding a ligand. Here, we demonstrate the possibility of altering a natural lysine-OFF riboswitch from Eschericia coli (ECRS) to a synthetic lysine-ON riboswitch and using it for metabolic control. To this end, a lysine-ON riboswitch library was constructed using tetA-based dual genetic selection. After screening the library, the functionality of the selected lysine-ON riboswitches was examined using a report gene, lacZ. Selected lysine-ON riboswitches were introduced into the lysE gene (encoding a lysine transport protein) of Corynebacterium glutamicum and used to achieve dynamic control of lysine transport in a recombinant lysine-producing strain, C. glutamicum LPECRS, which bears a deregulated aspartokinase and a lysine-OFF riboswitch for dynamic control of the enzyme citrate synthase. Batch fermentation results of the strains showed that the C. glutamicum LPECRS strain with an additional lysine-ON riboswitch for the control of lysE achieved a 21% increase in the yield of lysine compared to that of the C. glutamicum LPECRS strain and even a 89% increase in yield compared to that of the strain with deregulated aspartokinase. This work provides a useful approach to generate lysine-ON riboswitches for C. glutamicum metabolic engineering and demonstrates for the first time a synergetic effect of lysine-ON and -OFF riboswitches for improving lysine production in this industrially important microorganism. The approach can be used to dynamically control other genes and can be applied to other microorganisms.

  17. Aptamers in Virology: Recent advances and challenges

    Directory of Open Access Journals (Sweden)

    Jennifer M. Binning

    2012-02-01

    Full Text Available Aptamers generated from randomized libraries of nucleic acids have found utility in a wide variety of fields and in the clinic. Aptamers can be used to target both intracellular and extracellular components, including small molecules, proteins, cells, and viruses. With recent technological developments in stringent selection and rapid isolation strategies, it is likely that aptamers will continue to make an impact as useful tools and reagents. Although many recently developed aptamers are intended for use as therapeutic and diagnostic agents, use of aptamers for basic research, including target validation remains an active area with high potential to impact our understanding of molecular mechanisms and for drug discovery. In this brief review, we will discuss recent aptamer discoveries, their potential role in structural virology as well as challenges and future prospects.

  18. Array-Based Discovery of Aptamer Pairs

    Science.gov (United States)

    2014-12-11

    18460−18465. (25) Liu, Y.; Adams, J. D.; Turner, K.; Cochran, F. V.; Gambhir, S. S.; Soh, H. T. Lab Chip 2009, 9, 1033−1036. (26) Katilius, E.; Flores, C...discovery of aptamer pairs. We use microfluidic selection and high-throughput sequencing to obtain an enriched pool of aptamer sequences. Next, we...overcomes this problem to achieve efficient discovery of aptamer pairs. We use microfluidic selection and high- throughput sequencing to obtain an

  19. Functional detection of proteins by caged aptamers.

    Science.gov (United States)

    Pinto, Alessandro; Lennarz, Sabine; Rodrigues-Correia, Alexandre; Heckel, Alexander; O'Sullivan, Ciara K; Mayer, Günter

    2012-02-17

    While many diagnostic assay platforms enable the measurement of analytes with high sensitivity, most of them result in a disruption of the analyte's native structure and, thus, in loss of function. Consequently, the analyte can be used neither for further analytical assessment nor functional analysis. Herein we report the use of caged aptamers as templates during apta-PCR analysis of targets. Aptamers are short nucleic acids that fold into a well-defined three-dimensional structure in which they interact with target molecules with high affinity and specificity. Nucleic acid aptamers can also serve as templates for qPCR approaches and, thus, have been used as high affinity ligands to bind to target molecules and subsequently for quantification by qPCR, an assay format coined apta-PCR. Caged aptamers in turn refer to variants that bear one or more photolabile groups at strategic positions. The activity of caged aptamers can thus be turned on or off by light irradiation. The latter allows the mild elution of target-bound aptamers while the target's native structure and function remain intact. We demonstrate that this approach allows the quantitative and subsequently the functional assessment of analytes. Since caged aptamers can be generated emanating from virtually every available aptamer, the described approach can be generalized and adopted to any target-aptamer pair and, thus, have a broad applicability in proteomics and clinical diagnostics.

  20. Aptamers overview: selection, features and applications.

    Science.gov (United States)

    Hernandez, Luiza I; Machado, Isabel; Schafer, Thomas; Hernandez, Frank J

    2015-01-01

    Apatamer technology has been around for a quarter of a century and the field had matured enough to start seeing real applications, especially in the medical field. Since their discovery, aptamers rapidly emerged as key players in many fields, such as diagnostics, drug discovery, food science, drug delivery and therapeutics. Because of their synthetic nature, aptamers are evolving at an exponential rate gaining from the newest advances in chemistry, nanotechnology, biology and medicine. This review is meant to give an overview of the aptamer field, by including general aspects of aptamer identification and applications as well as highlighting certain features that contribute to their quick deployment in the biomedical field.

  1. A synthetic suicide riboswitch for the high-throughput screening of metabolite production in Saccharomyces cerevisiae.

    Science.gov (United States)

    Lee, Sang-Woo; Oh, Min-Kyu

    2015-03-01

    Artificial devices such as the synthetic riboswitch have shown potential to introduce unnatural phenotypic perturbation because its synthetic traits are distinct from that of innate metabolism. In this study, a riboswitch, a small regulatory element found in RNAs, was employed to reprogram microorganisms to produce valuable metabolites. A self-cleaving ribozyme glmS, found in gram-positive bacteria, cleaves its own transcript in response to the intracellular glucosamine 6-phosphate (GlcN6P) concentration. The glmS ribozyme was integrated into the 3'-untranslated region of FCY1, which encodes cytosine deaminase in Saccharomyces cerevisiae to construct a suicide riboswitch for evolutionary engineering. Growth of the strain harboring the suicide riboswitch was hampered by the addition of fluorocytosine, and was recovered as metabolite level increased. By using this riboswitch, we isolated a N-acetyl glucosamine (GlcNAc) producer strain by screening an efficient glutamine-fructose-6-phosphate transaminase (Gfa1p) and haloacid dehalogenase-like phosphatases (HAD phosphatases) originated from Escherichia coli. The suicide riboswitch was also applied to different metabolite by using artificial allosteric ribozyme. Since the mechanisms used in this work are universal in microorganisms, our synthetic suicide riboswitch can be applied to a wide range of organisms and can be exploited to the efficient and high-throughput screening of inconspicuous phenotypes.

  2. Mg(2+)-induced conformational changes in the btuB riboswitch from E. coli.

    Science.gov (United States)

    Choudhary, Pallavi K; Sigel, Roland K O

    2014-01-01

    Mg(2+) has been shown to modulate the function of riboswitches by facilitating the ligand-riboswitch interactions. The btuB riboswitch from Escherichia coli undergoes a conformational change upon binding to its ligand, coenzyme B12 (adenosyl-cobalamine, AdoCbl), and down-regulates the expression of the B12 transporter protein BtuB in order to control the cellular levels of AdoCbl. Here, we discuss the structural folding attained by the btuB riboswitch from E. coli in response to Mg(2+) and how it affects the ligand binding competent conformation of the RNA. The btuB riboswitch notably adopts different conformational states depending upon the concentration of Mg(2+). With the help of in-line probing, we show the existence of at least two specific conformations, one being achieved in the complete absence of Mg(2+) (or low Mg(2+) concentration) and the other appearing above ∼0.5 mM Mg(2+). Distinct regions of the riboswitch exhibit different dissociation constants toward Mg(2+), indicating a stepwise folding of the btuB RNA. Increasing the Mg(2+) concentration drives the transition from one conformation toward the other. The conformational state existing above 0.5 mM Mg(2+) defines the binding competent conformation of the btuB riboswitch which can productively interact with the ligand, coenzyme B12, and switch the RNA conformation. Moreover, raising the Mg(2+) concentration enhances the ratio of switched RNA in the presence of AdoCbl. The lack of a AdoCbl-induced conformational switch experienced by the btuB riboswitch in the absence of Mg(2+) indicates a crucial role played by Mg(2+) for defining an active conformation of the riboswitch.

  3. DNA-Aptamers Binding Aminoglycoside Antibiotics

    Directory of Open Access Journals (Sweden)

    Nadia Nikolaus

    2014-02-01

    Full Text Available Aptamers are short, single stranded DNA or RNA oligonucleotides that are able to bind specifically and with high affinity to their non-nucleic acid target molecules. This binding reaction enables their application as biorecognition elements in biosensors and assays. As antibiotic residues pose a problem contributing to the emergence of antibiotic-resistant pathogens and thereby reducing the effectiveness of the drug to fight human infections, we selected aptamers targeted against the aminoglycoside antibiotic kanamycin A with the aim of constructing a robust and functional assay that can be used for water analysis. With this work we show that aptamers that were derived from a Capture-SELEX procedure targeting against kanamycin A also display binding to related aminoglycoside antibiotics. The binding patterns differ among all tested aptamers so that there are highly substance specific aptamers and more group specific aptamers binding to a different variety of aminoglycoside antibiotics. Also the region of the aminoglycoside antibiotics responsible for aptamer binding can be estimated. Affinities of the different aptamers for their target substance, kanamycin A, are measured with different approaches and are in the micromolar range. Finally, the proof of principle of an assay for detection of kanamycin A in a real water sample is given.

  4. Selection and characterization of DNA aptamers

    NARCIS (Netherlands)

    Ruigrok, V.J.B.

    2013-01-01

    This thesis focusses on the selection and characterisation of DNA aptamers and the various aspects related to their selection from large pools of randomized oligonucleotides. Aptamers are affinity tools that can specifically recognize and bind predefined target molecules; this ability, however, is

  5. Post-SELEX optimization of aptamers.

    Science.gov (United States)

    Gao, Shunxiang; Zheng, Xin; Jiao, Binghua; Wang, Lianghua

    2016-07-01

    Aptamers are functional single-stranded DNA or RNA oligonucleotides, selected in vitro by SELEX (Systematic Evolution of Ligands by Exponential Enrichment), which can fold into stable unique three-dimensional structures that bind their target ligands with high affinity and specificity. Although aptamers show a number of favorable advantages such as better stability and easier modification when compared with the properties of antibodies, only a handful of aptamers have entered clinical trials and only one, pegaptanib, has received US Food and Drug Administration approval for clinical use. The main reasons that limit the practical application of aptamers are insufficient nuclease stability, bioavailability, thermal stability, or even affinity. Some aptamers obtained from modified libraries show better properties; however, polymerase amplification of nucleic acids containing non-natural bases is currently a primary drawback of the SELEX process. This review focuses on several post-SELEX optimization strategies of aptamers identified in recent years. We describe four common methods in detail: truncation, chemical modification, bivalent or multivalent aptamer construction, and mutagenesis. We believe that these optimization strategies should improve one or more specific properties of aptamers, and the type of feature(s) selected for improvement will be dependent on the application purpose.

  6. Selection and characterization of DNA aptamers

    NARCIS (Netherlands)

    Ruigrok, V.J.B.

    2013-01-01

    This thesis focusses on the selection and characterisation of DNA aptamers and the various aspects related to their selection from large pools of randomized oligonucleotides. Aptamers are affinity tools that can specifically recognize and bind predefined target molecules; this ability, however, is n

  7. Probing riboswitch-ligand interactions using thiamine pyrophosphate analogues.

    Science.gov (United States)

    Chen, Liuhong; Cressina, Elena; Dixon, Neil; Erixon, Karl; Agyei-Owusu, Kwasi; Micklefield, Jason; Smith, Alison G; Abell, Chris; Leeper, Finian J

    2012-08-14

    The Escherichia coli thiM riboswitch forms specific contacts with its natural ligand, thiamine pyrophosphate (TPP or thiamine diphosphate), allowing it to generate not only nanomolar binding affinity, but also a high degree of discrimination against similar small molecules. A range of synthetic TPP analogues have been used to probe each of the riboswitch-ligand interactions. The results show that the pyrimidine-sensing helix of thiM is exquisitely tuned to select for TPP by recognising the H-bonding donor and acceptors around its aminopyrimidine ring and also by forming π-stacking interactions that may be sensitive to the electronics of the ring. The central thiazolium ring of TPP appears to be more important for ligand recognition than previously thought. It may contribute to binding via long-range electrostatic interactions and/or by exerting an electron withdrawing effect on the pyrimidine ring, allowing its presence to be sensed indirectly and thereby allowing discrimination between thiamine (and its phosphate esters) and other aminopyrimidines found in vivo. The pyrophosphate moiety is essential for submicromolar binding affinity, but unexpectedly, it does not appear to be strictly necessary for modulation of gene expression.

  8. Metal Ion-Mediated Nucleobase Recognition by the ZTP Riboswitch.

    Science.gov (United States)

    Trausch, Jeremiah J; Marcano-Velázquez, Joan G; Matyjasik, Michal M; Batey, Robert T

    2015-07-23

    The ZTP riboswitch is a widespread family of regulatory RNAs that upregulate de novo purine synthesis in response to increased intracellular levels of ZTP or ZMP. As an important intermediate in purine biosynthesis, ZMP also serves as a proxy for the concentration of N10-formyl-tetrahydrofolate, a key component of one-carbon metabolism. Here, we report the structure of the ZTP riboswitch bound to ZMP at a resolution of 1.80 Å. The RNA contains two subdomains brought together through a long-range pseudoknot further stabilized through helix-helix packing. ZMP is bound at the subdomain interface of the RNA through a set of interactions with the base, ribose sugar, and phosphate moieties of the ligand. Unique to nucleobase recognition by RNAs, the Z base is inner-sphere coordinated to a magnesium cation bound by two backbone phosphates. This interaction, along with steric hindrance by the backbone, imparts specificity over chemically similar compounds such as ATP/AMP.

  9. Using Aptamers for Cancer Biomarker Discovery

    Directory of Open Access Journals (Sweden)

    Yun Min Chang

    2013-01-01

    Full Text Available Aptamers are single-stranded synthetic DNA- or RNA-based oligonucleotides that fold into various shapes to bind to a specific target, which includes proteins, metals, and molecules. Aptamers have high affinity and high specificity that are comparable to that of antibodies. They are obtained using iterative method, called (Systematic Evolution of Ligands by Exponential Enrichment SELEX and cell-based SELEX (cell-SELEX. Aptamers can be paired with recent advances in nanotechnology, microarray, microfluidics, and other technologies for applications in clinical medicine. One particular area that aptamers can shed a light on is biomarker discovery. Biomarkers are important in diagnosis and treatment of cancer. In this paper, we will describe ways in which aptamers can be used to discover biomarkers for cancer diagnosis and therapeutics.

  10. Aptamer Selection Technology and Recent Advances

    Directory of Open Access Journals (Sweden)

    Michael Blind

    2015-01-01

    Full Text Available Over the last decade, aptamers have begun to find their way from basic research to diverse commercial applications. The development of diagnostics is even more widespread than clinical applications because aptamers do not have to be extensively modified to enhance their in vivo stability and pharmacokinetics in diagnostic assays. The increasing attention has propelled the technical progress of the in vitro selection technology (SELEX to enhance the efficiency of developing aptamers for commercially interesting targets. This review highlights recent progress in the technical steps of a SELEX experiment with a focus on high-throughput next-generation sequencing and bioinformatics. Achievements have been made in the optimization of aptamer libraries, separation schemes, amplification of the selected libraries and the identification of aptamer sequences from enriched libraries.

  11. RNA and DNA aptamers in cytomics analysis.

    Science.gov (United States)

    Ulrich, Henning; Martins, Antonio Henrique B; Pesquero, João Bosco

    2005-08-01

    Using systematic evolution of ligands by exponential enrichment (SELEX), RNA or DNA molecules are selected from a combinatorial oligonucleotide library by their ability to bind their targets, i.e., cell surface antigens, with affinity and specificity similar to that of monoclonal antibodies. The generation of these high-affinity binders, also denominated aptamers, is carried out in vitro and does not involve animals. Therefore, aptamers can be developed against almost every molecule of biological importance, including toxins and nonimmunogenic targets, against which antibodies cannot be raised. The incorporation of modified pyrimidines resulting in nuclease-resistant RNA aptamers makes them promising candidates for studying protein interactions in vitro and in vivo. DNA aptamers do not need modifications for most applications. The protocols in this unit can be used for the development of fluorescent-tagged RNA or DNA aptamers for any cell surface protein in cytomics analysis.

  12. An ancient riboswitch class in bacteria regulates purine biosynthesis and one-carbon metabolism.

    Science.gov (United States)

    Kim, Peter B; Nelson, James W; Breaker, Ronald R

    2015-01-22

    Over 30 years ago, ZTP (5-aminoimidazole-4-carboxamide riboside 5'-triphosphate), a modified purine biosynthetic intermediate, was proposed to signal 10-formyl-tetrahydrofolate (10f-THF) deficiency in bacteria. However, the mechanisms by which this putative alarmone or its precursor ZMP (5-aminoimidazole-4-carboxamide ribonucleotide, also known as AICAR) brings about any metabolic changes remain unexplained. Herein, we report the existence of a widespread riboswitch class that is most commonly associated with genes related to de novo purine biosynthesis and one-carbon metabolism. Biochemical data confirm that members of this riboswitch class selectively bind ZMP and ZTP with nanomolar affinity while strongly rejecting numerous natural analogs. Indeed, increases in the ZMP/ZTP pool, caused by folate stress in bacterial cells, trigger changes in the expression of a reporter gene fused to representative ZTP riboswitches in vivo. The wide distribution of this riboswitch class suggests that ZMP/ZTP signaling is important for species in numerous bacterial lineages.

  13. Kinetic Studies of Lysine Riboswitch Folding Using Single-Molecule FRET

    Science.gov (United States)

    Fiegland, Larry; Garst, Andrew; Fiore, J.; Batey, Robert; Nesbitt, David

    2010-03-01

    Riboswitches regulate gene expression through conformational changes induced by metabolite binding. This regulation of gene expression depends on the kinetics of metabolite binding and structural changes. Therefore, an understanding of these dynamics is crucial to developing a compete knowledge of riboswitch functionality. To probe the binding of a metabolite and subsequent folding, a metabolite-binding domain of the Bacillus subtilis lysine riboswitch was transcribed and hybridized to a fluorescent-labeled RNA strand, which allows FRET monitoring of ligand-induced conformational changes. The RNA construct was studied using single-molecule FRET methods that allowed for characterization of the folding dynamics. In the presence of lysine, we observed two states, of which the relative populations are perturbed by lysine concentration. We measured the folding and unfolding rates of the inter-conversion between these states. We also observe that [Mg^2+] affects the lysine-free conformation and the lysine sensitivity of the riboswitch.

  14. Sequence, structure, and stacking: specifics of tRNA anchoring to the T box riboswitch.

    Science.gov (United States)

    Grigg, Jason C; Ke, Ailong

    2013-12-01

    The term riboswitch usually refers to small molecule sensing regulatory modules in the 5' untranslated regions of a mRNA. They are typically comprised of separate ligand binding and regulatory domains. The T box riboswitch is unique from other identified riboswitches because its effector is an essential macromolecule, tRNA. It senses the aminoacylation state of tRNA to regulate genes involved in a variety of functions relating to amino acid metabolism and tRNA aminoacylation. T box riboswitches performs an intuitively simple process using a complex structured RNA element and, until recently, the underlying mechanisms were poorly understood. Only two sequence-specific contacts had been previously identified: (1) between the specifier sequence (codon) and the tRNA anticodon and (2) between an anti-terminator stem loop and the tRNA acceptor arm CCA tail. tRNA aminoacylation blocks the latter interaction and therefore serves as the switch between termination and anti-termination. Outside of these two contacts, the structure and functions of T box riboswitches have come to light in some recent studies. We recently described the X-ray crystal structure of the highly conserved T box riboswitch distal Stem I region and demonstrated that this region interacts with the tRNA elbow to anchor it to the riboswitch. Independently, Lehmann et al. used sequence homology search to arrive at a similar model for Stem I-tRNA interactions. The model was further supported by two recent structures of the Stem I-tRNA complex, determined independently by our group and by Zhang and Ferré-D'Amaré. This article highlights some of these contributions to synthesize an updated model for tRNA recognition by the T box riboswitch.

  15. Structural Insights into Ligand Recognition by a Sensing Domain of the Cooperative Glycine Riboswitch

    Energy Technology Data Exchange (ETDEWEB)

    L Huang; A Serganov; D Patel

    2011-12-31

    Glycine riboswitches regulate gene expression by feedback modulation in response to cooperative binding to glycine. Here, we report on crystal structures of the second glycine-sensing domain from the Vibrio cholerae riboswitch in the ligand-bound and unbound states. This domain adopts a three-helical fold that centers on a three-way junction and accommodates glycine within a bulge-containing binding pocket above the junction. Glycine recognition is facilitated by a pair of bound Mg{sup 2+} cations and governed by specific interactions and shape complementarity with the pocket. A conserved adenine extrudes from the binding pocket and intercalates into the junction implying that glycine binding in the context of the complete riboswitch could impact on gene expression by stabilizing the riboswitch junction and regulatory P1 helix. Analysis of riboswitch interactions in the crystal and footprinting experiments indicates that adjacent glycine-sensing modules of the riboswitch could form specific interdomain interactions, thereby potentially contributing to the cooperative response.

  16. Application of supervised machine learning algorithms for the classification of regulatory RNA riboswitches.

    Science.gov (United States)

    Singh, Swadha; Singh, Raghvendra

    2016-04-03

    Riboswitches, the small structured RNA elements, were discovered about a decade ago. It has been the subject of intense interest to identify riboswitches, understand their mechanisms of action and use them in genetic engineering. The accumulation of genome and transcriptome sequence data and comparative genomics provide unprecedented opportunities to identify riboswitches in the genome. In the present study, we have evaluated the following six machine learning algorithms for their efficiency to classify riboswitches: J48, BayesNet, Naïve Bayes, Multilayer Perceptron, sequential minimal optimization, hidden Markov model (HMM). For determining effective classifier, the algorithms were compared on the statistical measures of specificity, sensitivity, accuracy, F-measure and receiver operating characteristic (ROC) plot analysis. The classifier Multilayer Perceptron achieved the best performance, with the highest specificity, sensitivity, F-score and accuracy, and with the largest area under the ROC curve, whereas HMM was the poorest performer. At present, the available tools for the prediction and classification of riboswitches are based on covariance model, support vector machine and HMM. The present study determines Multilayer Perceptron as a better classifier for the genome-wide riboswitch searches.

  17. Effects of FMN riboswitch on antioxidant activity in Deinococcus radiodurans under H₂O₂ stress.

    Science.gov (United States)

    Yang, Peng; Chen, Zhouwei; Shan, Zhan; Ding, Xianfeng; Liu, Lili; Guo, Jiangfeng

    2014-01-01

    The flavin mononucleotide (FMN) riboswitch is structured noncoding RNA domains that control gene expression by selectively binding FMN or sensing surrounding changes without protein factors, which are involved in the biosynthesis and transport of riboflavin and related compounds. We constructed the deletion mutant of FMN riboswitch to investigate its possible role in response to H₂O₂ stress in Deinococcus radiodurans. The results showed that the deletion of FMN riboswitch resulted in an obvious growth delay in D. radiodurans. Compared with the survival rate of 56% of D. radiodurans, only 40% of the mutant survived after treated with 50 mM of H₂O₂, indicating that deletion of FMN riboswitch obviously increased the susceptibility to H₂O₂. Compared with the wild type R1 strain of D. radiodurans, FMN riboswitch knockout cells accumulated a higher level of intracellular reactive oxygen species (ROS) while their total catalase activity reduced significantly. Results from quantitative real-time PCR analysis implies structural alterations of in response to H₂O₂ challenge. Our data suggest a critical role of FMN riboswitch in the oxidation tolerance system of D. radiodurans.

  18. Development of radiopharmaceuticals based on aptamers: selection and characterization of DNA aptamers for CEA

    Energy Technology Data Exchange (ETDEWEB)

    Correa, C.R.; Andrade, A.S.R., E-mail: antero@cdtn.br [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil); Augusto-Pinto, L. [BioAptus, Belo Horizonte, MG (Brazil); Goes, A.M., E-mail: goes@icb.ufmg.br [Departamento de Imunologia e Bioquimica. Instituto de Ciencias Biologicas. Universidade Federal de Minas Gerais. Belo Horizonte, MG (Brazil)

    2011-07-01

    Colorectal cancer is among the top four causes of cancer deaths worldwide. Carcinoembryonic antigen (CEA) is a complex intracellular glycoprotein produced by about 90% of colorectal cancers. CEA has been identified as an attractive target for cancer research because of its pattern of expression in the surface cell and its likely functional role in tumorigenesis. Research on the rapid selection of ligands based on the SELEX (systematic evolution of ligands by exponential enrichment) forms the basis for the development of high affinity and high specificity molecules, which can bind to surface determinants of tumour cells, like CEA. The oligonucleotides ligands generated in this technique are called aptamers. Aptamers can potentially find applications as therapeutic or diagnostic tools for many kind of diseases, like a tumor. Aptamers offer low immunogenicity, good tumour penetration, rapid uptake and fast systemic clearance, which favour their application as effective vehicles for radiotherapy. In addition aptamers can be labeled with different radioactive isotopes. The aim of this work was select aptamers binding to the CEA tumor marker. The aptamers are obtained through by SELEX, in which aptamers are selected from a library of random sequences of synthetic DNA by repetitive binding of the oligonucleotides to target molecule (CEA). Analyses of the secondary structure of the aptamers were determined using the m fold toll. Three aptamers were selected to binding assay with target cells. These aptamers were confirmed to have affinity and specific binding for T84 cell line (target cell), showed by confocal imaging. We are currently studying the potential efficacy of these aptamers as targeted radiopharmaceuticals, for use as imaging agents or therapeutic applications. The development of aptamers specific to CEA open new perspectives for colorectal cancer diagnosis and treatment. Acknowledgments: This investigation was supported by the Centro de Desenvolvimento da

  19. Aptamer Selection Express: A Novel Method for Rapid Single-Step Selection and Sensing of Aptamers

    Science.gov (United States)

    2008-12-01

    aptamers of Bacillus anthracis (Ba), Shiga toxin , botulinum neurotoxin (BoNT), and Francisella tularensis bacteria (all selected by SELEX) have been...This process has been used to select aptamers against different types of targets ( Bacillus anthracis spores, Bacillus thuringiensis spores, MS-2...studied by reselecting aptamers against different targets, Ba spores, Shiga toxin , and F. tularensis bacteria. In contrast to SELEX, the use of

  20. Future of aptamers in medicine

    CSIR Research Space (South Africa)

    Khati, M

    2010-06-01

    Full Text Available stream_source_info Khati_2010.pdf.txt stream_content_type text/plain stream_size 59703 Content-Encoding ISO-8859-1 stream_name Khati_2010.pdf.txt Content-Type text/plain; charset=ISO-8859-1 doi: 10.1136/jcp.2008....062786 2010 63: 480-487 originally published online April 1, 2010J Clin Pathol Makobetsa Khati The future of aptamers in medicine http://jcp.bmj.com/content/63/6/480.full.html Updated information and services can be found at: These include...

  1. Metabolomic analysis of riboswitch containing E. coli recombinant expression system.

    Science.gov (United States)

    Muhamadali, Howbeer; Xu, Yun; Morra, Rosa; Trivedi, Drupad K; Rattray, Nicholas J W; Dixon, Neil; Goodacre, Royston

    2016-02-01

    In this study we have employed metabolomics approaches to understand the metabolic effects of producing enhanced green fluorescent protein (eGFP) as a recombinant protein in Escherichia coli cells. This metabolic burden analysis was performed against a number of recombinant expression systems and control strains and included: (i) standard transcriptional recombinant expression control system BL21(DE3) with the expression plasmid pET-eGFP, (ii) the recently developed dual transcriptional-translational recombinant expression control strain BL21(IL3), with pET-eGFP, (iii) BL21(DE3) with an empty expression plasmid pET, (iv) BL21(IL3) with an empty expression plasmid, and (v) BL21(DE3) without an expression plasmid; all strains were cultured under various induction conditions. The growth profiles of all strains together with the results gathered by the analysis of the Fourier transform infrared (FT-IR) spectroscopy data, identified IPTG-dependent induction as the dominant factor hampering cellular growth and metabolism, which was in general agreement with the findings of GC-MS analysis of cell extracts and media samples. In addition, the exposure of host cells to the synthetic inducer ligand, pyrimido[4,5-d] pyrimidine-2,4-diamine (PPDA), of the orthogonal riboswitch containing expression system (BL21(IL3)) did not display any detrimental effects, and its detected levels in all the samples were at similar levels, emphasising the inability of the cells to metabolise PPDA. The overall results obtained in this study suggested that although the BL21(DE3)-EGFP and BL21(IL3)-EGFP strains produced comparable levels of recombinant eGFP, the presence of the orthogonal riboswitch seemed to be moderating the metabolic burden of eGFP production in the cells enabling higher biomass yield, whilst providing a greater level of control over protein expression.

  2. DNA Aptamers in the Diagnosis and Treatment of Human Diseases

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    Qinchang Zhu

    2015-11-01

    Full Text Available Aptamers have a promising role in the field of life science and have been extensively researched for application as analytical tools, therapeutic agents and as vehicles for targeted drug delivery. Compared with RNA aptamers, DNA aptamers have inherent advantages in stability and facility of generation and synthesis. To better understand the specific potential of DNA aptamers, an overview of the progress in the generation and application of DNA aptamers in human disease diagnosis and therapy are presented in this review. Special attention is given to researches that are relatively close to practical application. DNA aptamers are expected to have great potential in the diagnosis and treatment of human diseases.

  3. Phosphatase-inert glucosamine 6-phosphate mimics serve as actuators of the glmS riboswitch.

    Science.gov (United States)

    Fei, Xiang; Holmes, Thomas; Diddle, Julianna; Hintz, Lauren; Delaney, Dan; Stock, Alex; Renner, Danielle; McDevitt, Molly; Berkowitz, David B; Soukup, Juliane K

    2014-12-19

    The glmS riboswitch is unique among gene-regulating riboswitches and catalytic RNAs. This is because its own metabolite, glucosamine-6-phosphate (GlcN6P), binds to the riboswitch and catalytically participates in the RNA self-cleavage reaction, thereby providing a novel negative feedback mechanism. Given that a number of pathogens harbor the glmS riboswitch, artificial actuators of this potential RNA target are of great interest. Structural/kinetic studies point to the 2-amino and 6-phosphate ester functionalities in GlcN6P as being crucial for this actuation. As a first step toward developing artificial actuators, we have synthesized a series of nine GlcN6P analogs bearing phosphatase-inert surrogates in place of the natural phosphate ester. Self-cleavage assays with the Bacillus cereus glmS riboswitch give a broad SAR. Two analogs display significant activity, namely, the 6-deoxy-6-phosphonomethyl analog (5) and the 6-O-malonyl ether (13). Kinetic profiles show a 22-fold and a 27-fold higher catalytic efficiency, respectively, for these analogs vs glucosamine (GlcN). Given their nonhydrolyzable phosphate surrogate functionalities, these analogs are arguably the most robust artificial glmS riboswitch actuators yet reported. Interestingly, the malonyl ether (13, extra O atom) is much more effective than the simple malonate (17), and the "sterically true" phosphonate (5) is far superior to the chain-truncated (7) or chain-extended (11) analogs, suggesting that positioning via Mg coordination is important for activity. Docking results are consistent with this view. Indeed, the viability of the phosphonate and 6-O-malonyl ether mimics of GlcN6P points to a potential new strategy for artificial actuation of the glmS riboswitch in a biological setting, wherein phosphatase-resistance is paramount.

  4. Genetic analysis of riboswitch-mediated transcriptional regulation responding to Mn2+ in Salmonella.

    Science.gov (United States)

    Shi, Yixin; Zhao, Guang; Kong, Wei

    2014-04-18

    Riboswitches are a class of cis-acting regulatory RNAs normally characterized from the 5'-UTR of bacterial transcripts that bind a specific ligand to regulate expression of associated genes by forming alternative conformations. Here, we present a riboswitch that contributes to transcriptional regulation through sensing Mn(2+) in Salmonella typhimurium. We characterized a 5'-UTR (UTR1) from the mntH locus encoding a Mn(2+) transporter, which forms a Rho-independent terminator to implement transcription termination with a high Mn(2+) selectivity both in vivo and in vitro. Nucleotide substitutions that cause disruption of the terminator interfere with the regulatory function of UTR1. RNA probing analyses outlined a specific UTR1 conformation that favors the terminator structure in Mn(2+)-replete condition. Switch sequence GCUAUG can alternatively base pair duplicated hexanucleotide CAUAGC to form either a pseudoknot or terminator stem. Mn(2+), but not Mg(2+), and Ca(2+), can enhance cleavage at specific nucleotides in UTR1. We conclude that UTR1 is a riboswitch that senses cytoplasmic Mn(2+) and therefore participates in Mn(2+)-responsive mntH regulation in Salmonella. This riboswitch domain is also conserved in several Gram-negative enteric bacteria, indicating that this Mn(2+)-responsive mechanism could have broader implications in bacterial gene expression. Additionally, a high level of cytoplasmic Mn(2+) can down-regulate transcription of the Salmonella Mg(2+) transporter mgtA locus in a Mg(2+) riboswitch-dependent manner. On the other hand, these two types of cation riboswitches do not share similarity at the primary or secondary structural levels. Taken together, characterization of Mn(2+)-responsive riboswitches should expand the scope of RNA regulatory elements in response to inorganic ions.

  5. Cell-Specific Aptamers as Emerging Therapeutics

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    Cindy Meyer

    2011-01-01

    Full Text Available Aptamers are short nucleic acids that bind to defined targets with high affinity and specificity. The first aptamers have been selected about two decades ago by an in vitro process named SELEX (systematic evolution of ligands by exponential enrichment. Since then, numerous aptamers with specificities for a variety of targets from small molecules to proteins or even whole cells have been selected. Their applications range from biosensing and diagnostics to therapy and target-oriented drug delivery. More recently, selections using complex targets such as live cells have become feasible. This paper summarizes progress in cell-SELEX techniques and highlights recent developments, particularly in the field of medically relevant aptamers with a focus on therapeutic and drug-delivery applications.

  6. Aptamers: A Feasible Technology in Cancer Immunotherapy

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    M. M. Soldevilla

    2016-01-01

    Full Text Available Aptamers are single-chained RNA or DNA oligonucleotides (ODNs with three-dimensional folding structures which allow them to bind to their targets with high specificity. Aptamers normally show affinities comparable to or higher than that of antibodies. They are chemically synthesized and therefore less expensive to manufacture and produce. A variety of aptamers described to date have been shown to be reliable in modulating immune responses against cancer by either blocking or activating immune receptors. Some of them have been conjugated to other molecules to target the immune system and reduce off-target side effects. Despite the success of first-line treatments against cancer, the elevated number of relapsing cases and the tremendous side effects shown by the commonly used agents hinder conventional treatments against cancer. The advantages provided by aptamers could enhance the therapeutic index of a given strategy and therefore enhance the antitumor effect. Here we recapitulate the provided benefits of aptamers with immunomodulatory activity described to date in cancer therapy and the benefits that aptamer-based immunotherapy could provide either alone or combined with first-line treatments in cancer therapy.

  7. Aptamers: A Feasible Technology in Cancer Immunotherapy.

    Science.gov (United States)

    Soldevilla, M M; Villanueva, H; Pastor, F

    2016-01-01

    Aptamers are single-chained RNA or DNA oligonucleotides (ODNs) with three-dimensional folding structures which allow them to bind to their targets with high specificity. Aptamers normally show affinities comparable to or higher than that of antibodies. They are chemically synthesized and therefore less expensive to manufacture and produce. A variety of aptamers described to date have been shown to be reliable in modulating immune responses against cancer by either blocking or activating immune receptors. Some of them have been conjugated to other molecules to target the immune system and reduce off-target side effects. Despite the success of first-line treatments against cancer, the elevated number of relapsing cases and the tremendous side effects shown by the commonly used agents hinder conventional treatments against cancer. The advantages provided by aptamers could enhance the therapeutic index of a given strategy and therefore enhance the antitumor effect. Here we recapitulate the provided benefits of aptamers with immunomodulatory activity described to date in cancer therapy and the benefits that aptamer-based immunotherapy could provide either alone or combined with first-line treatments in cancer therapy.

  8. The dual aptamer approach: rational design of a high-affinity FAD aptamer.

    Science.gov (United States)

    Merkle, T; Holder, I T; Hartig, J S

    2016-01-14

    A design strategy for high-affinity aptamers of complex biomolecules is presented. We developed an RNA with FAD-binding properties by combining known ATP- and FMN-aptamers. Cooperative binding of FAD was shown by SPR spectroscopy and fluorescence assays. The strategy should be transferable to several other biomolecules.

  9. Crystal structure of a c-di-AMP riboswitch reveals an internally pseudo-dimeric RNA.

    Science.gov (United States)

    Jones, Christopher P; Ferré-D'Amaré, Adrian R

    2014-11-18

    Cyclic diadenosine monophosphate (c-di-AMP) is a second messenger that is essential for growth and homeostasis in bacteria. A recently discovered c-di-AMP-responsive riboswitch controls the expression of genes in a variety of bacteria, including important pathogens. To elucidate the molecular basis for specific binding of c-di-AMP by a gene-regulatory mRNA domain, we have determined the co-crystal structure of this riboswitch. Unexpectedly, the structure reveals an internally pseudo-symmetric RNA in which two similar three-helix-junction elements associate head-to-tail, creating a trough that cradles two c-di-AMP molecules making quasi-equivalent contacts with the riboswitch. The riboswitch selectively binds c-di-AMP and discriminates exquisitely against other cyclic dinucleotides, such as c-di-GMP and cyclic-AMP-GMP, via interactions with both the backbone and bases of its cognate second messenger. Small-angle X-ray scattering experiments indicate that global folding of the riboswitch is induced by the two bound cyclic dinucleotides, which bridge the two symmetric three-helix domains. This structural reorganization likely couples c-di-AMP binding to gene expression.

  10. Measuring riboswitch activity in vitro and in artificial cells with purified transcription-translation machinery.

    Science.gov (United States)

    Martini, Laura; Mansy, Sheref S

    2014-01-01

    We present a simple method to measure the real-time activity of riboswitches with purified components in vitro and inside of artificial cells. Typically, riboswitch activity is measured in vivo by exploiting β-galactosidase encoding constructs with a putative riboswitch sequence in the untranslated region. Additional in vitro characterization often makes use of in-line probing to explore conformational changes induced by ligand binding to the mRNA or analyses of transcript lengths in the presence and absence of ligand. However, riboswitches ultimately control protein levels and often times require accessory factors. Therefore, an in vitro system capable of monitoring protein production with fully defined components that can be supplemented with accessory factors would greatly aid riboswitch studies. Herein we present a system that is amenable to such analyses. Further, since the described system can be easily reconstituted within compartments to build artificial, cellular mimics with sensing capability, protocols are provided for building sense-response systems within water-in-oil emulsion compartments and lipid vesicles. Only standard laboratory equipment and commercially available material are exploited for the described assays, including DNA, purified transcription-translation machinery, i.e., the PURE system, and a spectrofluorometer.

  11. Rho and RNase play a central role in FMN riboswitch regulation in Corynebacterium glutamicum.

    Science.gov (United States)

    Takemoto, Norihiko; Tanaka, Yuya; Inui, Masayuki

    2015-01-01

    Riboswitches are RNA elements that regulate gene expression in response to their ligand. Although these regulations are thought to be performed without any aid of other factors, recent studies suggested the participation of protein factors such as transcriptional termination factor Rho and RNase in some riboswitch regulations. However, to what extent these protein factors contribute to the regulation was unclear. Here, we studied the regulatory mechanism of the flavin mononucleotide (FMN) riboswitch of Corynebacterium glutamicum which controls the expression of downstream ribM gene. Our results showed that this riboswitch downregulates both ribM mRNA and RibM protein levels in FMN-rich cells. Analysis of mRNA stability and chromatin immunoprecipitation-real-time PCR analysis targeting RNA polymerase suggested the involvement of the mRNA degradation and premature transcriptional termination in this regulation, respectively. Simultaneous disruption of RNase E/G and Rho function completely abolished the regulation at the mRNA level. Also, the regulation at the protein level was largely diminished. However, some FMN-dependent regulation at the protein level remained, suggesting the presence of other minor regulatory mechanisms. Altogether, we demonstrated for the first time that two protein factors, Rho and RNase E/G, play a central role in the riboswitch-mediated gene expression control.

  12. [Ribozyme riboswitch based gene expression regulation systems for gene therapy applications: progress and challenges].

    Science.gov (United States)

    Feng, Jing-Xian; Wang, Jia-wen; Lin, Jun-sheng; Diao, Yong

    2014-11-01

    Robust and efficient control of therapeutic gene expression is needed for timing and dosing of gene therapy drugs in clinical applications. Ribozyme riboswitch provides a promising building block for ligand-controlled gene-regulatory system, based on its property that exhibits tunable gene regulation, design modularity, and target specificity. Ribozyme riboswitch can be used in various gene delivery vectors. In recent years, there have been breakthroughs in extending ribozyme riboswitch's application from gene-expression control to cellular function and fate control. High throughput screening platforms were established, that allow not only rapid optimization of ribozyme riboswitch in a microbial host, but also straightforward transfer of selected devices exhibiting desired activities to mammalian cell lines in a predictable manner. Mathematical models were employed successfully to explore the performance of ribozyme riboswitch quantitively and its rational design predictably. However, to progress toward gene therapy relevant applications, both precision rational design of regulatory circuits and the biocompatibility of regulatory ligand are still of crucial importance.

  13. Computational prediction of riboswitch tertiary structures including pseudoknots by RAGTOP: a hierarchical graph sampling approach.

    Science.gov (United States)

    Kim, Namhee; Zahran, Mai; Schlick, Tamar

    2015-01-01

    The modular organization of RNA structure has been exploited in various computational and theoretical approaches to identify RNA tertiary (3D) motifs and assemble RNA structures. Riboswitches exemplify this modularity in terms of both structural and functional adaptability of RNA components. Here, we extend our computational approach based on tree graph sampling to the prediction of riboswitch topologies by defining additional edges to mimick pseudoknots. Starting from a secondary (2D) structure, we construct an initial graph deduced from predicted junction topologies by our data-mining algorithm RNAJAG trained on known RNAs; we sample these graphs in 3D space guided by knowledge-based statistical potentials derived from bending and torsion measures of internal loops as well as radii of gyration for known RNAs. We present graph sampling results for 10 representative riboswitches, 6 of them with pseudoknots, and compare our predictions to solved structures based on global and local RMSD measures. Our results indicate that the helical arrangements in riboswitches can be approximated using our combination of modified 3D tree graph representations for pseudoknots, junction prediction, graph moves, and scoring functions. Future challenges in the field of riboswitch prediction and design are also discussed.

  14. Identification of Spermidine Binding Site in T-box Riboswitch Antiterminator RNA.

    Science.gov (United States)

    Liu, Jia; Zeng, Chunxi; Hogan, Vivian; Zhou, Shu; Monwar, Md Masud; Hines, Jennifer V

    2016-02-01

    The T-box transcription antitermination riboswitch controls bacterial gene expression by structurally responding to uncharged, cognate tRNA. Previous studies indicated that cofactors, such as the polyamine spermidine, might serve a specific functional role in enhancing riboswitch efficacy. As riboswitch function depends on key RNA structural changes involving the antiterminator element, the interaction of spermidine with the T-box riboswitch antiterminator element was investigated. Spermidine binds antiterminator model RNA with high affinity (micromolar Kd ) based on isothermal titration calorimetry and fluorescence-monitored binding assays. NMR titration studies, molecular modeling, and inline and enzymatic probing studies indicate that spermidine binds at the 3' portion of the highly conserved seven-nucleotide bulge in the antiterminator. Together, these results support the conclusion that spermidine binds the T-box antiterminator RNA preferentially in a location important for antiterminator function. The implications of these findings are significant both for better understanding of the T-box riboswitch mechanism and for antiterminator-targeted drug discovery efforts.

  15. Dynamic energy landscapes of riboswitches help interpret conformational rearrangements and function.

    Directory of Open Access Journals (Sweden)

    Giulio Quarta

    Full Text Available Riboswitches are RNAs that modulate gene expression by ligand-induced conformational changes. However, the way in which sequence dictates alternative folding pathways of gene regulation remains unclear. In this study, we compute energy landscapes, which describe the accessible secondary structures for a range of sequence lengths, to analyze the transcriptional process as a given sequence elongates to full length. In line with experimental evidence, we find that most riboswitch landscapes can be characterized by three broad classes as a function of sequence length in terms of the distribution and barrier type of the conformational clusters: low-barrier landscape with an ensemble of different conformations in equilibrium before encountering a substrate; barrier-free landscape in which a direct, dominant "downhill" pathway to the minimum free energy structure is apparent; and a barrier-dominated landscape with two isolated conformational states, each associated with a different biological function. Sharing concepts with the "new view" of protein folding energy landscapes, we term the three sequence ranges above as the sensing, downhill folding, and functional windows, respectively. We find that these energy landscape patterns are conserved in various riboswitch classes, though the order of the windows may vary. In fact, the order of the three windows suggests either kinetic or thermodynamic control of ligand binding. These findings help understand riboswitch structure/function relationships and open new avenues to riboswitch design.

  16. From selection hits to clinical leads: progress in aptamer discovery

    Directory of Open Access Journals (Sweden)

    Keith E Maier

    2016-01-01

    Full Text Available Aptamers were discovered more than 25 years ago, yet only one has been approved by the US Food and Drug Administration to date. With some noteworthy advances in their chemical design and the enzymes we use to make them, aptamers and aptamer-based therapeutics have seen a resurgence in interest. New aptamer drugs are being approved for clinical evaluation, and it is certain that we will see increasingly more aptamers and aptamer-like drugs in the future. In this review, we will discuss the production of aptamers with an emphasis on the advances and modifications that enabled early aptamers to succeed in clinical trials as well as those that are likely to be important for future generations of these drugs.

  17. Cell-SELEX Identifies a "Sticky" RNA Aptamer Sequence.

    Science.gov (United States)

    Ray, Partha; White, Rebekah R

    2017-01-01

    Cell-SELEX is performed to select for cell binding aptamers. We employed an additional selection pressure by using RNAse to remove surface-binding aptamers and select for cell-internalizing aptamers. A common RNA sequence was identified from independent cell-SELEX procedures against two different pancreatic cancer cell lines, indicating a strong selection pressure towards this sequence from the large pool of other available sequences present in the aptamer library. The aptamer is not specific for the pancreatic cancer cell lines, and a similar sequence motif is present in previously published internalizing aptamers. The identified sequence forms a structural motif that binds to a surface protein, which either is highly abundant or has strong affinity for the selected aptamer sequence. Deselecting (removing) this sequence during cell-SELEX may increase the probability of identifying aptamers against cell type-specific targets on the cell surface.

  18. Screening of Aptamers on Microfluidic Systems for Clinical Applications

    OpenAIRE

    Gwo-Bin Lee; Chao-Jyun Huang; Chen-Hsun Weng

    2012-01-01

    The use of microfluidic systems for screening of aptamers and their biomedical applications are reviewed in this paper. Aptamers with different nucleic acid sequences have been extensively studied and the results demonstrated a strong binding affinity to target molecules such that they can be used as promising candidate biomarkers for diagnosis and therapeutics. Recently, the aptamer screening protocol has been conducted with microfluidic-based devices. Furthermore, aptamer affinity screening...

  19. Development of RNA aptamers for detection of Salmonella Enteritidis.

    Science.gov (United States)

    Hyeon, Ji-Yeon; Chon, Jung-Whan; Choi, In-Soo; Park, Chankyu; Kim, Dong-Eun; Seo, Kun-Ho

    2012-04-01

    We developed and evaluated RNA aptamers to analyze their potential for use in detecting Salmonella Enteritidis. The selected aptamer was observed to specifically bind to Salmonella Enteritidis without any cross-reactivity to other Salmonella serovars. Thus, this study suggests that aptamers specific to Salmonella Enteritidis have a high potential for use in presumptive presumptive screening methods or alternative serotyping methods.

  20. Oligonucleotide Aptamers: New Tools for Targeted Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Hongguang Sun

    2014-01-01

    Full Text Available Aptamers are a class of small nucleic acid ligands that are composed of RNA or single-stranded DNA oligonucleotides and have high specificity and affinity for their targets. Similar to antibodies, aptamers interact with their targets by recognizing a specific three-dimensional structure and are thus termed “chemical antibodies.” In contrast to protein antibodies, aptamers offer unique chemical and biological characteristics based on their oligonucleotide properties. Hence, they are more suitable for the development of novel clinical applications. Aptamer technology has been widely investigated in various biomedical fields for biomarker discovery, in vitro diagnosis, in vivo imaging, and targeted therapy. This review will discuss the potential applications of aptamer technology as a new tool for targeted cancer therapy with emphasis on the development of aptamers that are able to specifically target cell surface biomarkers. Additionally, we will describe several approaches for the use of aptamers in targeted therapeutics, including aptamer-drug conjugation, aptamer-nanoparticle conjugation, aptamer-mediated targeted gene therapy, aptamer-mediated immunotherapy, and aptamer-mediated biotherapy.

  1. Crystal Structure of the Lysine Riboswitch Regulatory mRNA Element

    Energy Technology Data Exchange (ETDEWEB)

    Garst, A.; Heroux, A; Rambo, R; Batey, R

    2008-01-01

    Riboswitches are metabolite-sensitive elements found in mRNAs that control gene expression through a regulatory secondary structural switch. Along with regulation of lysine biosynthetic genes, mutations within the lysine-responsive riboswitch (L-box) play a role in the acquisition of resistance to antimicrobial lysine analogs. To understand the structural basis for lysine binding, we have determined the 2.8{angstrom} resolution crystal structure of lysine bound to the Thermotoga maritima asd lysine riboswitch ligand-binding domain. The structure reveals a complex architecture scaffolding a binding pocket completely enveloping lysine. Mutations conferring antimicrobial resistance cluster around this site as well as highly conserved long range interactions, indicating that they disrupt lysine binding or proper folding of the RNA. Comparison of the free and bound forms by x-ray crystallography, small angle x-ray scattering, and chemical probing reveals almost identical structures, indicating that lysine induces only limited and local conformational changes upon binding.

  2. Synthesis of spin-labeled riboswitch RNAs using convertible nucleosides and DNA-catalyzed RNA ligation.

    Science.gov (United States)

    Büttner, Lea; Seikowski, Jan; Wawrzyniak, Katarzyna; Ochmann, Anne; Höbartner, Claudia

    2013-10-15

    Chemically stable nitroxide radicals that can be monitored by electron paramagnetic resonance (EPR) spectroscopy can provide information on structural and dynamic properties of functional RNA such as riboswitches. The convertible nucleoside approach is used to install 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO) and 2,2,5,5-tetramethylpyrrolidin-1-oxyl (proxyl) labels at the exocyclic N(4)-amino group of cytidine and 2'-O-methylcytidine nucleotides in RNA. To obtain site-specifically labeled long riboswitch RNAs beyond the limit of solid-phase synthesis, we report the ligation of spin-labeled RNA using an in vitro selected deoxyribozyme as catalyst, and demonstrate the synthesis of TEMPO-labeled 53 nt SAM-III and 118 nt SAM-I riboswitch domains (SAM=S-adenosylmethionine).

  3. Structural Basis for Gene Regulation by a Thiamine Pyrophosphate-Sensing Riboswitch

    Energy Technology Data Exchange (ETDEWEB)

    Serganov,A.; Polonskaia, A.; Phan, A.; Breaker, R.; Patel, D.

    2006-01-01

    Riboswitches are metabolite-sensing RNAs, typically located in the non-coding portions of messenger RNAs, that control the synthesis of metabolite-related proteins. Here we describe a 2.05 Angstroms crystal structure of a riboswitch domain from the Escherichia coli thiM mRNA4 that responds to the coenzyme thiamine pyrophosphate (TPP). TPP is an active form of vitamin B1, an essential participant in many protein-catalysed reactions. Organisms from all three domains of life including bacteria, plants and fungi, use TPP-sensing riboswitches to control genes responsible for importing or synthesizing thiamine and its phosphorylated derivatives, making this riboswitch class the most widely distributed member of the metabolite-sensing RNA regulatory system. The structure reveals a complex folded RNA in which one subdomain forms an intercalation pocket for the 4-amino-5-hydroxymethyl-2-methylpyrimidine moiety of TPP, whereas another subdomain forms a wider pocket that uses bivalent metal ions and water molecules to make bridging contacts to the pyrophosphate moiety of the ligand. The two pockets are positioned to function as a molecular measuring device that recognizes TPP in an extended conformation. The central thiazole moiety is not recognized by the RNA, which explains why the antimicrobial compound pyrithiamine pyrophosphate targets this riboswitch and downregulates the expression of thiamine metabolic genes. Both the natural ligand and its drug-like analogue stabilize secondary and tertiary structure elements that are harnessed by the riboswitch to modulate the synthesis of the proteins coded by the mRNA. In addition, this structure provides insight into how folded RNAs can form precision binding pockets that rival those formed by protein genetic factors.

  4. In Vitro Selection for Small-Molecule-Triggered Strand Displacement and Riboswitch Activity.

    Science.gov (United States)

    Martini, Laura; Meyer, Adam J; Ellefson, Jared W; Milligan, John N; Forlin, Michele; Ellington, Andrew D; Mansy, Sheref S

    2015-10-16

    An in vitro selection method for ligand-responsive RNA sensors was developed that exploited strand displacement reactions. The RNA library was based on the thiamine pyrophosphate (TPP) riboswitch, and RNA sequences capable of hybridizing to a target duplex DNA in a TPP regulated manner were identified. After three rounds of selection, RNA molecules that mediated a strand exchange reaction upon TPP binding were enriched. The enriched sequences also showed riboswitch activity. Our results demonstrated that small-molecule-responsive nucleic acid sensors can be selected to control the activity of target nucleic acid circuitry.

  5. Aptamer-Functionalized Nano-Biosensors

    Directory of Open Access Journals (Sweden)

    Tai-Chia Chiu

    2009-12-01

    Full Text Available Nanomaterials have become one of the most interesting sensing materials because of their unique size- and shape-dependent optical properties, high surface energy and surface-to-volume ratio, and tunable surface properties. Aptamers are oligonucleotides that can bind their target ligands with high affinity. The use of nanomaterials that are bioconjugated with aptamers for selective and sensitive detection of analytes such as small molecules, metal ions, proteins, and cells has been demonstrated. This review focuses on recent progress in the development of biosensors by integrating functional aptamers with different types of nanomaterials, including quantum dots, magnetic nanoparticles (NPs, metallic NPs, and carbon nanotubes. Colorimetry, fluorescence, electrochemistry, surface plasmon resonance, surface-enhanced Raman scattering, and magnetic resonance imaging are common detection modes for a broad range of analytes with high sensitivity and selectivity when using aptamer bioconjugated nanomaterials (Apt-NMs. We highlight the important roles that the size and concentration of nanomaterials, the secondary structure and density of aptamers, and the multivalent interactions play in determining the specificity and sensitivity of the nanosensors towards analytes. Advantages and disadvantages of the Apt-NMs for bioapplications are focused.

  6. Nucleic acid aptamers: an emerging frontier in cancer therapy.

    Science.gov (United States)

    Zhu, Guizhi; Ye, Mao; Donovan, Michael J; Song, Erqun; Zhao, Zilong; Tan, Weihong

    2012-11-04

    The last two decades have witnessed the development and application of nucleic acid aptamers in a variety of fields, including target analysis, disease therapy, and molecular and cellular engineering. The efficient and widely applicable aptamer selection, reproducible chemical synthesis and modification, generally impressive target binding selectivity and affinity, relatively rapid tissue penetration, low immunogenicity, and rapid systemic clearance make aptamers ideal recognition elements for use as therapeutics or for in vivo delivery of therapeutics. In this feature article, we discuss the development and biomedical application of nucleic acid aptamers, with emphasis on cancer cell aptamer isolation, targeted cancer therapy, oncology biomarker identification and drug discovery.

  7. Function and dynamics of aptamers: A case study on the malachite green aptamer

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Tianjiao [Iowa State Univ., Ames, IA (United States)

    2008-01-01

    Aptamers are short single-stranded nucleic acids that can bind to their targets with high specificity and high affinity. To study aptamer function and dynamics, the malachite green aptamer was chosen as a model. Malachite green (MG) bleaching, in which an OH- attacks the central carbon (C1) of MG, was inhibited in the presence of the malachite green aptamer (MGA). The inhibition of MG bleaching by MGA could be reversed by an antisense oligonucleotide (AS) complementary to the MGA binding pocket. Computational cavity analysis of the NMR structure of the MGA-MG complex predicted that the OH- is sterically excluded from the C1 of MG. The prediction was confirmed experimentally using variants of the MGA with changes in the MG binding pocket. This work shows that molecular reactivity can be reversibly regulated by an aptamer-AS pair based on steric hindrance. In addition to demonstrate that aptamers could control molecular reactivity, aptamer dynamics was studied with a strategy combining molecular dynamics (MD) simulation and experimental verification. MD simulation predicted that the MG binding pocket of the MGA is largely pre-organized and that binding of MG involves reorganization of the pocket and a simultaneous twisting of the MGA terminal stems around the pocket. MD simulation also provided a 3D-structure model of unoccupied MGA that has not yet been obtained by biophysical measurements. These predictions were consistent with biochemical and biophysical measurements of the MGA-MG interaction including RNase I footprinting, melting curves, thermodynamic and kinetic constants measurement. This work shows that MD simulation can be used to extend our understanding of the dynamics of aptamer-target interaction which is not evident from static 3D-structures. To conclude, I have developed a novel concept to control molecular reactivity by an aptamer based on steric protection and a strategy to study the dynamics of aptamer-target interaction by combining MD

  8. Aptamer nanomedicine for cancer therapeutics: barriers and potential for translation.

    Science.gov (United States)

    Lao, Yeh-Hsing; Phua, Kyle K L; Leong, Kam W

    2015-03-24

    Aptamer nanomedicine, including therapeutic aptamers and aptamer nanocomplexes, is beginning to fulfill its potential in both clinical trials and preclinical studies. Especially in oncology, aptamer nanomedicine may perform better than conventional or antibody-based chemotherapeutics due to specificity compared to the former and stability compared to the latter. Many proof-of-concept studies on applying aptamers to drug delivery, gene therapy, and cancer imaging have shown promising efficacy and impressive safety in vivo toward translation. Yet, there remains ample room for improvement and critical barriers to be addressed. In this review, we will first introduce the recent progress in clinical trials of aptamer nanomedicine, followed by a discussion of the barriers at the design and in vivo application stages. We will then highlight recent advances and engineering strategies proposed to tackle these barriers. Aptamer cancer nanomedicine has the potential to address one of the most important healthcare issues of the society.

  9. Recent progresses in biomedical applications of aptamer-functionalized systems.

    Science.gov (United States)

    Ding, Fei; Gao, Yangguang; He, Xianran

    2017-09-15

    Aptamers, known as "chemical antibodies" are screened via a combinational technology of systematic evolution of ligands by exponential enrichment (SELEX). Due to their specific targeting ability, high binding affinity, low immunogenicity and easy modification, aptamer-functionalized systems have been extensively applied in various fields and exhibit favorable results. However, there is still a long way for them to be commercialized, and few aptamer-functionalized systems have yet successfully entered clinical and industrial use. Thus, it is necessary to overview the recent research progresses of aptamer-functionalized systems for the researchers to improve or design novel and better aptamer-functionalized systems. In this review, we first introduce the recent progresses of aptamer-functionalized systems' applications in biosensing, targeted drug delivery, gene therapy and cancer cell imaging, followed by a discussion of the challenges faced with extensive applications of aptamer-functionalized systems and speculation of the future prospects of them. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Generation of aptamer for biosensing applications

    Science.gov (United States)

    Gopinath, Subash C. B.; Hashim, U.; Arshad, M. K. Md.; Ruslinda, A. R.

    2016-07-01

    Systematic evolution of ligands by exponential enrichment (SELEX), an in vitro strategy which involves generation of aptamer. Aptamer is an artificial antibody, behave very similar to antibody and several instances reported to be better than antibodies. In this study, an attempt has been made to generate aptamer against factor IX, a potential candidate involve in human blood coagulation cascade. Totally, 10 selection cycles have been performed and molecules from 10th cycle have shown higher binding affinity with factor IX as 56 and 68% against the factor IX concentrations of 100 and 200 nM, respectively. With these higher binding affinities, it is clear that these molecules have higher potential for sensing applications.

  11. Developing Aptamers by Cell-Based SELEX.

    Science.gov (United States)

    Catuogno, Silvia; Esposito, Carla Lucia; de Franciscis, Vittorio

    2016-01-01

    The reliable targeting of cell surface disease-associated proteins is a major challenge in chemical biology and molecular medicine. In this regard, aptamers represent a very attractive and innovative class of ligand molecules. Aptamers are generated by a reiterated in vitro procedure, named SELEX (Systematic Evolution of Ligands by Exponential enrichment). In order to generate aptamers for heavily modified cell surface-bound proteins and transmembrane receptors, the SELEX procedure has been recently adapted to the use of living cells as complex targets (referred as "cell-SELEX"). Here we give an overview on the most recent advances in the field of cell-SELEX technology, providing a detailed description of the differential cell-SELEX approach that has been developed in our laboratory to identify specific signatures for human malignant glioma and non-small-cell lung cancer. The procedures used for the evaluation of binding specificity and for the preliminary identification of potential target receptors will be also described.

  12. Aptamer Based Microsphere Biosensor for Thrombin Detection

    Directory of Open Access Journals (Sweden)

    Xudong Fan

    2006-08-01

    Full Text Available We have developed an optical microsphere resonator biosensor using aptamer asreceptor for the measurement of the important biomolecule thrombin. The sphere surface ismodified with anti-thrombin aptamer, which has excellent binding affinity and selectivityfor thrombin. Binding of the thrombin at the sphere surface is monitored by the spectralposition of the microsphere’s whispering gallery mode resonances. A detection limit on theorder of 1 NIH Unit/mL is demonstrated. Control experiments with non-aptameroligonucleotide and BSA are also carried out to confirm the specific binding betweenaptamer and thrombin. We expect that this demonstration will lead to the development ofhighly sensitive biomarker sensors based on aptamer with lower cost and higher throughputthan current technology.

  13. An aptamer beacon responsive to botulinum toxins.

    Science.gov (United States)

    Bruno, John G; Richarte, Alicia M; Carrillo, Maria P; Edge, Allison

    2012-01-15

    Sixty candidate DNA aptamers were developed against botulinum neurotoxin (BoNT) type A light chain (LC) from ten rounds of selection, resulting in several identical sequences. Secondary structures of the identical aptamers were compared to structures of previously reported BoNT A DNA aptamers. A series of ten candidate loop structures were selected from this comparison as potential binding pockets and aptamer beacons. These candidate beacons were synthesized with 5'-TYE 665 and 3'-Iowa Black quencher labels for comparison of fluorescence levels as a function of BoNT A LC concentration. Only three of the ten candidates exhibited any fluorescence response to increasing levels of BoNT A LC. However, of the two most responsive candidates, one represented a subset loop of the larger more intensely fluorescent double-looped structure, designated Beacon 10. This beacon yielded a lower limit of detection of 1 ng/mL in buffer using a spectrofluorometer and a portable handheld fluorometer, but also responded substantially to BoNT A, B, E holotoxins and heavy or light chain components even in a dilute soil suspension, but not in 50% human serum. Beacon 10 did not respond strongly to a variety of other divergent peptides, suggesting that it is relatively specific to the level of botulinum toxins and is only useful for environmental testing. Beacon 10 also shared short sequence segments with other published BoNT aptamer DNA sequences, suggesting that these may be points of physical contact between the aptamers and BoNTs.

  14. Twister ribozymes as highly versatile expression platforms for artificial riboswitches

    Science.gov (United States)

    Felletti, Michele; Stifel, Julia; Wurmthaler, Lena A.; Geiger, Sophie; Hartig, Jörg S.

    2016-01-01

    The utilization of ribozyme-based synthetic switches in biotechnology has many advantages such as an increased robustness due to in cis regulation, small coding space and a high degree of modularity. The report of small endonucleolytic twister ribozymes provides new opportunities for the development of advanced tools for engineering synthetic genetic switches. Here we show that the twister ribozyme is distinguished as an outstandingly flexible expression platform, which in conjugation with three different aptamer domains, enables the construction of many different one- and two-input regulators of gene expression in both bacteria and yeast. Besides important implications in biotechnology and synthetic biology, the observed versatility in artificial genetic control set-ups hints at possible natural roles of this widespread ribozyme class. PMID:27670347

  15. RNA fluorescence with light-up aptamers

    Science.gov (United States)

    Ouellet, Jonathan

    2016-06-01

    Seeing is not only believing; it also includes understanding. Cellular imaging with GFP in live cells has been transformative in many research fields. Modulation of cellular regulation is tightly regulated and innovative imaging technologies contribute to further understand cellular signaling and physiology. New types of genetically encoded biosensors have been developed over the last decade. They are RNA aptamers that bind with their cognate fluorogen ligands and activate their fluorescence. The emergence and the evolution of these RNA aptamers as well as their conversion into a wide spectrum of applications are examined in a global way.

  16. 新型基因表达调控元件——人工核糖开关的构建及筛选%Engineering and screening of artificial riboswitch as a novel gene control element

    Institute of Scientific and Technical Information of China (English)

    杨会勇; 刁勇; 林俊生; 许瑞安

    2012-01-01

    Various artificial riboswitches have been constructed by utilization of designed aptamers or by modification of natural riboswitch systems, because they can regulate gene expression in a highly efficient, precise and fast way, and promise to supply simple cis-acting, modular, and non-immunogenic system for use in future gene therapy applications. In this review, we present an overview of currently available technologies to design and select engineered riboswitches, and discuss some possible technologies that would allow them highly responsive to non-natural ligands, and dynamic control of gene expression in mammalian cells. Though how to bring custom-designed riboswitches as a novel and versatile tool box to gene control system is still a great challenge, the combination of structure-activity relationship information, computer based molecular design, in vitro selection, and high-through screening will serve as powerful tools for further development of riboswitch based gene regulatory systems.%核糖开关作为一种新发现的RNA元件,可以高效、准确、快速地执行基因调控任务,且免疫原性低,有可能在将来以顺式模块的方式应用于未来的基因治疗.近年来已经成功构建了多种人造核糖开关,构建方法主要是利用人工适体元件与基因表达调控元件组装,或者是在天然核糖开关基础上进行改造.文中全面综述了涉及人工核糖开关设计及筛选的技术,讨论了可以用于哺乳细胞、响应非天然配体信号、调控特征为热力学和动力学控制的核糖开关的设计新策略,并对核糖开关的筛选构建策略及其在基因治疗及新型药物开发领域的应用前景进行了展望.尽管目前将核糖开关设计成为功能强大的新型基因调控系统还面临很大的困难,但通过构效关系的研究、计算机辅助设计、体外筛选及细胞内筛选技术、高通量优化筛选等技术的综合应用,核糖开关一定可以

  17. Fluoride ion encapsulation by Mg[superscript 2+] ions and phosphates in a fluoride riboswitch

    Energy Technology Data Exchange (ETDEWEB)

    Ren, Aiming; Rajashankar, Kanagalaghatta R.; Patel, Dinshaw J. (Cornell); (MSKCC)

    2012-06-26

    Significant advances in our understanding of RNA architecture, folding and recognition have emerged from structure-function studies on riboswitches, non-coding RNAs whose sensing domains bind small ligands and whose adjacent expression platforms contain RNA elements involved in the control of gene regulation. We now report on the ligand-bound structure of the Thermotoga petrophila fluoride riboswitch, which adopts a higher-order RNA architecture stabilized by pseudoknot and long-range reversed Watson-Crick and Hoogsteen A {sm_bullet} U pair formation. The bound fluoride ion is encapsulated within the junctional architecture, anchored in place through direct coordination to three Mg{sup 2+} ions, which in turn are octahedrally coordinated to water molecules and five inwardly pointing backbone phosphates. Our structure of the fluoride riboswitch in the bound state shows how RNA can form a binding pocket selective for fluoride, while discriminating against larger halide ions. The T. petrophila fluoride riboswitch probably functions in gene regulation through a transcription termination mechanism.

  18. Fluoride ion encapsulation by Mg2+ ions and phosphates in a fluoride riboswitch.

    Science.gov (United States)

    Ren, Aiming; Rajashankar, Kanagalaghatta R; Patel, Dinshaw J

    2012-05-13

    Significant advances in our understanding of RNA architecture, folding and recognition have emerged from structure-function studies on riboswitches, non-coding RNAs whose sensing domains bind small ligands and whose adjacent expression platforms contain RNA elements involved in the control of gene regulation. We now report on the ligand-bound structure of the Thermotoga petrophila fluoride riboswitch, which adopts a higher-order RNA architecture stabilized by pseudoknot and long-range reversed Watson-Crick and Hoogsteen A•U pair formation. The bound fluoride ion is encapsulated within the junctional architecture, anchored in place through direct coordination to three Mg(2+) ions, which in turn are octahedrally coordinated to water molecules and five inwardly pointing backbone phosphates. Our structure of the fluoride riboswitch in the bound state shows how RNA can form a binding pocket selective for fluoride, while discriminating against larger halide ions. The T. petrophila fluoride riboswitch probably functions in gene regulation through a transcription termination mechanism.

  19. The re-design of a theophylline riboswitch for DNT sensing

    Science.gov (United States)

    Chushak, Yaroslav; Kelley-Loughnane, Nancy; Harbaugh, Svetlana; Stone, Morley

    2008-03-01

    Riboswitches are noncoding elements of mRNA that recognize and bind to small molecules and regulate the translation process of downstream genes. As an initial study, we used a theophylline riboswitch that regulates the expression of the Tobacco etch virus (TEV) protease placed downstream of the switch as a controlling element. Upon expression of TEV protease, an optical reporter is cleaved producing change in fluorescence resonance energy transfer (FRET) between BFP and eGFP. We altered the sensing domain of the original construct to create a synthetic riboswitch that responds to the presence of 2,4-dinitrotoluene (DNT) molecules. Computational analysis using Autodock4 and AMBER9 software packages showed that U24A mutant has a significantly higher binding affinity for DNT molecule compared to the original theophylline. Cells expressing the re-designed riboswitch showed a marked optical difference in fluorescence emission in the presence of DNT molecules, leading to the potential of using this construct in biosentinel applications of highly nitrated compounds.

  20. A riboswitch sensor to determine vitamin B12 in fermented foods.

    Science.gov (United States)

    Zhu, Xuan; Wang, Xiaofeng; Zhang, Chen; Wang, Xiaoqi; Gu, Qing

    2015-05-15

    We describe a sensitive and selective method for determination of vitamin B12 content in fermented foods using riboswitch sensor. A riboswitch amplicon from Propionibacterium freudenreichii was cloned in p519NGFP vector in Escherichia coli BL21 (DE3). The expression of green fluorescence protein was revers correlated to the concentrations of adenosylcobalamin. Adenosylcobalamin directly binds to riboswitch region leading to conformational changes in the secondary structure of mRNA, thus inhibiting expression. After various examinations, a standard curve was obtained from 10 to 1000 ng/mL of cyanocobalamin. The limit of determination is 10 ng/mL. The inter-assay coefficients of variation were 7.5% for the range of 10-1000 ng/mL. The recovery of this method was 92.3%. This method has no or less responses to nucleic acid, pseudovitamin B12, vitamin B12 bound to intrinsic factor and haptocorrin. The riboswitch sensor results were similar with HPLC, but they were Ca. 24% lower than the microbiological assay results.

  1. Analysis of riboswitch structure and ligand binding using small-angle X-ray scattering (SAXS).

    Science.gov (United States)

    Baird, Nathan J; Ferré-D'Amaré, Adrian R

    2014-01-01

    Small-angle X-ray scattering (SAXS) is a powerful tool for examining the global conformation of riboswitches in solution, and how this is modulated by binding of divalent cations and small molecule ligands. SAXS experiments, which typically require only minutes per sample, directly yield two quantities describing the size and shape of the RNA: the radius of gyration (Rg) and the maximum linear dimension (Dmax). Examination of these quantities can reveal if a riboswitch undergoes cation-induced compaction. Comparison of the Rg and Dmax values between samples containing different concentrations of ligand reveals the overall structural response of the riboswitch to ligand. The Kratky plot (a graphical representation that emphasizes the higher-resolution SAXS data) and the P(r) plot or pair-probability distribution (an indirect Fourier transform, or power spectrum of the data) can provide additional evidence of riboswitch conformational changes. Simulation methods have been developed for generating three-dimensional reconstructions consistent with the one-dimensional SAXS data. These low-resolution molecular envelopes can aid in deciphering the relative helical arrangement within the RNA.

  2. Construction and application of riboswitch-based sensors that detect metabolites within bacterial cells.

    Science.gov (United States)

    Fowler, Casey C; Li, Yingfu

    2014-01-01

    A riboswitch is an RNA element that detects the level of a specific metabolite within the cell and regulates the expression of co-transcribed genes. By fusing a riboswitch to a reporter protein in a carefully designed and tested construct, this ability can be exploited to create an intracellular sensor that detects the level of a particular small molecule within live bacterial cells. There is a great deal of flexibility in the design of such a sensor and factors such as the molecule to be detected and the downstream experiments in which the sensor will be applied should guide the specific blueprint of the final construct. The completed sensor plasmid needs to be rigorously tested with appropriate controls to ensure that its dynamic range, signal strength, sensitivity and specificity are suitable for its intended applications. In this chapter, methods for the design, assessment and use of riboswitch sensors are provided along with those for one example application for which riboswitch sensors are ideally suited.

  3. Recognition of the bacterial second messenger cyclic diguanylate by its cognate riboswitch

    Energy Technology Data Exchange (ETDEWEB)

    Kulshina, Nadia; Baird, Nathan J.; Ferré-D' Amaré, Adrian R.; (UWASH); (FHCRC)

    2009-12-03

    The cyclic diguanylate (bis-(3'-5')-cyclic dimeric guanosine monophosphate, c-di-GMP) riboswitch is the first known example of a gene-regulatory RNA that binds a second messenger. c-di-GMP is widely used by bacteria to regulate processes ranging from biofilm formation to the expression of virulence genes. The cocrystal structure of the c-di-GMP responsive GEMM riboswitch upstream of the tfoX gene of Vibrio cholerae reveals the second messenger binding the RNA at a three-helix junction. The two-fold symmetric second messenger is recognized asymmetrically by the monomeric riboswitch using canonical and noncanonical base-pairing as well as intercalation. These interactions explain how the RNA discriminates against cyclic diadenylate (c-di-AMP), a putative bacterial second messenger. Small-angle X-ray scattering and biochemical analyses indicate that the RNA undergoes compaction and large-scale structural rearrangement in response to ligand binding, consistent with organization of the core three-helix junction of the riboswitch concomitant with binding of c-di-GMP.

  4. A DNA sequence obtained by replacement of the dopamine RNA aptamer bases is not an aptamer.

    Science.gov (United States)

    Álvarez-Martos, Isabel; Ferapontova, Elena E

    2017-08-05

    A unique specificity of the aptamer-ligand biorecognition and binding facilitates bioanalysis and biosensor development, contributing to discrimination of structurally related molecules, such as dopamine and other catecholamine neurotransmitters. The aptamer sequence capable of specific binding of dopamine is a 57 nucleotides long RNA sequence reported in 1997 (Biochemistry, 1997, 36, 9726). Later, it was suggested that the DNA homologue of the RNA aptamer retains the specificity of dopamine binding (Biochem. Biophys. Res. Commun., 2009, 388, 732). Here, we show that the DNA sequence obtained by the replacement of the RNA aptamer bases for their DNA analogues is not able of specific biorecognition of dopamine, in contrast to the original RNA aptamer sequence. This DNA sequence binds dopamine and structurally related catecholamine neurotransmitters non-specifically, as any DNA sequence, and, thus, is not an aptamer and cannot be used neither for in vivo nor in situ analysis of dopamine in the presence of structurally related neurotransmitters. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Method for Confirming Cytoplasmic Delivery of RNA Aptamers

    Science.gov (United States)

    Dickey, David D; Dassie, Justin P; Giangrande, Paloma H

    2016-01-01

    RNA aptamers are single-stranded RNA oligos that represent a powerful emerging technology with potential for treating numerous diseases. More recently, cell-targeted RNA aptamers have been developed for delivering RNA interference (RNAi) modulators (siRNAs and miRNAs) to specific diseased cells (e.g., cancer cells or HIV infected cells) in vitro and in vivo. However, despite initial promising reports, the broad application of this aptamer delivery technology awaits the development of methods that can verify and confirm delivery of aptamers to the cytoplasm of target cells where the RNAi machinery resides. We recently developed a functional assay (RIP assay) to confirm cellular uptake and subsequent cytoplasmic release of an RNA aptamer which binds to a cell surface receptor expressed on prostate cancer cells (PSMA). To assess cytoplasmic delivery, the aptamer was chemically conjugated to saporin, a ribosome inactivating protein toxin that is toxic to cells only when delivered to the cytoplasm (where it inhibits the ribosome) by a cell-targeting ligand (e.g., aptamer). Here, we describe the chemistry used to conjugate the aptamer to saporin and discuss a gel-based method to verify conjugation efficiency. We also detail an in vitro functional assay to confirm that the aptamer retains function following conjugation to saporin and describe a cellular assay to measure aptamer-mediated saporin-induced cytotoxicity. PMID:26472453

  6. Nucleic acid-based aptamers: applications, development and clinical trials.

    Science.gov (United States)

    Kanwar, Jagat R; Roy, Kislay; Maremanda, Nihal G; Subramanian, Krishnakumar; Veedu, Rakesh N; Bawa, Raj; Kanwar, Rupinder K

    2015-01-01

    Short single-stranded oligonucleotides called aptamers, often termed as chemical antibodies, have been developed as powerful alternatives to traditional antibodies with respect to their obvious advantages like high specificity and affinity, longer shelf-life, easier manufacturing protocol, freedom to introduce chemical modifications for further improvement, etc. Reiterative selection process of aptamers over 10-15 cycles starting from a large initial pool of random nucleotide sequences renders them with high binding affinity, thereby making them extremely specific for their targets. Aptamer-based detection systems are well investigated and likely to displace primitive detection systems. Aptamer chimeras (combination of aptamers with another aptamer or biomacromolecule or chemical moiety) have the potential activity of both the parent molecules, and thus hold the capability to perform diverse functions at the same time. Owing to their extremely high specificity and lack of immunogenicity or pathogenicity, a number of other aptamers have recently entered clinical trials and have garnered favorable attention from pharmaceutical companies. Promising results from the clinical trials provide new hope to change the conventional style of therapy. Aptamers have attained high therapeutic relevance in a short time as compared to synthetic drugs and/or other modes of therapy. This review follows the various trends in aptamer technology including production, selection, modifications and success in clinical fields. It focusses largely on the various applications of aptamers which mainly depend upon their selection procedures. The review also sheds light on various modifications and chimerizations that have been implemented in order to improve the stability and functioning of the aptamers, including introduction of locked nucleic acids (LNAs). The application of various aptamers in detection systems has been discussed elaborately in order to stress on their role as efficient

  7. What a Difference an OH Makes: Conformational Dynamics as the Basis for the Ligand Specificity of the Neomycin-Sensing Riboswitch.

    Science.gov (United States)

    Duchardt-Ferner, Elke; Gottstein-Schmidtke, Sina R; Weigand, Julia E; Ohlenschläger, Oliver; Wurm, Jan-Philip; Hammann, Christian; Suess, Beatrix; Wöhnert, Jens

    2016-01-22

    To ensure appropriate metabolic regulation, riboswitches must discriminate efficiently between their target ligands and chemically similar molecules that are also present in the cell. A remarkable example of efficient ligand discrimination is a synthetic neomycin-sensing riboswitch. Paromomycin, which differs from neomycin only by the substitution of a single amino group with a hydroxy group, also binds but does not flip the riboswitch. Interestingly, the solution structures of the two riboswitch-ligand complexes are virtually identical. In this work, we demonstrate that the local loss of key intermolecular interactions at the substitution site is translated through a defined network of intramolecular interactions into global changes in RNA conformational dynamics. The remarkable specificity of this riboswitch is thus based on structural dynamics rather than static structural differences. In this respect, the neomycin riboswitch is a model for many of its natural counterparts.

  8. Diagnosis of active TB using aptamers

    CSIR Research Space (South Africa)

    Khati, M

    2013-08-01

    Full Text Available ) and the 6-kDa early secreted antigen target (ESAT-6), which are potent T-cell antigens that are recognised by over 70% of TB patients. We then used these aptamers to develop a TB diagnostic tool that can be used at point-of-care for early and rapid detection...

  9. RAPID-SELEX for RNA Aptamers

    Science.gov (United States)

    Ozer, Abdullah; Pagano, John M.; White, Brian S.; Shalloway, David; Lis, John T.; Craighead, Harold G.

    2013-01-01

    Aptamers are high-affinity ligands selected from DNA or RNA libraries via SELEX, a repetitive in vitro process of sequential selection and amplification steps. RNA SELEX is more complicated than DNA SELEX because of the additional transcription and reverse transcription steps. Here, we report a new selection scheme, RAPID-SELEX (RNA Aptamer Isolation via Dual-cycles SELEX), that simplifies this process by systematically skipping unnecessary amplification steps. Using affinity microcolumns, we were able to complete a multiplex selection for protein targets, CHK2 and UBLCP1, in a third of the time required for analogous selections using a conventional SELEX approach. High-throughput sequencing of the enriched pools from both RAPID and SELEX revealed many identical candidate aptamers from the starting pool of 5×1015 sequences. For CHK2, the same sequence was preferentially enriched in both selections as the top candidate and was found to bind to its respective target. These results demonstrate the efficiency and, most importantly, the robustness of our selection scheme. RAPID provides a generalized approach that can be used with any selection technology to accelerate the rate of aptamer discovery, without compromising selection performance. PMID:24376564

  10. RAPID-SELEX for RNA aptamers.

    Science.gov (United States)

    Szeto, Kylan; Latulippe, David R; Ozer, Abdullah; Pagano, John M; White, Brian S; Shalloway, David; Lis, John T; Craighead, Harold G

    2013-01-01

    Aptamers are high-affinity ligands selected from DNA or RNA libraries via SELEX, a repetitive in vitro process of sequential selection and amplification steps. RNA SELEX is more complicated than DNA SELEX because of the additional transcription and reverse transcription steps. Here, we report a new selection scheme, RAPID-SELEX (RNA Aptamer Isolation via Dual-cycles SELEX), that simplifies this process by systematically skipping unnecessary amplification steps. Using affinity microcolumns, we were able to complete a multiplex selection for protein targets, CHK2 and UBLCP1, in a third of the time required for analogous selections using a conventional SELEX approach. High-throughput sequencing of the enriched pools from both RAPID and SELEX revealed many identical candidate aptamers from the starting pool of 5 × 10(15) sequences. For CHK2, the same sequence was preferentially enriched in both selections as the top candidate and was found to bind to its respective target. These results demonstrate the efficiency and, most importantly, the robustness of our selection scheme. RAPID provides a generalized approach that can be used with any selection technology to accelerate the rate of aptamer discovery, without compromising selection performance.

  11. Crystallographic analysis of TPP riboswitch binding by small-molecule ligands discovered through fragment-based drug discovery approaches.

    Science.gov (United States)

    Warner, Katherine Deigan; Ferré-D'Amaré, Adrian R

    2014-01-01

    Riboswitches are structured mRNA elements that regulate gene expression in response to metabolite or second-messenger binding and are promising targets for drug discovery. Fragment-based drug discovery methods have identified weakly binding small molecule "fragments" that bind a thiamine pyrophosphate (TPP) riboswitch. However, these fragments require substantial chemical elaboration into more potent, drug-like molecules. Structure determination of the fragments bound to the riboswitch is the necessary next step. In this chapter, we describe the methods for co-crystallization and structure determination of fragment-bound TPP riboswitch structures. We focus on considerations for screening crystallization conditions across multiple crystal forms and provide guidance for building the fragment into the refined crystallographic model. These methods are broadly applicable for crystallographic analyses of any small molecules that bind structured RNAs.

  12. The ribB FMN riboswitch from Escherichia coli operates at the transcriptional and translational level and regulates riboflavin biosynthesis.

    Science.gov (United States)

    Pedrolli, Danielle; Langer, Simone; Hobl, Birgit; Schwarz, Julia; Hashimoto, Masayuki; Mack, Matthias

    2015-08-01

    FMN riboswitches are genetic elements that, in many bacteria, control genes responsible for biosynthesis and/or transport of riboflavin (vitamin B2 ). We report that the Escherichia coli ribB FMN riboswitch controls expression of the essential gene ribB coding for the riboflavin biosynthetic enzyme 3,4-dihydroxy-2-butanone-4-phosphate synthase (RibB; EC 4.1.99.12). Our data show that the E. coli ribB FMN riboswitch is unusual because it operates at the transcriptional and also at the translational level. Expression of ribB is negatively affected by FMN and by the FMN analog roseoflavin mononucleotide, which is synthesized enzymatically from roseoflavin and ATP. Consequently, in addition to flavoenzymes, the E. coli ribB FMN riboswitch constitutes a target for the antibiotic roseoflavin produced by Streptomyces davawensis.

  13. Nucleic acid aptamers: research tools in disease diagnostics and therapeutics.

    Science.gov (United States)

    Santosh, Baby; Yadava, Pramod K

    2014-01-01

    Aptamers are short sequences of nucleic acid (DNA or RNA) or peptide molecules which adopt a conformation and bind cognate ligands with high affinity and specificity in a manner akin to antibody-antigen interactions. It has been globally acknowledged that aptamers promise a plethora of diagnostic and therapeutic applications. Although use of nucleic acid aptamers as targeted therapeutics or mediators of targeted drug delivery is a relatively new avenue of research, one aptamer-based drug "Macugen" is FDA approved and a series of aptamer-based drugs are in clinical pipelines. The present review discusses the aspects of design, unique properties, applications, and development of different aptamers to aid in cancer diagnosis, prevention, and/or treatment under defined conditions.

  14. Small-Molecule Binding Aptamers: Selection Strategies, Characterization, and Applications

    Science.gov (United States)

    Ruscito, Annamaria; DeRosa, Maria

    2016-05-01

    Aptamers are single-stranded, synthetic oligonucleotides that fold into 3-dimensional shapes capable of binding non-covalently with high affinity and specificity to a target molecule. They are generated via an in vitro process known as the Systematic Evolution of Ligands by EXponential enrichment, from which candidates are screened and characterized, and then applied in aptamer-based biosensors for target detection. Aptamers for small molecule targets such as toxins, antibiotics, molecular markers, drugs, and heavy metals will be the focus of this review. Their accurate detection is ultimately needed for the protection and wellbeing of humans and animals. However, issues such as the drastic difference in size of the aptamer and small molecule make it challenging to select, characterize, and apply aptamers for the detection of small molecules. Thus, recent (since 2012) notable advances in small molecule aptamers, which have overcome some of these challenges, are presented here, while defining challenges that still exist are discussed

  15. Development of universal antidotes to control aptamer activity.

    Science.gov (United States)

    Oney, Sabah; Lam, Ruby T S; Bompiani, Kristin M; Blake, Charlene M; Quick, George; Heidel, Jeremy D; Liu, Joanna Yi-Ching; Mack, Brendan C; Davis, Mark E; Leong, Kam W; Sullenger, Bruce A

    2009-10-01

    With an ever increasing number of people taking numerous medications, the need to safely administer drugs and limit unintended side effects has never been greater. Antidote control remains the most direct means to counteract acute side effects of drugs, but, unfortunately, it has been challenging and cost prohibitive to generate antidotes for most therapeutic agents. Here we describe the development of a set of antidote molecules that are capable of counteracting the effects of an entire class of therapeutic agents based upon aptamers. These universal antidotes exploit the fact that, when systemically administered, aptamers are the only free extracellular oligonucleotides found in circulation. We show that protein- and polymer-based molecules that capture oligonucleotides can reverse the activity of several aptamers in vitro and counteract aptamer activity in vivo. The availability of universal antidotes to control the activity of any aptamer suggests that aptamers may be a particularly safe class of therapeutics.

  16. Small-Molecule Binding Aptamers: Selection Strategies, Characterization, and Applications

    Directory of Open Access Journals (Sweden)

    Annamaria eRuscito

    2016-05-01

    Full Text Available Aptamers are single-stranded, synthetic oligonucleotides that fold into 3-dimensional shapes capable of binding non-covalently with high affinity and specificity to a target molecule. They are generated via an in vitro process known as the Systematic Evolution of Ligands by EXponential enrichment, from which candidates are screened and characterized, and then applied in aptamer-based biosensors for target detection. Aptamers for small molecule targets such as toxins, antibiotics, molecular markers, drugs, and heavy metals will be the focus of this review. Their accurate detection is ultimately needed for the protection and wellbeing of humans and animals. However, issues such as the drastic difference in size of the aptamer and small molecule make it challenging to select, characterize, and apply aptamers for the detection of small molecules. Thus, recent (since 2012 notable advances in small molecule aptamers, which have overcome some of these challenges, are presented here, while defining challenges that still exist are discussed

  17. Crystal structures of the SAM-III/S[subscript MK] riboswitch reveal the SAM-dependent translation inhibition mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Lu, C.; Smith, A.M.; Fuchs, R.T.; Ding, F.; Rajashankar, K.; Henkin, T.M.; Ke, A. (Cornell); (OSU)

    2010-01-07

    Three distinct classes of S-adenosyl-L-methionine (SAM)-responsive riboswitches have been identified that regulate bacterial gene expression at the levels of transcription attenuation or translation inhibition. The SMK box (SAM-III) translational riboswitch has been identified in the SAM synthetase gene in members of the Lactobacillales. Here we report the 2.2-{angstrom} crystal structure of the Enterococcus faecalis SMK box riboswitch. The Y-shaped riboswitch organizes its conserved nucleotides around a three-way junction for SAM recognition. The Shine-Dalgarno sequence, which is sequestered by base-pairing with the anti-Shine-Dalgarno sequence in response to SAM binding, also directly participates in SAM recognition. The riboswitch makes extensive interactions with the adenosine and sulfonium moieties of SAM but does not appear to recognize the tail of the methionine moiety. We captured a structural snapshot of the SMK box riboswitch sampling the near-cognate ligand S-adenosyl-L-homocysteine (SAH) in which SAH was found to adopt an alternative conformation and fails to make several key interactions.

  18. Atomic resolution mechanistic studies of ribocil: A highly selective unnatural ligand mimic of the E. coli FMN riboswitch

    Science.gov (United States)

    Howe, John A.; Xiao, Li; Fischmann, Thierry O.; Wang, Hao; Tang, Haifeng; Villafania, Artjohn; Zhang, Rumin; Barbieri, Christopher M.; Roemer, Terry

    2016-01-01

    ABSTRACT Bacterial riboswitches are non-coding RNA structural elements that direct gene expression in numerous metabolic pathways. The key regulatory roles of riboswitches, and the urgent need for new classes of antibiotics to treat multi-drug resistant bacteria, has led to efforts to develop small-molecules that mimic natural riboswitch ligands to inhibit metabolic pathways and bacterial growth. Recently, we reported the results of a phenotypic screen targeting the riboflavin biosynthesis pathway in the Gram-negative bacteria Escherichia coli that led to the identification of ribocil, a small molecule inhibitor of the flavin mononucleotide (FMN) riboswitch controlling expression of this biosynthetic pathway. Although ribocil is structurally distinct from FMN, ribocil functions as a potent and highly selective synthetic mimic of the natural ligand to repress riboswitch-mediated ribB gene expression and inhibit bacterial growth both in vitro and in vivo. Herein, we expand our analysis of ribocil; including mode of binding in the FMN binding pocket of the riboswitch, mechanisms of resistance and structure-activity relationship guided efforts to generate more potent analogs. PMID:27485612

  19. Exploring lysine riboswitch for metabolic flux control and improvement of L-lysine synthesis in Corynebacterium glutamicum.

    Science.gov (United States)

    Zhou, Li-Bang; Zeng, An-Ping

    2015-06-19

    Riboswitch, a regulatory part of an mRNA molecule that can specifically bind a metabolite and regulate gene expression, is attractive for engineering biological systems, especially for the control of metabolic fluxes in industrial microorganisms. Here, we demonstrate the use of lysine riboswitch and intracellular l-lysine as a signal to control the competing but essential metabolic by-pathways of lysine biosynthesis. To this end, we first examined the natural lysine riboswitches of Eschericia coli (ECRS) and Bacillus subtilis (BSRS) to control the expression of citrate synthase (gltA) and thus the metabolic flux in the tricarboxylic acid (TCA) cycle in E. coli. ECRS and BSRS were then successfully used to control the gltA gene and TCA cycle activity in a lysine producing strain Corynebacterium glutamicum LP917, respectively. Compared with the strain LP917, the growth of both lysine riboswitch-gltA mutants was slower, suggesting a reduced TCA cycle activity. The lysine production was 63% higher in the mutant ECRS-gltA and 38% higher in the mutant BSRS-gltA, indicating a higher metabolic flux into the lysine synthesis pathway. This is the first report on using an amino acid riboswitch for improvement of lysine biosynthesis. The lysine riboswitches can be easily adapted to dynamically control other essential but competing metabolic pathways or even be engineered as an "on-switch" to enhance the metabolic fluxes of desired metabolic pathways.

  20. Aptamers Against Viral Hepatitis: from Rational Design to Practical Application

    Institute of Scientific and Technical Information of China (English)

    Hui FENG; Kang-hong HU

    2008-01-01

    Aptamers are short nucleic acids or peptides that strongly bind to a protein of interest and functionally inhibit a given target protein at the intracellular level. Besides high affinity and specificity, aptamers have several advantages over traditional antibodies. Hence, they have been broadly selected to develop antiviral agents for therapeutic applications against hepatitis B and C viruses (HBV, HCV). This review provides a summary of in vitro selection and characterization of aptamers against viral hepatitis, which is of practical significance in drug discovery.

  1. Cell-targeting aptamers act as intracellular delivery vehicles.

    Science.gov (United States)

    Gopinath, Subash C B; Lakshmipriya, Thangavel; Chen, Yeng; Arshad, M K Md; Kerishnan, Jesinda P; Ruslinda, A R; Al-Douri, Yarub; Voon, C H; Hashim, Uda

    2016-08-01

    Aptamers are single-stranded nucleic acids or peptides identified from a randomized combinatorial library through specific interaction with the target of interest. Targets can be of any size, from small molecules to whole cells, attesting to the versatility of aptamers for binding a wide range of targets. Aptamers show drug properties that are analogous to antibodies, with high specificity and affinity to their target molecules. Aptamers can penetrate disease-causing microbial and mammalian cells. Generated aptamers that target surface biomarkers act as cell-targeting agents and intracellular delivery vehicles. Within this context, the "cell-internalizing aptamers" are widely investigated via the process of cell uptake with selective binding during in vivo systematic evolution of ligands by exponential enrichment (SELEX) or by cell-internalization SELEX, which targets cell surface antigens to be receptors. These internalizing aptamers are highly preferable for the localization and functional analyses of multiple targets. In this overview, we discuss the ways by which internalizing aptamers are generated and their successful applications. Furthermore, theranostic approaches featuring cell-internalized aptamers are discussed with the purpose of analyzing and diagnosing disease-causing pathogens.

  2. Generation of Aptamers from A Primer-Free Randomized ssDNA Library Using Magnetic-Assisted Rapid Aptamer Selection

    Science.gov (United States)

    Tsao, Shih-Ming; Lai, Ji-Ching; Horng, Horng-Er; Liu, Tu-Chen; Hong, Chin-Yih

    2017-04-01

    Aptamers are oligonucleotides that can bind to specific target molecules. Most aptamers are generated using random libraries in the standard systematic evolution of ligands by exponential enrichment (SELEX). Each random library contains oligonucleotides with a randomized central region and two fixed primer regions at both ends. The fixed primer regions are necessary for amplifying target-bound sequences by PCR. However, these extra-sequences may cause non-specific bindings, which potentially interfere with good binding for random sequences. The Magnetic-Assisted Rapid Aptamer Selection (MARAS) is a newly developed protocol for generating single-strand DNA aptamers. No repeat selection cycle is required in the protocol. This study proposes and demonstrates a method to isolate aptamers for C-reactive proteins (CRP) from a randomized ssDNA library containing no fixed sequences at 5‧ and 3‧ termini using the MARAS platform. Furthermore, the isolated primer-free aptamer was sequenced and binding affinity for CRP was analyzed. The specificity of the obtained aptamer was validated using blind serum samples. The result was consistent with monoclonal antibody-based nephelometry analysis, which indicated that a primer-free aptamer has high specificity toward targets. MARAS is a feasible platform for efficiently generating primer-free aptamers for clinical diagnoses.

  3. Transcriptional pausing at the translation start site operates as a critical checkpoint for riboswitch regulation

    Science.gov (United States)

    Chauvier, Adrien; Picard-Jean, Frédéric; Berger-Dancause, Jean-Christophe; Bastet, Laurène; Naghdi, Mohammad Reza; Dubé, Audrey; Turcotte, Pierre; Perreault, Jonathan; Lafontaine, Daniel A.

    2017-01-01

    On the basis of nascent transcript sequencing, it has been postulated but never demonstrated that transcriptional pausing at translation start sites is important for gene regulation. Here we show that the Escherichia coli thiamin pyrophosphate (TPP) thiC riboswitch contains a regulatory pause site in the translation initiation region that acts as a checkpoint for thiC expression. By biochemically probing nascent transcription complexes halted at defined positions, we find a narrow transcriptional window for metabolite binding, in which the downstream boundary is delimited by the checkpoint. We show that transcription complexes at the regulatory pause site favour the formation of a riboswitch intramolecular lock that strongly prevents TPP binding. In contrast, cotranscriptional metabolite binding increases RNA polymerase pausing and induces Rho-dependent transcription termination at the checkpoint. Early transcriptional pausing may provide a general mechanism, whereby transient transcriptional windows directly coordinate the sensing of environmental cues and bacterial mRNA regulation. PMID:28071751

  4. Structural stability, acidity, and halide selectivity of the fluoride riboswitch recognition site

    KAUST Repository

    Chawla, Mohit

    2015-01-14

    Using static and dynamics DFT methods we show that the Mg2+/F-/phosphate/water cluster at the center of the fluoride riboswitch is stable by its own and, once assembled, does not rely on any additional factor from the overall RNA fold. Further, we predict that the pKa of the water molecule bridging two Mg cations is around 8.4. We also demonstrate that the halide selectivity of the fluoride riboswitch is determined by the stronger Mg-F bond, which is capable of keeping together the cluster. Replacing F- with Cl- results in a cluster that is unstable under dynamic conditions. Similar conclusions on the structure and energetics of the cluster in the binding pocket of fluoride-inhibited pyrophosphatase suggest that the peculiarity of fluoride is in its ability to establish much stronger metal-halide bonds.

  5. Discovery and development of anticancer aptamers.

    Science.gov (United States)

    Ireson, Christopher R; Kelland, Lloyd R

    2006-12-01

    Aptamers, also termed as decoys or "chemical antibodies," represent an emerging class of therapeutics. They are short DNA or RNA oligonucleotides or peptides that assume a specific and stable three-dimensional shape in vivo, thereby providing specific tight binding to protein targets. In some cases and as opposed to antisense oligonucleotides, effects can be mediated against extracellular targets, thereby preventing a need for intracellular transportation. The first aptamer approved for use in man is a RNA-based molecule (Macugen, pegaptanib) that is administered locally (intravitreally) to treat age-related macular degeneration by targeting vascular endothelial growth factor. The most advanced aptamer in the cancer setting is AS1411, formerly known as AGRO100, which is being administered systemically in clinical trials. AS1411 is a 26-mer unmodified guanosine-rich oligonucleotide, which induces growth inhibition in vitro, and has shown activity against human tumor xenografts in vivo. The mechanism underlying its antiproliferative effects in cancer cells seems to involve initial binding to cell surface nucleolin and internalization, leading to an inhibition of DNA replication. In contrast to other unmodified oligonucleotides, AS1411 is relatively stable in serum-containing medium, probably as a result of the formation of dimers and a quartet structure. In a dose escalation phase I study in patients with advanced solid tumors, doses up to 10 mg/kg/d (using a four or seven continuous infusion regime) have been studied. Promising signs of activity have been reported (multiple cases of stable disease and one near complete response in a patient with renal cancer) in the absence of any significant adverse effects. Further trials are ongoing in renal and non-small cell lung cancers. In preclinical studies, additional aptamers have been described against several cancer targets, such as tenascin-C, the transcription factor signal transducer and activator of transcription 3

  6. Modern affinity reagents: Recombinant antibodies and aptamers.

    Science.gov (United States)

    Groff, Katherine; Brown, Jeffrey; Clippinger, Amy J

    2015-12-01

    Affinity reagents are essential tools in both basic and applied research; however, there is a growing concern about the reproducibility of animal-derived monoclonal antibodies. The need for higher quality affinity reagents has prompted the development of methods that provide scientific, economic, and time-saving advantages and do not require the use of animals. This review describes two types of affinity reagents, recombinant antibodies and aptamers, which are non-animal technologies that can replace the use of animal-derived monoclonal antibodies. Recombinant antibodies are protein-based reagents, while aptamers are nucleic-acid-based. In light of the scientific advantages of these technologies, this review also discusses ways to gain momentum in the use of modern affinity reagents, including an update to the 1999 National Academy of Sciences monoclonal antibody production report and federal incentives for recombinant antibody and aptamer efforts. In the long-term, these efforts have the potential to improve the overall quality and decrease the cost of scientific research. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Aptamer Stainings for Super-resolution Microscopy.

    Science.gov (United States)

    de Castro, Maria Angela Gomes; Rammner, Burkhard; Opazo, Felipe

    2016-01-01

    Fluorescence microscopy is an invaluable tool to visualize molecules in their biological context with ease and flexibility. However, studies using conventional light microscopy have been limited to the resolution that light diffraction allows (i.e., ~200 nm). This limitation has been recently circumvented by several types of advanced fluorescence microscopy techniques, which have achieved resolutions of up to ~10 nm. The resulting enhanced imaging precision has helped to find important cellular details that were not visible using diffraction-limited instruments. However, it has also revealed that conventional stainings using large affinity tags, such as antibodies, are not accurate enough for these imaging techniques. Since aptamers are substantially smaller than antibodies, they could provide a real advantage in super-resolution imaging. Here we compare the live staining of transferrin receptors (TfnR) obtained with different fluorescently labeled affinity probes: aptamers, specific monoclonal antibodies, or the natural receptor ligand transferrin. We observed negligible differences between these staining strategies when imaging is performed with conventional light microscopy (i.e., laser scanning confocal microscopy). However, a clear superiority of the aptamer tag over antibodies became apparent in super-resolved images obtained with stimulated emission depletion (STED) microscopy.

  8. Rapid one-step selection method for generating nucleic acid aptamers: development of a DNA aptamer against α-bungarotoxin.

    Directory of Open Access Journals (Sweden)

    Lasse H Lauridsen

    Full Text Available BACKGROUND: Nucleic acids based therapeutic approaches have gained significant interest in recent years towards the development of therapeutics against many diseases. Recently, research on aptamers led to the marketing of Macugen®, an inhibitor of vascular endothelial growth factor (VEGF for the treatment of age related macular degeneration (AMD. Aptamer technology may prove useful as a therapeutic alternative against an array of human maladies. Considering the increased interest in aptamer technology globally that rival antibody mediated therapeutic approaches, a simplified selection, possibly in one-step, technique is required for developing aptamers in limited time period. PRINCIPAL FINDINGS: Herein, we present a simple one-step selection of DNA aptamers against α-bungarotoxin. A toxin immobilized glass coverslip was subjected to nucleic acid pool binding and extensive washing followed by PCR enrichment of the selected aptamers. One round of selection successfully identified a DNA aptamer sequence with a binding affinity of 7.58 µM. CONCLUSION: We have demonstrated a one-step method for rapid production of nucleic acid aptamers. Although the reported binding affinity is in the low micromolar range, we believe that this could be further improved by using larger targets, increasing the stringency of selection and also by combining a capillary electrophoresis separation prior to the one-step selection. Furthermore, the method presented here is a user-friendly, cheap and an easy way of deriving an aptamer unlike the time consuming conventional SELEX-based approach. The most important application of this method is that chemically-modified nucleic acid libraries can also be used for aptamer selection as it requires only one enzymatic step. This method could equally be suitable for developing RNA aptamers.

  9. Rapid one-step selection method for generating nucleic acid aptamers: development of a DNA aptamer against α-bungarotoxin.

    Science.gov (United States)

    Lauridsen, Lasse H; Shamaileh, Hadi A; Edwards, Stacey L; Taran, Elena; Veedu, Rakesh N

    2012-01-01

    Nucleic acids based therapeutic approaches have gained significant interest in recent years towards the development of therapeutics against many diseases. Recently, research on aptamers led to the marketing of Macugen®, an inhibitor of vascular endothelial growth factor (VEGF) for the treatment of age related macular degeneration (AMD). Aptamer technology may prove useful as a therapeutic alternative against an array of human maladies. Considering the increased interest in aptamer technology globally that rival antibody mediated therapeutic approaches, a simplified selection, possibly in one-step, technique is required for developing aptamers in limited time period. Herein, we present a simple one-step selection of DNA aptamers against α-bungarotoxin. A toxin immobilized glass coverslip was subjected to nucleic acid pool binding and extensive washing followed by PCR enrichment of the selected aptamers. One round of selection successfully identified a DNA aptamer sequence with a binding affinity of 7.58 µM. We have demonstrated a one-step method for rapid production of nucleic acid aptamers. Although the reported binding affinity is in the low micromolar range, we believe that this could be further improved by using larger targets, increasing the stringency of selection and also by combining a capillary electrophoresis separation prior to the one-step selection. Furthermore, the method presented here is a user-friendly, cheap and an easy way of deriving an aptamer unlike the time consuming conventional SELEX-based approach. The most important application of this method is that chemically-modified nucleic acid libraries can also be used for aptamer selection as it requires only one enzymatic step. This method could equally be suitable for developing RNA aptamers.

  10. Fluorescence assays for monitoring RNA-ligand interactions and riboswitch-targeted drug discovery screening.

    Science.gov (United States)

    Liu, J; Zeng, C; Zhou, S; Means, J A; Hines, J V

    2015-01-01

    Riboswitches and other noncoding regulatory RNA are intriguing targets for the development of therapeutic agents. A significant challenge in the drug discovery process, however, is the identification of potent compounds that bind the target RNA specifically and disrupt its function. Essential to this process is an effectively designed cascade of screening assays. A screening cascade for identifying compounds that target the T box riboswitch antiterminator element is described. In the primary assays, moderate to higher throughput screening of compound libraries is achieved by combining the sensitivity of fluorescence techniques with functionally relevant assays. Active compounds are then validated and the binding to target RNA further characterized in secondary assays. The cascade of assays monitor ligand-induced changes in the steady-state fluorescence of an attached dye or internally incorporated 2-aminopurine; the fluorescence anisotropy of an RNA complex; and, the thermal denaturation fluorescence profile of a fluorophore-quencher labeled RNA. While the assays described have been developed for T box riboswitch-targeted drug discovery, the fluorescence methods and screening cascade design principles can be applied to drug discovery efforts targeted toward other medicinally relevant noncoding RNA.

  11. Aptamers as Both Drugs and Drug-Carriers

    Directory of Open Access Journals (Sweden)

    Md. Ashrafuzzaman

    2014-01-01

    Full Text Available Aptamers are short nucleic acid oligos. They may serve as both drugs and drug-carriers. Their use as diagnostic tools is also evident. They can be generated using various experimental, theoretical, and computational techniques. The systematic evolution of ligands by exponential enrichment which uses iterative screening of nucleic acid libraries is a popular experimental technique. Theory inspired methodology entropy-based seed-and-grow strategy that designs aptamer templates to bind specifically to targets is another one. Aptamers are predicted to be highly useful in producing general drugs and theranostic drugs occasionally for certain diseases like cancer, Alzheimer’s disease, and so on. They bind to various targets like lipids, nucleic acids, proteins, small organic compounds, and even entire organisms. Aptamers may also serve as drug-carriers or nanoparticles helping drugs to get released in specific target regions. Due to better target specific physical binding properties aptamers cause less off-target toxicity effects. Therefore, search for aptamer based drugs, drug-carriers, and even diagnostic tools is expanding fast. The biophysical properties in relation to the target specific binding phenomena of aptamers, energetics behind the aptamer transport of drugs, and the consequent biological implications will be discussed. This review will open up avenues leading to novel drug discovery and drug delivery.

  12. Application of aptamers in diagnostics, drug-delivery and imaging

    Indian Academy of Sciences (India)

    CHETAN CHANDOLA; SHEETAL KALME; MARCO G CASTELEIJN; ARTO URTTI; MUNIASAMY NEERATHILINGAM

    2016-09-01

    Aptamers are small, single-stranded oligonucleotides (DNA or RNA) that bind to their target with high specificity andaffinity. Although aptamers are analogous to antibodies for a wide range of target recognition and variety ofapplications, they have significant advantages over antibodies. Since aptamers have recently emerged as a class ofbiomolecules with an application in a wide array of fields, we need to summarize the latest developments herein. Inthis review we will discuss about the latest developments in using aptamers in diagnostics, drug delivery and imaging.We begin with diagnostics, discussing the application of aptamers for the detection of infective agents itself, antigens/toxins (bacteria), biomarkers (cancer), or a combination. The ease of conjugation and labelling of aptamers makesthem a potential tool for diagnostics. Also, due to the reduced off-target effects of aptamers, their use as a potentialdrug delivery tool is emerging rapidly. Hence, we discuss their use in targeted delivery in conjugation with siRNAs,nanoparticles, liposomes, drugs and antibodies. Finally, we discuss about the conjugation strategies applicable forRNA and DNA aptamers for imaging. Their stability and self-assembly after heating makes them superior overprotein-based binding molecules in terms of labelling and conjugation strategies.

  13. Screening of aptamers on microfluidic systems for clinical applications.

    Science.gov (United States)

    Weng, Chen-Hsun; Huang, Chao-Jyun; Lee, Gwo-Bin

    2012-01-01

    The use of microfluidic systems for screening of aptamers and their biomedical applications are reviewed in this paper. Aptamers with different nucleic acid sequences have been extensively studied and the results demonstrated a strong binding affinity to target molecules such that they can be used as promising candidate biomarkers for diagnosis and therapeutics. Recently, the aptamer screening protocol has been conducted with microfluidic-based devices. Furthermore, aptamer affinity screening by a microfluidic-based method has demonstrated remarkable advantages over competing traditional methods. In this paper, we first reviewed microfluidic systems which demonstrated efficient and rapid screening of a specific aptamer. Then, the clinical applications of screened aptamers, also performed by microfluidic systems, are further reviewed. These automated microfluidic systems can provide advantages over their conventional counterparts including more compactness, faster analysis, less sample/reagent consumption and automation. An aptamer-based compact microfluidic system for diagnosis may even lead to a point-of-care device. The use of microfluidic systems for aptamer screening and diagnosis is expected to continue growing in the near future and may make a substantial impact on biomedical applications.

  14. Screening of Aptamers on Microfluidic Systems for Clinical Applications

    Directory of Open Access Journals (Sweden)

    Gwo-Bin Lee

    2012-07-01

    Full Text Available The use of microfluidic systems for screening of aptamers and their biomedical applications are reviewed in this paper. Aptamers with different nucleic acid sequences have been extensively studied and the results demonstrated a strong binding affinity to target molecules such that they can be used as promising candidate biomarkers for diagnosis and therapeutics. Recently, the aptamer screening protocol has been conducted with microfluidic-based devices. Furthermore, aptamer affinity screening by a microfluidic-based method has demonstrated remarkable advantages over competing traditional methods. In this paper, we first reviewed microfluidic systems which demonstrated efficient and rapid screening of a specific aptamer. Then, the clinical applications of screened aptamers, also performed by microfluidic systems, are further reviewed. These automated microfluidic systems can provide advantages over their conventional counterparts including more compactness, faster analysis, less sample/reagent consumption and automation. An aptamer-based compact microfluidic system for diagnosis may even lead to a point-of-care device. The use of microfluidic systems for aptamer screening and diagnosis is expected to continue growing in the near future and may make a substantial impact on biomedical applications.

  15. Optimized light-directed synthesis of aptamer microarrays.

    Science.gov (United States)

    Franssen-van Hal, Nicole L W; van der Putte, Pepijn; Hellmuth, Klaus; Matysiak, Stefan; Kretschy, Nicole; Somoza, Mark M

    2013-06-18

    Aptamer microarrays are a promising high-throughput method for ultrasensitive detection of multiple analytes, but although much is known about the optimal synthesis of oligonucleotide microarrays used in hybridization-based genomics applications, the bioaffinity interactions between aptamers and their targets is qualitatively different and requires significant changes to synthesis parameters. Focusing on streptavidin-binding DNA aptamers, we employed light-directed in situ synthesis of microarrays to analyze the effects of sequence fidelity, linker length, surface probe density, and substrate functionalization on detection sensitivity. Direct comparison with oligonucleotide hybridization experiments indicates that aptamer microarrays are significantly more sensitive to sequence fidelity and substrate functionalization and have different optimal linker length and surface probe density requirements. Whereas microarray hybridization probes generate maximum signal with multiple deletions, aptamer sequences with the same deletion rate result in a 3-fold binding signal reduction compared with the same sequences synthesized for maximized sequence fidelity. The highest hybridization signal was obtained with dT 5mer linkers, and the highest aptamer signal was obtained with dT 11mers, with shorter aptamer linkers significantly reducing the binding signal. The probe hybridization signal was found to be more sensitive to molecular crowding, whereas the aptamer probe signal does not appear to be constrained within the density of functional surface groups commonly used to synthesize microarrays.

  16. Extending the lifetime of anticoagulant oligodeoxynucleotide aptamers in blood

    Energy Technology Data Exchange (ETDEWEB)

    Dougan, Hayes E-mail: dougan@triumf.ca; Lyster, Donald M.; Vo, Can V.; Stafford, Alan; Weitz, Jeffrey I.; Hobbs, John B

    2000-04-01

    We have investigated {sup 123}I and {sup 125}I DNA aptamer analogs of anticoagulant DNA aptamers to thrombin exosite 1 and exosite 2 for thrombus imaging potential. Two severe problems are rapid clearance from circulating blood and blood nuclease. With aptamers (unlike antisense) the nucleotide analogs used in polymerase chain reaction-selection cycles also must be used in the radiotracer. We investigated 3'-biotin-streptavidin (SA) bioconjugates of the aptamers to alleviate these problems. Blood nuclease assays and biodistribution analysis were used in the mouse and rabbit. We found that 3'-biotin protected the aptamers significantly from blood nuclease in vitro, but it did not slow in vivo clearance. In contrast, the 3'-biotin-SA bioconjugates were resistant to blood nuclease in vitro and were also longer-lived (10-20 times) in vivo. Bioconjugate aptamers retained affinity for thrombin. Two solutions emerge: 1) In noncirculating blood (within a thrombus) 3'-biotin extends aptamer lifetime, whereas 2) in circulating blood (the transport medium), where more aggressive clearance is encountered, 3'-SA extends aptamer lifetime.

  17. In vivo SELEX for Identification of Brain-penetrating Aptamers

    Directory of Open Access Journals (Sweden)

    Congsheng Cheng

    2013-01-01

    Full Text Available The physiological barriers of the brain impair drug delivery for treatment of many neurological disorders. One delivery approach that has not been investigated for their ability to penetrate the brain is RNA-based aptamers. These molecules can impart delivery to peripheral tissues and circulating immune cells, where they act as ligand mimics or can be modified to carry payloads. We developed a library of aptamers and an in vivo evolution protocol to determine whether specific aptamers could be identified that would home to the brain after injection into the peripheral vasculature. Unlike biopanning with recombinant bacteriophage libraries, we found that the aptamer library employed here required more than 15 rounds of in vivo selection for convergence to specific sequences. The aptamer species identified through this approach bound to brain capillary endothelia and penetrated into the parenchyma. The methods described may find general utility for targeting various payloads to the brain.

  18. Aptamer-assembled nanomaterials for fluorescent sensing and imaging

    Directory of Open Access Journals (Sweden)

    Lu Danqing

    2017-01-01

    Full Text Available Aptamers, which are selected in vitro by a technology known as the systematic evolution of ligands by exponential enrichment (SELEX, represent a crucial recognition element in molecular sensing. With advantages such as good biocompatibility, facile functionalization, and special optical and physical properties, various nanomaterials can protect aptamers from enzymatic degradation and nonspecific binding in living systems and thus provide a preeminent platform for biochemical applications. Coupling aptamers with various nanomaterials offers many opportunities for developing highly sensitive and selective sensing systems. Here, we focus on the recent applications of aptamer-assembled nanomaterials in fluorescent sensing and imaging. Different types of nanomaterials are examined along with their advantages and disadvantages. Finally, we look toward the future of aptamer-assembled nanomaterials.

  19. Generating Cell Targeting Aptamers for Nanotheranostics Using Cell-SELEX.

    Science.gov (United States)

    Lyu, Yifan; Chen, Guang; Shangguan, Dihua; Zhang, Liqin; Wan, Shuo; Wu, Yuan; Zhang, Hui; Duan, Lian; Liu, Chao; You, Mingxu; Wang, Jie; Tan, Weihong

    2016-01-01

    Detecting and understanding changes in cell conditions on the molecular level is of great importance for the accurate diagnosis and timely therapy of diseases. Cell-based SELEX (Systematic Evolution of Ligands by EXponential enrichment), a foundational technology used to generate highly-specific, cell-targeting aptamers, has been increasingly employed in studies of molecular medicine, including biomarker discovery and early diagnosis/targeting therapy of cancer. In this review, we begin with a mechanical description of the cell-SELEX process, covering aptamer selection, identification and identification, and aptamer characterization; following this introduction is a comprehensive discussion of the potential for aptamers as targeting moieties in the construction of various nanotheranostics. Challenges and prospects for cell-SELEX and aptamer-based nanotheranostic are also discussed.

  20. High Efficiency Acetylcholinesterase Immobilization on DNA Aptamer Modified Surfaces

    Directory of Open Access Journals (Sweden)

    Orada Chumphukam

    2014-04-01

    Full Text Available We report here the in vitro selection of DNA aptamers for electric eel acetylcholinesterase (AChE. One selected aptamer sequence (R15/19 has a high affinity towards the enzyme (Kd = 157 ± 42 pM. Characterization of the aptamer showed its binding is not affected by low ionic strength (~20 mM, however significant reduction in affinity occurred at high ionic strength (~1.2 M. In addition, this aptamer does not inhibit the catalytic activity of AChE that we exploit through immobilization of the DNA on a streptavidin-coated surface. Subsequent immobilization of AChE by the aptamer results in a 4-fold higher catalytic activity when compared to adsorption directly on to plastic.

  1. Aptamer-assembled nanomaterials for fluorescent sensing and imaging

    Science.gov (United States)

    Lu, Danqing; He, Lei; Zhang, Ge; Lv, Aiping; Wang, Ruowen; Zhang, Xiaobing; Tan, Weihong

    2017-01-01

    Aptamers, which are selected in vitro by a technology known as the systematic evolution of ligands by exponential enrichment (SELEX), represent a crucial recognition element in molecular sensing. With advantages such as good biocompatibility, facile functionalization, and special optical and physical properties, various nanomaterials can protect aptamers from enzymatic degradation and nonspecific binding in living systems and thus provide a preeminent platform for biochemical applications. Coupling aptamers with various nanomaterials offers many opportunities for developing highly sensitive and selective sensing systems. Here, we focus on the recent applications of aptamer-assembled nanomaterials in fluorescent sensing and imaging. Different types of nanomaterials are examined along with their advantages and disadvantages. Finally, we look toward the future of aptamer-assembled nanomaterials.

  2. Aptamers Against Immunologic Targets: Diagnostic and Therapeutic Prospects.

    Science.gov (United States)

    Vorobyeva, Mariya; Timoshenko, Valentina; Vorobjev, Pavel; Venyaminova, Alya

    2016-02-01

    The concept of in vitro selection of nucleic acid aptamers emerged 25 years ago, and since then tremendous progress has been achieved in the development of different aptamers and their applications for various bioanalytical and therapeutic purposes. Among other protein targets of aptamers, immune system proteins are of particular interest both as diagnostic markers and therapeutic targets. The present review summarizes up-to-date articles concerning the selection and design of DNA and RNA aptamers against immunologic targets such as antibodies, cytokines, and T-cell and B-cell receptors. We also discuss the prospects of employing aptamers as recognizing modules of diagnostic aptasensors, potential therapeutic candidates for the treatment of autoimmune diseases and cancer, and specific tools for functional studies of immune system proteins.

  3. In vitro evaluation of radiolabeled aptamers for colon carcinoma diagnosis

    Energy Technology Data Exchange (ETDEWEB)

    Correa, C.R.; Ferreira, I.M; Santos, S.R.; Faria, L.S.; Andrade, A.S.R., E-mail: crisrcorrea@gmail.com, E-mail: antero@cdtn.br [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil); Goes, A.M., E-mail: goes@icb.ufmg.br [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Inst. de Ciencias Biologicas. Dept. de Imunologia e Bioquimica

    2013-07-01

    Cancer is a leading cause of death worldwide, representing a major public health problem worldwide. Colorectal cancers accounts around 8% of all deaths for cancer in 2008, is the fourth most lethal. Many colorectal cancer markers, such as carcinoembryonic antigen (CEA), A33, and CSA-p, have been studied as the therapeutic targets in preclinical or clinical settings. CEA is a complex intracellular glycoprotein produced by about 90% of colorectal cancers. Since its discovery in 1965, a very large number of studies have been carried out to determine the effectiveness of CEA as clinically useful tumor markers. Aptamers are short single-stranded nucleic acid oligomers (DNA or RNA) that can form specific and complex three-dimensional structures which can bind with high affinity to specific targets, they are functionally equivalent of antibodies. Aptamers have the advantage of being highly specific, relatively small size, and non-immunogenic. The aim of this study was develop anti-CEA aptamers for use as imaging agents. The aptamers are obtained through by SELEX (systematic evolution of ligands by exponential enrichment), in which aptamers are selected from a library of random sequences of synthetic DNA by repetitive binding of the oligonucleotides to target molecule. These aptamers were confirmed to have affinity and specific binding for T84 cell line (target cell), showed by fluorescence microscopic images. Individual aptamers sequences that bound T84 cells were {sup 32}P-radiolabeled and incubated at different concentrations on cell monolayers, to monitor the aptamers affinity binding. The selected aptamers can identify colon cancer cell line. This aptamers could be further developed for early disease detection as radiopharmaceuticals, as well as prognostic markers, of colorectal cancers. (author)

  4. Discovery of Aptamer Ligands for Hepatic Stellate Cells Using SELEX.

    Science.gov (United States)

    Chen, Zhijin; Liu, Hao; Jain, Akshay; Zhang, Li; Liu, Chang; Cheng, Kun

    2017-01-01

    Insulin like growth factor II receptor (IGFIIR) is a transmembrane protein overexpressed in activated hepatic stellate cells (HSCs), which are the major target for the treatment of liver fibrosis. In this study, we aim to discover an IGFIIR-specific aptamer that can be potentially used as a targeting ligand for the treatment and diagnosis of liver fibrosis. Systematic evolution of ligands by exponential enrichment (SELEX) was conducted on recombinant human IGFIIR to identify IGFIIR-specific aptamers. The binding affinity and specificity of the discovered aptamers to IGFIIR and hepatic stellate cells were studied using flow cytometry and Surface Plasmon Resonance (SPR). Aptamer-20 showed the highest affinity to recombinant human IGFIIR protein with a Kd of 35.5 nM, as determined by SPR. Aptamer-20 also has a high affinity (apparent Kd 45.12 nM) to LX-2 human hepatic stellate cells. Binding of aptamer-20 to hepatic stellate cells could be inhibited by knockdown of IGFIIR using siRNA, indicating a high specificity of the aptamer. The aptamer formed a chimera with an anti-fibrotic PCBP2 siRNA and delivered the siRNA to HSC-T6 cells to trigger silencing activity. In Vivo biodistribution study of the siRNA-aptamer chimera also demonstrated a high and specific uptake in the liver of the rats with CCl4-induced liver fibrosis. These data suggest that aptamer-20 is a high-affinity ligand for antifibrotic and diagnostic agents for liver fibrosis.

  5. Conformational heterogeneity of the SAM-I riboswitch transcriptional ON state: a chaperone-like role for S-adenosyl methionine.

    Science.gov (United States)

    Huang, Wei; Kim, Joohyun; Jha, Shantenu; Aboul-Ela, Fareed

    2012-05-18

    Riboswitches are promising targets for the design of novel antibiotics and engineering of portable genetic regulatory elements. There is evidence that variability in riboswitch properties allows tuning of expression for genes involved in different stages of biosynthetic pathways by mechanisms that are not currently understood. Here, we explore the mechanism for tuning of S-adenosyl methionine (SAM)-I riboswitch folding. Most SAM-I riboswitches function at the transcriptional level by sensing the cognate ligand SAM. SAM-I riboswitches orchestrate the biosynthetic pathways of cysteine, methionine, SAM, and so forth. We use base-pair probability predictions to examine the secondary-structure folding landscape of several SAM-I riboswitch sequences. We predict different folding behaviors for different SAM-I riboswitch sequences. We identify several "decoy" base-pairing interactions involving 5' riboswitch residues that can compete with the formation of a P1 helix, a component of the ligand-bound "transcription OFF" state, in the absence of SAM. We hypothesize that blockage of these interactions through SAM contacts contributes to stabilization of the OFF state in the presence of ligand. We also probe folding patterns for a SAM-I riboswitch RNA using constructs with different 3' truncation points experimentally. Folding was monitored through fluorescence, susceptibility to base-catalyzed cleavage, nuclear magnetic resonance, and indirectly through SAM binding. We identify key decision windows at which SAM can affect the folding pathway towards the OFF state. The presence of decoy conformations and differential sensitivities to SAM at different transcript lengths is crucial for SAM-I riboswitches to modulate gene expression in the context of global cellular metabolism.

  6. A highly specialized flavin mononucleotide riboswitch responds differently to similar ligands and confers roseoflavin resistance to Streptomyces davawensis.

    Science.gov (United States)

    Pedrolli, Danielle Biscaro; Matern, Andreas; Wang, Joy; Ester, Miriam; Siedler, Kathrin; Breaker, Ronald; Mack, Matthias

    2012-09-01

    Streptomyces davawensis is the only organism known to synthesize the antibiotic roseoflavin, a riboflavin (vitamin B2) analog. Roseoflavin is converted to roseoflavin mononucleotide (RoFMN) and roseoflavin adenine dinucleotide in the cytoplasm of target cells. (Ribo-)Flavin mononucleotide (FMN) riboswitches are genetic elements, which in many bacteria control genes responsible for the biosynthesis and transport of riboflavin. Streptomyces davawensis is roseoflavin resistant, and the closely related bacterium Streptomyces coelicolor is roseoflavin sensitive. The two bacteria served as models to investigate roseoflavin resistance of S. davawensis and to analyze the mode of action of roseoflavin in S. coelicolor. Our experiments demonstrate that the ribB FMN riboswitch of S. davawensis (in contrast to the corresponding riboswitch of S. coelicolor) is able to discriminate between the two very similar flavins FMN and RoFMN and shows opposite responses to the latter ligands.

  7. Efficient suppression of biofilm formation by a nucleic acid aptamer.

    Science.gov (United States)

    Ning, Yi; Cheng, Lijuan; Ling, Min; Feng, Xinru; Chen, Lingli; Wu, Minxi; Deng, Le

    2015-08-01

    Biofilms are microbial communities that are attached to a solid surface using extracellular polymeric substances. Motility and initial attachment mediated by flagella are required for biofilm formation. Therefore, blocking the motility of flagella is a potential strategy to inhibit biofilm formation. In this study, single-stranded DNA aptamers specific to the Salmonella choleraesuis were selected after 14 cycles of the systematic evolution of ligands by exponential enrichment. Among the selected aptamers, the aptamer 3 showed the highest affinity for S. choleraesuis with a dissociation constant (Kd) of 41 ± 2 nM. Aptamer 3, conjugated with magnetic beads, was then used to capture its binding target on the bacteria. After mass spectrometry and specific binding analysis, the flagellin was identified as the target captured by aptamer 3. Furthermore, inhibition experiments, inverted microscopy and atomic force microscopy demonstrated that aptamer 3 was able to control the biofilm formation and promote the inhibitory effect of an antibiotic on bacterial biofilms. Single-stranded DNA aptamers therefore have great potential as inhibitors of biofilm formation.

  8. Aptamers: active targeting ligands for cancer diagnosis and therapy.

    Science.gov (United States)

    Wu, Xu; Chen, Jiao; Wu, Min; Zhao, Julia Xiaojun

    2015-01-01

    Aptamers, including DNA, RNA and peptide aptamers, are a group of promising recognition units that can specifically bind to target molecules and cells. Due to their excellent specificity and high affinity to targets, aptamers have attracted great attention in various fields in which selective recognition units are required. They have been used in biosensing, drug delivery, disease diagnosis and therapy (especially for cancer treatment). In this review, we summarized recent applications of DNA and RNA aptamers in cancer theranostics. The specific binding ability of aptamers to cancer-related markers and cancer cells ensured their high performance for early diagnosis of cancer. Meanwhile, the efficient targeting ability of aptamers to cancer cells and tissues provided a promising way to deliver imaging agents and drugs for cancer imaging and therapy. Furthermore, with the development of nanoscience and nanotechnology, the conjugation of aptamers with functional nanomaterials paved an exciting way for the fabrication of theranostic agents for different types of cancers, which might be a powerful tool for cancer treatment.

  9. Nanostructure shape effects on response of plasmonic aptamer sensors.

    Science.gov (United States)

    Balamurugan, Subramanian; Mayer, Kathryn M; Lee, Seunghyun; Soper, Steven A; Hafner, Jason H; Spivak, David A

    2013-09-01

    A localized surface plasmon resonance (LSPR) sensor surface was fabricated by the deposition of gold nanorods on a glass substrate and subsequent immobilization of the DNA aptamer, which specifically bind to thrombin. This LSPR aptamer sensor showed a response of 6-nm λ(max) shift for protein binding with the detection limit of at least 10 pM, indicating one of the highest sensitivities achieved for thrombin detection by optical extinction LSPR. We also tested the LSPR sensor fabricated using gold bipyramid, which showed higher refractive index sensitivity than the gold nanorods, but the overall response of gold bipyramid sensor appears to be 25% less than that of the gold nanorod substrate, despite the approximately twofold higher refractive index sensitivity. XPS analysis showed that this is due to the low surface density of aptamers on the gold bipyramid compared with gold nanorods. The low surface density of the aptamers on the gold bipyramid surface may be due to the effect of shape of the nanostructure on the kinetics of aptamer monolayer formation. The small size of aptamers relative to other bioreceptors is the key to achieving high sensitivity by biosensors on the basis of LSPR, demonstrated here for protein binding. The generality of aptamer sensors for protein detection using gold nanorod and gold nanobipyramid substrates is anticipated to have a large impact in the important development of sensors toward biomarkers, environmental toxins, and warfare agents.

  10. Comprehensive characterization of a theophylline riboswitch reveals two pivotal features of Shine-Dalgarno influencing activated translation property.

    Science.gov (United States)

    Cui, Wenjing; Cheng, Jintao; Miao, Shengnan; Zhou, Li; Liu, Zhongmei; Guo, Junling; Zhou, Zhemin

    2017-03-01

    Tuneable gene expression controlled by synthetic biological elements is of great importance to biotechnology and synthetic biology. The synthetic riboswitch is a pivotal type of elements that can easily control the heterologous gene expression in diverse bacteria. In this study, the theophylline-dependent synthetic riboswitch and the corresponding variants with varied spacings between Shine-Dalgarno (SD) sequence and start codon were employed to comprehensively characterize the induction and regulation properties through combining a strong promoter aprE in Bacillus subtilis. Amongst the sets of newly constructed expression elements, the expression element with 9-bp spacing exhibited the higher expression level, a superior induction fold performance, and a considerably lower leaky expression than those with longer or shorter spacings. The riboswitch expression element with 9-bp spacing showed an approximately linear dose dependence from 0 to 8 mM of theophylline. Modification of the SD sequence through the insertion of a single A base prior to the native sequence enables the increase of the expression level post induction while decreasing the induction fold as a result of the elevated leaky level. The riboswitch elements with the engineered SD and the optimal 9-bp spacing exhibit an altered dose dependency in which the approximately linear range shifts to 0-4 mM, although it has a similar profile to the induction process. These results not only provide comprehensive data for the induced expression by a theophylline riboswitch combined with a strong native promoter from B. subtilis but also provide the two pivotal features of SD essential to the modular design of other synthetic riboswitches.

  11. Aptamer-Gated Nanoparticles for Smart Drug Delivery

    Directory of Open Access Journals (Sweden)

    Huseyin Avni Oktem

    2011-08-01

    Full Text Available Aptamers are functional nucleic acid sequences which can bind specific targets. An artificial combinatorial methodology can identify aptamer sequences for any target molecule, from ions to whole cells. Drug delivery systems seek to increase efficacy and reduce side-effects by concentrating the therapeutic agents at specific disease sites in the body. This is generally achieved by specific targeting of inactivated drug molecules. Aptamers which can bind to various cancer cell types selectively and with high affinity have been exploited in a variety of drug delivery systems for therapeutic purposes. Recent progress in selection of cell-specific aptamers has provided new opportunities in targeted drug delivery. Especially functionalization of nanoparticles with such aptamers has drawn major attention in the biosensor and biomedical areas. Moreover, nucleic acids are recognized as an attractive building materials in nanomachines because of their unique molecular recognition properties and structural features. A active controlled delivery of drugs once targeted to a disease site is a major research challenge. Stimuli-responsive gating is one way of achieving controlled release of nanoparticle cargoes. Recent reports incorporate the structural properties of aptamers in controlled release systems of drug delivering nanoparticles. In this review, the strategies for using functional nucleic acids in creating smart drug delivery devices will be explained. The main focus will be on aptamer-incorporated nanoparticle systems for drug delivery purposes in order to assess the future potential of aptamers in the therapeutic area. Special emphasis will be given to the very recent progress in controlled drug release based on molecular gating achieved with aptamers.

  12. Aptamer modification improves the adenoviral transduction of malignant glioma cells.

    Science.gov (United States)

    Chen, Hao; Zheng, Xiaojing; Di, BingYan; Wang, Dongyang; Zhang, Yaling; Xia, Haibin; Mao, Qinwen

    2013-12-01

    Adenovirus has shown increasing promise in the gene-viral therapy for glioblastoma, a treatment strategy that relies on the delivery of viruses or transgenes into tumor cells. However, targeting of adenovirus to human glioblastoma remains a challenge due to the low expression level of coxsackie and adenovirus receptor (CAR) in glioma cells. Aptamers are small and highly structured single-stranded oligonucleotides that bind at high affinity to a target molecule, and are good candidates for targeted imaging and therapy. In this study, to construct an aptamer-modified Ad5, we first genetically modified the HVR5 of Ad hexon by biotin acceptor peptide (BAP), which would be metabolically biotinylated during production in HEK293 cells, and then attached the biotin labeled aptamer to the modified Ad through avidin–biotin binding. The aptamers used in this study includes AS1411 and GBI-10. The former is a DNA aptamer that can bind to nucleolin, a nuclear matrix protein found on the surface of cancer cells. The latter is a DNA aptamer that can recognize the extracellular matrix protein tenascin-C on the surface of human glioblastoma cells. To examine if aptamer-modification of the hexon protein could improve the adenoviral transduction efficiency, a glioblastoma cell line, U251, was transduced with aptamer-modified Ads. The transduction efficiency of AS1411- or GBI-10-modified Ad was approximately 4.1-fold or 5.2-fold higher than that of the control. The data indicated that aptamer modified adenovirus would be a useful tool for cancer gene therapy.

  13. 核糖开关与基因表达调控%Riboswitch in Gene Expression Regulation

    Institute of Scientific and Technical Information of China (English)

    韩祥东; 刘薇; 吴存祥; 冯永君

    2011-01-01

    Riboswitch is a growing interest in the research of gene expression regulation in recent years. Located in the untranslated regions ( UTR) of mRNAs, the riboswitch directly responses to signals and changes its stem-loop structure to regulate the expression of the downstream gene at the transcription or post-transcription level. Both the signal sensing and conformational changing processes of riboswitches are independent of protein factors. Four classes of riboswitches are categorized based on their sensed signals, which include small-molecule metabolites, metal ions, environmental factors (temperature, pH) , and uncharged tRNA sensitive riboswitches. The most interested class of riboswitches which has the largest number of members is the ones to sense small-molecule metabolites. As more riboswitches are being identified, their significance in molecular evolution and environmental adaptation will be better understood, and could be used as helpful tools for basic biological research or industrial drug development.%核糖开关(riboswitch)是近几年基因表达调控研究的一个热点.核糖开关位于mRNA的非翻译区(untranslated regions,UTR),能够直接感受胞内外信号并引起自身二级结构的变化,在转录或后转录(翻译和mRNA稳定性)水平实现对下游相关基因的表达调控,该过程不依赖于包括蛋白质在内的其它任何因子的作用.根据现已发现的核糖开关所能识别的信号因子类型,可以将其分为4类,即小分子代谢物、金属离子、环境因素及空载tRNA敏感的核糖开关;其中,小分子代谢物敏感的核糖开关是发现和研究最多且最深入的一类.随着研究的深入,将会有更多的核糖开关被发现,这不仅有助于理解生物进化与环境适应性,而且在生物学基础研究,新型药物的开发以及工业生产领域都将发挥重要作用.

  14. Aptamer-targeted DNA nanostructures for therapeutic delivery.

    Science.gov (United States)

    Charoenphol, Phapanin; Bermudez, Harry

    2014-05-05

    DNA-based nanostructures have been widely used in various applications due to their structural diversity, programmability, and uniform structures. Their intrinsic biocompatibility and biodegradability further motivates the investigation of DNA-based nanostructures as delivery vehicles. Incorporating AS1411 aptamers into DNA pyramids leads to enhanced intracellular uptake and selectively inhibits the growth of cancer cells, achieved without the use of transfection reagents. Furthermore, aptamer-displaying pyramids are found to be substantially more resistant to nuclease degradation than single-stranded aptamers. These findings, along with their modularity, reinforce the potential of DNA-based nanostructures for therapeutic applications.

  15. CELL-SELEX: Novel Perspectives of Aptamer-Based Therapeutics

    Directory of Open Access Journals (Sweden)

    Hans P. Wendel

    2008-04-01

    Full Text Available Aptamers, single stranded DNA or RNA molecules, generated by a method called SELEX (systematic evolution of ligands by exponential enrichment have been widely used in various biomedical applications. The newly developed Cell-SELEX (cell based-SELEX targeting whole living cells has raised great expectations for cancer biology, -therapy and regenerative medicine. Combining nanobiotechnology with aptamers, this technology opens the way to more sophisticated applications in molecular diagnosis. This paper gives a review of recent developments in SELEX technologies and new applications of aptamers.

  16. Biosensor platform based on carbon nanotubes covalently modified with aptamers

    Science.gov (United States)

    Komarov, I. A.; Rubtsova, E. I.; Golovin, A. V.; Bobrinetskiy, I. I.

    2016-12-01

    We developed a new platform for biosensing applications. Aptamers as sensitive agents have a great potential and gives us possibility to have highest possible selectivity among other sensing agents like enzymes or antibodies. We covalently bound aptamers to the functional groups of c-CNTs and then put this system on the surface of polymer substrate. Thus we got high sensitive flexible transparent biological sensors. We also suggest that by varying aptamer type we can make set of biosensors for disease detection which can be integrated into self-healthcare systems and gadgets.

  17. Comparative sequence and structure analysis reveals the conservation and diversity of nucleotide positions and their associated tertiary interactions in the riboswitches.

    Directory of Open Access Journals (Sweden)

    Sri D Appasamy

    Full Text Available The tertiary motifs in complex RNA molecules play vital roles to either stabilize the formation of RNA 3D structure or to provide important biological functionality to the molecule. In order to better understand the roles of these tertiary motifs in riboswitches, we examined 11 representative riboswitch PDB structures for potential agreement of both motif occurrences and conservations. A total of 61 unique tertiary interactions were found in the reference structures. In addition to the expected common A-minor motifs and base-triples mainly involved in linking distant regions the riboswitch structures three highly conserved variants of A-minor interactions called G-minors were found in the SAM-I and FMN riboswitches where they appear to be involved in the recognition of the respective ligand's functional groups. From our structural survey as well as corresponding structure and sequence alignments, the agreement between motif occurrences and conservations are very prominent across the representative riboswitches. Our analysis provide evidence that some of these tertiary interactions are essential components to form the structure where their sequence positions are conserved despite a high degree of diversity in other parts of the respective riboswitches sequences. This is indicative of a vital role for these tertiary interactions in determining the specific biological function of riboswitch.

  18. Small molecules that interact with RNA: riboswitch-based gene control and its involvement in metabolic regulation in plants and algae.

    Science.gov (United States)

    Bocobza, Samuel E; Aharoni, Asaph

    2014-08-01

    Riboswitches are RNA elements that bind small molecules and in turn regulate gene expression. This mechanism allows the cell to sense the intracellular concentration of these small molecules. A particular riboswitch typically regulates its adjacent gene by altering the transcription, the translation or the splicing of this gene. Recently, a riboswitch that binds thiamin pyrophosphate (TPP) was characterized and found to regulate thiamin biosynthesis in plants and algae. Furthermore, it appears that this element is an essential regulator of primary metabolism in plants. Manipulation of endogenous riboswitch activity resulted in metabolic phenotypes that underlined the role of these elements and their ligands in preserving metabolic homeostasis. This situation supports the hypothesis that riboswitches could be remnants of the most ancient metabolic regulators. Here, we review the mode of action of the plant and algal TPP riboswitch and its relevance to the metabolic network. We also discuss the potential engineering of riboswitches as metabolite sensors in plants and platforms for gene control. Whether additional such RNA-based mechanisms exist in plants and in algae is still an open question, yet, the importance of these elements to metabolic regulation is beyond doubt.

  19. Aptamer-Based Electrochemical Sensing of Lysozyme

    Directory of Open Access Journals (Sweden)

    Alina Vasilescu

    2016-06-01

    Full Text Available Protein analysis and quantification are required daily by thousands of laboratories worldwide for activities ranging from protein characterization to clinical diagnostics. Multiple factors have to be considered when selecting the best detection and quantification assay, including the amount of protein available, its concentration, the presence of interfering molecules, as well as costs and rapidity. This is also the case for lysozyme, a 14.3-kDa protein ubiquitously present in many organisms, that has been identified with a variety of functions: antibacterial activity, a biomarker of several serious medical conditions, a potential allergen in foods or a model of amyloid-type protein aggregation. Since the design of the first lysozyme aptamer in 2001, lysozyme became one of the most intensively-investigated biological target analytes for the design of novel biosensing concepts, particularly with regards to electrochemical aptasensors. In this review, we discuss the state of the art of aptamer-based electrochemical sensing of lysozyme, with emphasis on sensing in serum and real samples.

  20. Graphene- and aptamer-based electrochemical biosensor.

    Science.gov (United States)

    Xu, Ke; Meshik, Xenia; Nichols, Barbara M; Zakar, Eugene; Dutta, Mitra; Stroscio, Michael A

    2014-05-23

    This study investigated the effectiveness of a graphene- and aptamer-based field-effect-transistor-like (FET-like) sensor in detecting lead and potassium ions. The sensor consists of a graphene-covered Si/SiO2 wafer with thrombin binding aptamer (TBA) attached to the graphene layer and terminated by a methylene blue (MB) molecule. K(+) and Pb(2+) both bind to TBA and cause a conformational change, which results in MB moving closer to the graphene surface and donating an electron. Thus, the abundance of K(+) and Pb(2+) can be determined by monitoring the current across the source and drain channel. Device transfer curves were obtained with ambipolar field effect observed. Current readings were taken for K(+) concentrations of 100 μM to 50 mM and Pb(2+) concentrations of 10 μM to 10 mM. As expected, I d decreased as ion concentration increased. In addition, there was a negative shift in V Dirac in response to increased ion concentration.

  1. Aptamer Binding Studies Using MicroScale Thermophoresis.

    Science.gov (United States)

    Breitsprecher, Dennis; Schlinck, Nina; Witte, David; Duhr, Stefan; Baaske, Philipp; Schubert, Thomas

    2016-01-01

    The characterization and development of highly specific aptamers requires the analysis of the interaction strength between aptamer and target. MicroScale Thermophoresis (MST) is a rapid and precise method to quantify biomolecular interactions in solution at microliter scale. The basis of this technology is a physical effect referred to as thermophoresis, which describes the directed movement of molecules through temperature gradients. The thermophoretic properties of a molecule depend on its size, charge, and hydration shell. Since at least one of these parameters is altered upon binding of a ligand, this method can be used to analyze virtually any biomolecular interaction in any buffer or complex bioliquid. This section provides a detailed protocol describing how MST is used to obtain quantitative binding parameters for aptamer-target interactions. The two DNA-aptamers HD1 and HD22, which are targeted against human thrombin, are used as model systems to demonstrate a rapid and straightforward screening approach to determine optimal buffer conditions.

  2. Aptamer based electrochemical sensors for emerging environmental pollutants

    Directory of Open Access Journals (Sweden)

    Akhtar eHAYAT

    2014-06-01

    Full Text Available Environmental contaminants monitoring is one of the key issues in understanding and managing hazards to human health and ecosystems. In this context, aptamer based electrochemical sensors have achieved intense significance because of their capability to resolve a potentially large number of problems and challenges in environmental contamination. An aptasensor is a compact analytical device incorporating an aptamer (oligonulceotide as the sensing element either integrated within or intimately associated with a physiochemical transducer surface. Nucleic acid is well known for the function of carrying and passing genetic information, however, it has found a key role in analytical monitoring during recent years. Aptamer based sensors represent a novelty in environmental analytical science and there are great expectations for their promising performance as alternative to conventional analytical tools. This review paper focuses on the recent advances in the development of aptamer based electrochemical sensors for environmental applications with special emphasis on emerging pollutants.

  3. Aptamer based electrochemical sensors for emerging environmental pollutants

    Science.gov (United States)

    Hayat, Akhtar; Marty, Jean Louis

    2014-06-01

    Environmental contaminants monitoring is one of the key issues in understanding and managing hazards to human health and ecosystems. In this context, aptamer based electrochemical sensors have achieved intense significance because of their capability to resolve a potentially large number of problems and challenges in environmental contamination. An aptasensor is a compact analytical device incorporating an aptamer (oligonulceotide) as the sensing element either integrated within or intimately associated with a physiochemical transducer surface. Nucleic acid is well known for the function of carrying and passing genetic information, however, it has found a key role in analytical monitoring during recent years. Aptamer based sensors represent a novelty in environmental analytical science and there are great expectations for their promising performance as alternative to conventional analytical tools. This review paper focuses on the recent advances in the development of aptamer based electrochemical sensors for environmental applications with special emphasis on emerging pollutants.

  4. Probing the Structure of DNA Aptamers with a Classic Heterocycle.

    Directory of Open Access Journals (Sweden)

    G. Reid Bishop

    2004-02-01

    Full Text Available DNA aptamers are synthetic, single-stranded DNA oligonucleotides selectedby SELEX methods for their binding with specific ligands. Here we present ethidiumbinding results for three related DNA aptamers (PDB code: 1OLD, 1DB6, and 2ARGthat bind L-argininamide (L-Arm. The ligand bound form of each aptamer's structurehas been reported and each are found to be composed primarily of two domainsconsisting of a stem helical region and a loop domain that forms a binding pocket for thecognate ligand. Previous thermodynamic experiments demonstrated that the DNAaptamer 1OLD undergoes a large conformational ordering upon binding to L-Arm. Herewe extend those linkage binding studies by examining the binding of the heterocyclicintercalator ethidium to each of the three aptamers by fluorescence and absorptionspectrophotometric titrations. Our results reveal that ethidium binds to each aptamer with∆Go's in the range of -8.7 to -9.4 kcal/mol. The stoichiometry of binding is 2:1 for eachaptamer and is quantitatively diminished in the presence of L-Arm as is the overallfluorescence intensity of ethidium. Together, these results demonstrate that a portion ofthe bound ethidium is excluded from the aptamer in the presence of a saturating amountof L-Arm. These results demonstrate the utility of ethidium and related compounds forthe probing of non-conventional DNA structures and reveal an interesting fundamentalthermodynamic linkage in DNA aptamers. Results are discussed in the context of thethermodynamic stability and structure of each of the aptamers examined.

  5. Methods To Identify Aptamers against Cell Surface Biomarkers

    Directory of Open Access Journals (Sweden)

    Frédéric Ducongé

    2011-09-01

    Full Text Available Aptamers are nucleic acid-based ligands identified through a process of molecular evolution named SELEX (Systematic Evolution of Ligands by Exponential enrichment. During the last 10-15 years, numerous aptamers have been developed specifically against targets present on or associated with the surface of human cells or infectious pathogens such as viruses, bacteria, fungi or parasites. Several of the aptamers have been described as potent probes, rivalling antibodies, for use in flow cytometry or microscopy. Some have also been used as drugs by inhibiting or activating functions of their targets in a manner similar to neutralizing or agonistic antibodies. Additionally, it is straightforward to conjugate aptamers to other agents without losing their affinity and they have successfully been used in vitro and in vivo to deliver drugs, siRNA, nanoparticles or contrast agents to target cells. Hence, aptamers identified against cell surface biomarkers represent a promising class of ligands. This review presents the different strategies of SELEX that have been developed to identify aptamers for cell surface-associated proteins as well as some of the methods that are used to study their binding on living cells.

  6. Integrated Microfluidic Isolation of Aptamers Using Electrophoretic Oligonucleotide Manipulation

    Science.gov (United States)

    Kim, Jinho; Olsen, Timothy R.; Zhu, Jing; Hilton, John P.; Yang, Kyung-Ae; Pei, Renjun; Stojanovic, Milan N.; Lin, Qiao

    2016-05-01

    We present a microfluidic approach to integrated isolation of DNA aptamers via systematic evolution of ligands by exponential enrichment (SELEX). The approach employs a microbead-based protocol for the processes of affinity selection and amplification of target-binding oligonucleotides, and an electrophoretic DNA manipulation scheme for the coupling of these processes, which are required to occur in different buffers. This achieves the full microfluidic integration of SELEX, thereby enabling highly efficient isolation of aptamers in drastically reduced times and with minimized consumption of biological material. The approach as such also offers broad target applicability by allowing selection of aptamers with respect to targets that are either surface-immobilized or solution-borne, potentially allowing aptamers to be developed as readily available affinity reagents for a wide range of targets. We demonstrate the utility of this approach on two different procedures, respectively for isolating aptamers against a surface-immobilized protein (immunoglobulin E) and a solution-phase small molecule (bisboronic acid in the presence of glucose). In both cases aptamer candidates were isolated in three rounds of SELEX within a total process time of approximately 10 hours.

  7. CD28 Aptamers as Powerful Immune Response Modulators

    Directory of Open Access Journals (Sweden)

    Fernando Pastor

    2013-01-01

    Full Text Available CD28 is one of the main costimulatory receptors responsible for the proper activation of T lymphocytes. We have isolated two aptamers that bind to the CD28 receptor. As a monomer, one of them interfered with the binding of CD28 to its ligand (B7, precluding the costimulatory signal, whereas the other one was inactive. However, dimerization of any of the anti-CD28 aptamers was sufficient to provide an artificial costimulatory signal. No antibody has featured a dual function (i.e., the ability to work as agonist and antagonist to date. Two different agonistic structures were engineered for each anti-CD28 aptamer. One showed remarkably improved costimulatory properties, surpassing the agonistic effect of an anti-CD28 antibody. Moreover, we showed in vivo that the CD28 agonistic aptamer is capable of enhancing the cellular immune response against a lymphoma idiotype and of prolonging survival of mice which receive the aptamer together with an idiotype vaccine. The CD28 aptamers described in this work could be used to modulate the immune response either blocking the interaction with B7 or enhancing vaccine-induced immune responses in cancer immunotherapy.

  8. Aptamer-targeted RNAi for HIV-1 therapy.

    Science.gov (United States)

    Zhou, Jiehua; Rossi, John J

    2011-01-01

    The highly specific mechanism of RNA (RNAi) that inhibits the expression of disease genes is increasingly being harnessed to develop a new class of therapeutics for a wide variety of human maladies. The successful use of small interfering RNAs (siRNAs) for therapeutic purposes requires safe and efficient delivery to specific cells and tissues. Herein, we demonstrate novel cell type-specific dual inhibitory function anti-gp120 aptamer-siRNA delivery systems for HIV-1 therapy, in which both the aptamer and the siRNA portions have potent anti-HIV activities. The envelope glycoprotein is expressed on the surface of HIV-1 infected cells, allowing binding and internalization of the aptamer-siRNA chimeric molecules. The Dicer substrate siRNA delivered by the aptamers is functionally processed by Dicer, resulting in specific inhibition of HIV-1 replication and infectivity in cultured CEM T-cells and primary blood mononuclear cells. Our results provide a set of novel aptamer-targeted RNAi therapeutics to combat HIV and further validate the use of anti-gp120 aptamers for delivery of Dicer substrate siRNAs.

  9. Aptamer-Modified Semiconductor Quantum Dots for Biosensing Applications

    Directory of Open Access Journals (Sweden)

    Lin Wen

    2017-07-01

    Full Text Available Semiconductor quantum dots have attracted extensive interest in the biosensing area because of their properties, such as narrow and symmetric emission with tunable colors, high quantum yield, high stability and controllable morphology. The introduction of various reactive functional groups on the surface of semiconductor quantum dots allows one to conjugate a spectrum of ligands, antibodies, peptides, or nucleic acids for broader and smarter applications. Among these ligands, aptamers exhibit many advantages including small size, high chemical stability, simple synthesis with high batch-to-batch consistency and convenient modification. More importantly, it is easy to introduce nucleic acid amplification strategies and/or nanomaterials to improve the sensitivity of aptamer-based sensing systems. Therefore, the combination of semiconductor quantum dots and aptamers brings more opportunities in bioanalysis. Here we summarize recent advances on aptamer-functionalized semiconductor quantum dots in biosensing applications. Firstly, we discuss the properties and structure of semiconductor quantum dots and aptamers. Then, the applications of biosensors based on aptamer-modified semiconductor quantum dots by different signal transducing mechanisms, including optical, electrochemical and electrogenerated chemiluminescence approaches, is discussed. Finally, our perspectives on the challenges and opportunities in this promising field are provided.

  10. APTAMER-BASED SERRS SENSOR FOR THROMBIN DETECTION

    Energy Technology Data Exchange (ETDEWEB)

    Cho, H; Baker, B R; Wachsmann-Hogiu, S; Pagba, C V; Laurence, T A; Lane, S M; Lee, L P; Tok, J B

    2008-07-02

    We describe an aptamer-based Surface Enhanced Resonance Raman Scattering (SERRS) sensor with high sensitivity, specificity, and stability for the detection of a coagulation protein, human a-thrombin. The sensor achieves high sensitivity and a limit of detection of 100 pM by monitoring the SERRS signal change upon the single step of thrombin binding to immobilized thrombin binding aptamer. The selectivity of the sensor is demonstrated by the specific discrimination of thrombin from other protein analytes. The specific recognition and binding of thrombin by the thrombin binding aptamer is essential to the mechanism of the aptamer-based sensor, as shown through measurements using negative control oligonucleotides. In addition, the sensor can detect 1 nM thrombin in the presence of complex biofluids, such as 10% fetal calf serum, demonstrating that the immobilized, 5{prime}-capped, 3{prime}-capped aptamer is sufficiently robust for clinical diagnostic applications. Furthermore, the proposed sensor may be implemented for multiplexed detection using different aptamer-Raman probe complexes.

  11. Aptamers Binding to c-Met Inhibiting Tumor Cell Migration.

    Directory of Open Access Journals (Sweden)

    Birgit Piater

    Full Text Available The human receptor tyrosine kinase c-Met plays an important role in the control of critical cellular processes. Since c-Met is frequently over expressed or deregulated in human malignancies, blocking its activation is of special interest for therapy. In normal conditions, the c-Met receptor is activated by its bivalent ligand hepatocyte growth factor (HGF. Also bivalent antibodies can activate the receptor by cross linking, limiting therapeutic applications. We report the generation of the RNA aptamer CLN64 containing 2'-fluoro pyrimidine modifications by systematic evolution of ligands by exponential enrichment (SELEX. CLN64 and a previously described single-stranded DNA (ssDNA aptamer CLN3 exhibited high specificities and affinities to recombinant and cellular expressed c-Met. Both aptamers effectively inhibited HGF-dependent c-Met activation, signaling and cell migration. We showed that these aptamers did not induce c-Met activation, revealing an advantage over bivalent therapeutic molecules. Both aptamers were shown to bind overlapping epitopes but only CLN3 competed with HGF binding to cMet. In addition to their therapeutic and diagnostic potential, CLN3 and CLN64 aptamers exhibit valuable tools to further understand the structural and functional basis for c-Met activation or inhibition by synthetic ligands and their interplay with HGF binding.

  12. Aptamer-Modified Semiconductor Quantum Dots for Biosensing Applications.

    Science.gov (United States)

    Wen, Lin; Qiu, Liping; Wu, Yongxiang; Hu, Xiaoxiao; Zhang, Xiaobing

    2017-07-28

    Semiconductor quantum dots have attracted extensive interest in the biosensing area because of their properties, such as narrow and symmetric emission with tunable colors, high quantum yield, high stability and controllable morphology. The introduction of various reactive functional groups on the surface of semiconductor quantum dots allows one to conjugate a spectrum of ligands, antibodies, peptides, or nucleic acids for broader and smarter applications. Among these ligands, aptamers exhibit many advantages including small size, high chemical stability, simple synthesis with high batch-to-batch consistency and convenient modification. More importantly, it is easy to introduce nucleic acid amplification strategies and/or nanomaterials to improve the sensitivity of aptamer-based sensing systems. Therefore, the combination of semiconductor quantum dots and aptamers brings more opportunities in bioanalysis. Here we summarize recent advances on aptamer-functionalized semiconductor quantum dots in biosensing applications. Firstly, we discuss the properties and structure of semiconductor quantum dots and aptamers. Then, the applications of biosensors based on aptamer-modified semiconductor quantum dots by different signal transducing mechanisms, including optical, electrochemical and electrogenerated chemiluminescence approaches, is discussed. Finally, our perspectives on the challenges and opportunities in this promising field are provided.

  13. Dual-Targeting Small-Molecule Inhibitors of the Staphylococcus aureus FMN Riboswitch Disrupt Riboflavin Homeostasis in an Infectious Setting.

    Science.gov (United States)

    Wang, Hao; Mann, Paul A; Xiao, Li; Gill, Charles; Galgoci, Andrew M; Howe, John A; Villafania, Artjohn; Barbieri, Christopher M; Malinverni, Juliana C; Sher, Xinwei; Mayhood, Todd; McCurry, Megan D; Murgolo, Nicholas; Flattery, Amy; Mack, Matthias; Roemer, Terry

    2017-05-18

    Riboswitches are bacterial-specific, broadly conserved, non-coding RNA structural elements that control gene expression of numerous metabolic pathways and transport functions essential for cell growth. As such, riboswitch inhibitors represent a new class of potential antibacterial agents. Recently, we identified ribocil-C, a highly selective inhibitor of the flavin mononucleotide (FMN) riboswitch that controls expression of de novo riboflavin (RF, vitamin B2) biosynthesis in Escherichia coli. Here, we provide a mechanistic characterization of the antibacterial effects of ribocil-C as well as of roseoflavin (RoF), an antimetabolite analog of RF, among medically significant Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecalis. We provide genetic, biophysical, computational, biochemical, and pharmacological evidence that ribocil-C and RoF specifically inhibit dual FMN riboswitches, separately controlling RF biosynthesis and uptake processes essential for MRSA growth and pathogenesis. Such a dual-targeting mechanism is specifically required to develop broad-spectrum Gram-positive antibacterial agents targeting RF metabolism. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. c-di-AMP binds the ydaO riboswitch in two pseudo-symmetry-related pockets.

    Science.gov (United States)

    Ren, Aiming; Patel, Dinshaw J

    2014-09-01

    The ydaO riboswitch, involved in sporulation, osmotic stress responses and cell wall metabolism, targets the second messenger cyclic-di-AMP with subnanomolar affinity. We have solved the structure of c-di-AMP bound to the Thermoanaerobacter tengcongensis ydaO riboswitch, thereby identifying a five-helical scaffold containing a zippered-up bubble, a pseudoknot and long-range tertiary base pairs. Highlights include the identification of two c-di-AMP binding pockets on the same face of the riboswitch, related by pseudo-two-fold symmetry, with potential for cross-talk between sites mediated by adjacently positioned base-stacking alignments connecting pockets. The adenine rings of bound c-di-AMP molecules are wedged between bases and stabilized by stacking, base-sugar and sugar-sugar intermolecular hydrogen bonding interactions. The structural studies are complemented by isothermal titration calorimetry-based binding studies of mutants mediating key tertiary intermolecular contacts. The T. tengcongensis ydaO riboswitch, like its Bacillus subtilis counterpart, most likely functions through a transcription termination mechanism, with the c-di-AMP bound state representing an 'off' switch.

  15. Riboswitch-mediated Attenuation of Transgene Cytotoxicity Increases Adeno-associated Virus Vector Yields in HEK-293 Cells.

    Science.gov (United States)

    Strobel, Benjamin; Klauser, Benedikt; Hartig, Jörg S; Lamla, Thorsten; Gantner, Florian; Kreuz, Sebastian

    2015-10-01

    Cytotoxicity of transgenes carried by adeno-associated virus (AAV) vectors might be desired, for instance, in oncolytic virotherapy or occur unexpectedly in exploratory research when studying sparsely characterized genes. To date, most AAV-based studies use constitutively active promoters (e.g., the CMV promoter) to drive transgene expression, which often hampers efficient AAV production due to cytotoxic, antiproliferative, or unknown transgene effects interfering with producer cell performance. Therefore, we explored artificial riboswitches as novel tools to control transgene expression during AAV production in mammalian cells. Our results demonstrate that the guanine-responsive GuaM8HDV aptazyme efficiently attenuates transgene expression and associated detrimental effects, thereby boosting AAV vector yields up to 23-fold after a single addition of guanine. Importantly, riboswitch-harboring vectors preserved their ability to express functional transgene at high levels in the absence of ligand, as demonstrated in a mouse model of AAV-TGFβ1-induced pulmonary fibrosis. Thus, our study provides the first application-ready biotechnological system-based on aptazymes, which should enable high viral vector yields largely independent of the transgene used. Moreover, the RNA-intrinsic, small-molecule regulatable mode of action of riboswitches provides key advantages over conventional transcription factor-based regulatory systems. Therefore, such riboswitch vectors might be ultimately applied to temporally control therapeutic transgene expression in vivo.

  16. Rapid One-Step Selection Method for Generating Nucleic Acid Aptamers: Development of a DNA Aptamer against alpha-Bungarotoxin

    DEFF Research Database (Denmark)

    Lauridsen, Lasse Holm; Shamaileh, Hadi A.; Edwards, Stacey L.

    2012-01-01

    Background: Nucleic acids based therapeutic approaches have gained significant interest in recent years towards the development of therapeutics against many diseases. Recently, research on aptamers led to the marketing of Macugen (R), an inhibitor of vascular endothelial growth factor (VEGF...... by PCR enrichment of the selected aptamers. One round of selection successfully identified a DNA aptamer sequence with a binding affinity of 7.58 mu M. Conclusion: We have demonstrated a one-step method for rapid production of nucleic acid aptamers. Although the reported binding affinity is in the low...... micromolar range, we believe that this could be further improved by using larger targets, increasing the stringency of selection and also by combining a capillary electrophoresis separation prior to the one-step selection. Furthermore, the method presented here is a user-friendly, cheap and an easy way...

  17. Molecule-binding dependent assembly of split aptamer and γ-cyclodextrin: A sensitive excimer signaling approach for aptamer biosensors

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Fen [State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 (China); Hubei Key Laboratory of Mine Environmental Pollution Control and Remediation, Environmental Science and Engineering College, Hubei Polytechnic University, Huangshi 435003 (China); Lian, Yan; Li, Jishan; Zheng, Jing; Hu, Yaping; Liu, Jinhua; Huang, Jin [State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 (China); Yang, Ronghua, E-mail: Yangrh@pku.edu.cn [State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 (China)

    2013-10-17

    Graphical abstract: Adenosine-binding aptamer was splitted into two fragments P2 and P3 which labeled pyrene molecules, mainly produce monomer signal. γ-CD cavity brings P2 and P3 in close proximity, allowing for weak excimer emission. In the presence of target, P2 and P3 are expected to bind ATP and form an aptamer/target complex, leads to large increase of the pyrene excimer fluorescence. -- Highlights: •We assembled split aptamer and γ-cyclodextrin fluorescence biosensors for ATP detection. •The biosensor increased quantum yield and emission lifetime of the excimer. •Time-resolved fluorescence is effective for ATP assay in complicated environment. -- Abstract: A highly sensitive and selective fluorescence aptamer biosensors for the determination of adenosine triphosphate (ATP) was developed. Binding of a target with splitting aptamers labeled with pyrene molecules form stable pyrene dimer in the γ-cyclodextrin (γ-CD) cavity, yielding a strong excimer emission. We have found that inclusion of pyrene dimer in γ-cyclodextrin cavity not only exhibits additive increases in quantum yield and emission lifetime of the excimer, but also facilitates target-induced fusion of the splitting aptamers to form the aptamer/target complex. As proof-of-principle, the approach was applied to fluorescence detection of adenosine triphosphate. With an anti-ATP aptamer, the approach exhibits excimer fluorescence response toward ATP with a maximum signal-to-background ratio of 32.1 and remarkably low detection limit of 80 nM ATP in buffer solution. Moreover, due to the additive fluorescence lifetime of excimer induced by γ-cyclodextrin, time-resolved measurements could be conveniently used to detect as low as 0.5 μM ATP in blood serum quantitatively.

  18. Analysis of riboswitch structure and function by an energy landscape framework.

    Science.gov (United States)

    Quarta, Giulio; Kim, Namhee; Izzo, Joseph A; Schlick, Tamar

    2009-11-06

    The thiamine pyrophosphate (TPP) riboswitch employs modular domains for binding TPP to form a platform for gene expression regulation. Specifically, TPP binding triggers a conformational switch in the RNA from a transcriptionally active "on" state to an inactive "off" state that concomitantly causes the formation of a terminator hairpin and halting of transcription. Here, clustering analysis of energy landscapes at different nucleotide lengths suggests a novel computational tool for analysis of the mechanics of transcription elongation in the presence or absence of the ligand. Namely, we suggest that the riboswitch's kinetics are tightly governed by a length-dependent switch, whereby the energy landscape has two clusters available during transcription elongation and where TPP's binding shifts the preference to one form. Significantly, the biologically active and inactive structures determined experimentally matched well the structures predominant in each computational set. These clustering/structural analyses combined with modular computational design suggest design principles that exploit the above features to analyze as well as create new functions and structures of RNA systems.

  19. GEMM-I riboswitches from Geobacter sense the bacterial second messenger cyclic AMP-GMP.

    Science.gov (United States)

    Kellenberger, Colleen A; Wilson, Stephen C; Hickey, Scott F; Gonzalez, Tania L; Su, Yichi; Hallberg, Zachary F; Brewer, Thomas F; Iavarone, Anthony T; Carlson, Hans K; Hsieh, Yu-Fang; Hammond, Ming C

    2015-04-28

    Cyclic dinucleotides are an expanding class of signaling molecules that control many aspects of bacterial physiology. A synthase for cyclic AMP-GMP (cAG, also referenced as 3'-5', 3'-5' cGAMP) called DncV is associated with hyperinfectivity of Vibrio cholerae but has not been found in many bacteria, raising questions about the prevalence and function of cAG signaling. We have discovered that the environmental bacterium Geobacter sulfurreducens produces cAG and uses a subset of GEMM-I class riboswitches (GEMM-Ib, Genes for the Environment, Membranes, and Motility) as specific receptors for cAG. GEMM-Ib riboswitches regulate genes associated with extracellular electron transfer; thus cAG signaling may control aspects of bacterial electrophysiology. These findings expand the role of cAG beyond organisms that harbor DncV and beyond pathogenesis to microbial geochemistry, which is important to environmental remediation and microbial fuel cell development. Finally, we have developed an RNA-based fluorescent biosensor for live-cell imaging of cAG. This selective, genetically encodable biosensor will be useful to probe the biochemistry and cell biology of cAG signaling in diverse bacteria.

  20. Rational design of aptazyme riboswitches for efficient control of gene expression in mammalian cells

    Science.gov (United States)

    Zhong, Guocai; Wang, Haimin; Bailey, Charles C; Gao, Guangping; Farzan, Michael

    2016-01-01

    Efforts to control mammalian gene expression with ligand-responsive riboswitches have been hindered by lack of a general method for generating efficient switches in mammalian systems. Here we describe a rational-design approach that enables rapid development of efficient cis-acting aptazyme riboswitches. We identified communication-module characteristics associated with aptazyme functionality through analysis of a 32-aptazyme test panel. We then developed a scoring system that predicts an aptazymes’s activity by integrating three characteristics of communication-module bases: hydrogen bonding, base stacking, and distance to the enzymatic core. We validated the power and generality of this approach by designing aptazymes responsive to three distinct ligands, each with markedly wider dynamic ranges than any previously reported. These aptayzmes efficiently regulated adeno-associated virus (AAV)-vectored transgene expression in cultured mammalian cells and mice, highlighting one application of these broadly usable regulatory switches. Our approach enables efficient, protein-independent control of gene expression by a range of small molecules. DOI: http://dx.doi.org/10.7554/eLife.18858.001 PMID:27805569

  1. Riboswitch control of induction of aminoglycoside resistance acetyl and adenyl-transferases.

    Science.gov (United States)

    He, Weizhi; Zhang, Xuhui; Zhang, Jun; Jia, Xu; Zhang, Jing; Sun, Wenxia; Jiang, Hengyi; Chen, Dongrong; Murchie, Alastair I H

    2013-08-01

    The acquisition of antibiotic resistance by human pathogens poses a significant threat to public health. The mechanisms that control the proliferation and expression of antibiotic resistance genes are not yet completely understood. The aminoglycosides are a historically important class of antibiotics that were introduced in the 1940s. Aminoglycoside resistance is conferred most commonly through enzymatic modification of the drug or enzymatic modification of the target rRNA through methylation or through the overexpression of efflux pumps. In our recent paper, we reported that expression of the aminoglycoside resistance genes encoding the aminoglycoside acetyl transferase (AAC) and aminoglycoside adenyl transferase (AAD) enzymes was controlled by an aminoglycoside-sensing riboswitch RNA. This riboswitch is embedded in the leader RNA of the aac/aad genes and is associated with the integron cassette system. The leader RNA can sense and bind specific aminoglycosides such that the binding causes a structural transition in the leader RNA, which leads to the induction of aminoglycoside antibiotic resistance. Specific aminoglycosides induce reporter gene expression mediated by the leader RNA. Aminoglycoside RNA binding was measured directly and, aminoglycoside-induced changes in RNA structure monitored by chemical probing. UV cross-linking and mutational analysis identified potential aminoglycoside binding sites on the RNA.

  2. Endo-S-c-di-GMP Analogues-Polymorphism and Binding Studies with Class I Riboswitch

    Directory of Open Access Journals (Sweden)

    Herman O. Sintim

    2012-11-01

    Full Text Available C-di-GMP, a cyclic guanine dinucleotide, has been shown to regulate biofilm formation as well as virulence gene expression in a variety of bacteria. Analogues of c-di-GMP have the potential to be used as chemical probes to study c-di-GMP signaling and could even become drug leads for the development of anti-biofilm compounds. Herein we report the synthesis and biophysical studies of a series of c-di-GMP analogues, which have both phosphate and sugar moieties simultaneously modified (called endo-S-c-di-GMP analogues. We used computational methods to predict the relative orientation of the guanine nucleobases in c-di-GMP and analogues. DOSY NMR of the endo-S-c-di-GMP series showed that the polymorphism of c-di-GMP can be tuned with conservative modifications to the phosphate and sugar moieties (conformational steering. Binding studies with Vc2 RNA (a class I c-di-GMP riboswitch revealed that conservative modifications to the phosphate and 2'-positions of c-di-GMP dramatically affected binding to class I riboswitch.

  3. A microfluidic surface enhanced Raman spectroscopic biosensor using aptamer functionalized nanopillars

    DEFF Research Database (Denmark)

    Yang, J.; Palla, M.; Bosco, F. G.

    2013-01-01

    This paper presents a microchip incorporating an aptamer-functionalized nanopillar substrate, enabling the specific detection of low-abundance biomolecules using surface enhanced Raman spectroscopy (SERS). In a temperature controlled microchamber, aptamers immobilized on the nanostructure surface...

  4. Cell-SELEX Identifies a “Sticky” RNA Aptamer Sequence

    Science.gov (United States)

    2017-01-01

    Cell-SELEX is performed to select for cell binding aptamers. We employed an additional selection pressure by using RNAse to remove surface-binding aptamers and select for cell-internalizing aptamers. A common RNA sequence was identified from independent cell-SELEX procedures against two different pancreatic cancer cell lines, indicating a strong selection pressure towards this sequence from the large pool of other available sequences present in the aptamer library. The aptamer is not specific for the pancreatic cancer cell lines, and a similar sequence motif is present in previously published internalizing aptamers. The identified sequence forms a structural motif that binds to a surface protein, which either is highly abundant or has strong affinity for the selected aptamer sequence. Deselecting (removing) this sequence during cell-SELEX may increase the probability of identifying aptamers against cell type-specific targets on the cell surface. PMID:28194280

  5. Cell-SELEX Identifies a “Sticky” RNA Aptamer Sequence

    Directory of Open Access Journals (Sweden)

    Partha Ray

    2017-01-01

    Full Text Available Cell-SELEX is performed to select for cell binding aptamers. We employed an additional selection pressure by using RNAse to remove surface-binding aptamers and select for cell-internalizing aptamers. A common RNA sequence was identified from independent cell-SELEX procedures against two different pancreatic cancer cell lines, indicating a strong selection pressure towards this sequence from the large pool of other available sequences present in the aptamer library. The aptamer is not specific for the pancreatic cancer cell lines, and a similar sequence motif is present in previously published internalizing aptamers. The identified sequence forms a structural motif that binds to a surface protein, which either is highly abundant or has strong affinity for the selected aptamer sequence. Deselecting (removing this sequence during cell-SELEX may increase the probability of identifying aptamers against cell type-specific targets on the cell surface.

  6. Aptamer-initiated on-particle template-independent enzymatic polymerization (aptamer-OTEP) for electrochemical analysis of tumor biomarkers.

    Science.gov (United States)

    Wang, Pengjuan; Wan, Ying; Deng, Shengyuan; Yang, Shulin; Su, Yan; Fan, Chunhai; Aldalbahi, Ali; Zuo, Xiaolei

    2016-12-15

    Herein, an aptamer-initiated on-particle template-independent enzymatic polymerization (aptamer-OTEP) strategy for electrochemical aptasensor (E-aptasensor) is developed for analysis of cancer biomarker carcino-embryonic antigen (CEA). A pair of DNA aptamers is employed which can be specifically bond with CEA simultaneously. One of the aptamer is thiolated at 3'-terminal and immobilized onto the gold electrode as a capture probe, while the other one has a thiol group at its 5'-terminal and is modified onto the gold nanoparticles surface to form a nanoprobe. In the present of target, the two aptamers can "sandwich" the target, thus the nanoprobe is attached to the electrode. Then terminal deoxynucleotidyl transferase (TdT) is employed to catalyze the incorporation of biotin labeled dNTPs into the 3'-OH terminals of the DNA aptamer on the nanoprobe. The as-generated long DNA oligo tentacles allow specific binding of numerous avidin modified horseradish peroxidase (Av-HRP), resulting in tens of thousands of HRP catalyzed reduction of hydrogen peroxide and sharply increasing electrochemical signals. Taking advantage of the enzyme based nucleic acid amplification and nanoprobe, this strategy is demonstrated to possess the outstanding amplification efficiency.

  7. Novel riboswitch-binding flavin analog that protects mice against Clostridium difficile infection without inhibiting cecal flora.

    Science.gov (United States)

    Blount, Kenneth F; Megyola, Cynthia; Plummer, Mark; Osterman, David; O'Connell, Tim; Aristoff, Paul; Quinn, Cheryl; Chrusciel, R Alan; Poel, Toni J; Schostarez, Heinrich J; Stewart, Catherine A; Walker, Daniel P; Wuts, Peter G M; Breaker, Ronald R

    2015-09-01

    Novel mechanisms of action and new chemical scaffolds are needed to rejuvenate antibacterial drug discovery, and riboswitch regulators of bacterial gene expression are a promising class of targets for the discovery of new leads. Herein, we report the characterization of 5-(3-(4-fluorophenyl)butyl)-7,8-dimethylpyrido[3,4-b]quinoxaline-1,3(2H,5H)-dione (5FDQD)-an analog of riboflavin that was designed to bind riboswitches that naturally recognize the essential coenzyme flavin mononucleotide (FMN) and regulate FMN and riboflavin homeostasis. In vitro, 5FDQD and FMN bind to and trigger the function of an FMN riboswitch with equipotent activity. MIC and time-kill studies demonstrated that 5FDQD has potent and rapidly bactericidal activity against Clostridium difficile. In C57BL/6 mice, 5FDQD completely prevented the onset of lethal antibiotic-induced C. difficile infection (CDI). Against a panel of bacteria representative of healthy bowel flora, the antibacterial selectivity of 5FDQD was superior to currently marketed CDI therapeutics, with very little activity against representative strains from the Bacteroides, Lactobacillus, Bifidobacterium, Actinomyces, and Prevotella genera. Accordingly, a single oral dose of 5FDQD caused less alteration of culturable cecal flora in mice than the comparators. Collectively, these data suggest that 5FDQD or closely related analogs could potentially provide a high rate of CDI cure with a low likelihood of infection recurrence. Future studies will seek to assess the role of FMN riboswitch binding to the mechanism of 5FDQD antibacterial action. In aggregate, our results indicate that riboswitch-binding antibacterial compounds can be discovered and optimized to exhibit activity profiles that merit preclinical and clinical development as potential antibacterial therapeutic agents.

  8. tPA-binding RNA Aptamers

    DEFF Research Database (Denmark)

    Bjerregaard, Nils

    2015-01-01

    The serine protease Tissue-type Plasminogen Activator (tPA) is the principal initiator of fibrinolysis in mammalian physiology. Recombinant tPA is employed in the pharmacological resolution of vessel occlusions caused by pathological thrombosis, the originating cause of cerebral ischaemic strokes....... Globally, stroke is the leading cause of disability and the third leading cause of mortality. Fibrinolytic therapy with recombinant tPA rescues suboptimally perfused penumbral nervous tissue, but has also been implicated in detrimental neurotoxic effects, which are chiefly mediated by the Low......, and upon conjugation to serum albumin. K18v2 was able to inhibit tPA-induced fibrinogen depletion in vitro, which may provide additional benefits in stroke treatment. A conjugate of both aptamers separated by a linker encompassed the activities of both constituent sequences, and additionally possessed...

  9. Modified AS1411 Aptamer Suppresses Hepatocellular Carcinoma by Up-Regulating Galectin-14

    OpenAIRE

    Cho, Yuri; Lee, Yun Bin; Lee, Jeong-Hoon; Lee, Dong Hyeon; Cho, Eun Ju; Yu, Su Jong; Kim, Yoon Jun; Kim, Jong In; Im, Jong Hun; Lee, Jung Hwan; Oh, Eun Ju; Yoon, Jung-Hwan

    2016-01-01

    Aptamers are small synthetic oligonucleotides that bind to target proteins with high specificity and affinity. AS1411 is an aptamer that binds to nucleolin, which is overexpressed in the cytoplasm and occurs on the surface of cancer cells. We investigated the therapeutic potential of aptamers in hepatocellular carcinoma (HCC) by evaluating anti-tumor effects and confirming the affinity and specificity of AS1411- and modified AS1411-aptamers in HCC cells. Cell growth was assessed using the MTS...

  10. General approach for engineering small-molecule-binding DNA split aptamers.

    Science.gov (United States)

    Kent, Alexandra D; Spiropulos, Nicholas G; Heemstra, Jennifer M

    2013-10-15

    Here we report a general method for engineering three-way junction DNA aptamers into split aptamers. Split aptamers show significant potential for use as recognition elements in biosensing applications, but reliable methods for generating these sequences are currently lacking. We hypothesize that the three-way junction is a "privileged architecture" for the elaboration of aptamers into split aptamers, as it provides two potential splitting sites that are distal from the target binding pocket. We propose a general method for split aptamer engineering that involves removing one loop region, then systematically modifying the number of base pairs in the remaining stem regions in order to achieve selective assembly only in the presence of the target small molecule. We screen putative split aptamer sequence pairs using split aptamer proximity ligation (StAPL) technology developed by our laboratory, but we validate that the results obtained using StAPL translate directly to systems in which the aptamer fragments are assembling noncovalently. We introduce four new split aptamer sequences, which triples the number of small-molecule-binding DNA split aptamers reported to date, and the methods described herein provide a reliable route for the engineering of additional split aptamers, dramatically advancing the potential substrate scope of DNA assembly based biosensors.

  11. Use of magnetic beads in selection and detection of biotoxin aptamers by electrochemiluminescence and enzymatic methods.

    Science.gov (United States)

    Bruno, John G; Kiel, Johnathan L

    2002-01-01

    Systematic evolution of ligands by exponential enrichment (SELEX) was used to develop DNA ligands (aptamers) to cholera whole toxin and staphylococcal enterotoxin B (SEB). Affinity selection of aptamers was accomplished by conjugating the biotoxins to tosyl-activated magnetic beads. The use of magnetic beads reduces the volumes needed to perform aptamer selection, thus obviating alcohol precipitation and allowing direct PCR amplification from the bead surface. Following five rounds of SELEX, 5'-biotinylated aptamers were bound to streptavidin-coated magnetic beads and used for the detection of ruthenium trisbypyridine [Ru(bpy)3(2+)]-labeled cholera toxin and SEB by an electrochemiluminescence methodology. A comparison of control (double-stranded) aptamer binding was made with aptamers that were heat denatured at 96 degrees C (single-stranded) and allowed to cool (conform) in the presence of biotoxin-conjugated magnetic beads. Results suggest that control aptamers performed equally well when compared to heat-denatured DNA aptamers in the cholera toxin electrochemiluminescence assay and a colorimetric microplate assay employing peroxidase-labeled cholera toxin and 5'-amino terminated aptamers conjugated to N-oxysuccinimide-activated microtiter wells. Interestingly, however, in the SEB electrochemiluminescence assay, double-stranded aptamers exceeded the performance of single-stranded aptamers. The detection limits of all aptamer assays were in the low nanogram to low picogram ranges.

  12. Increased anticoagulant activity of thrombin-binding DNA aptamers by nanoscale organization on DNA nanostructures

    DEFF Research Database (Denmark)

    Rangnekar, Abhijit; Zhang, Alex M.; Shiyuan Li, Susan;

    2012-01-01

    Control over thrombin activity is much desired to regulate blood clotting in surgical and therapeutic situations. Thrombin-binding RNA and DNA aptamers have been used to inhibit thrombin activity and thus the coagulation cascade. Soluble DNA aptamers, as well as two different aptamers tethered by...

  13. Theophylline-dependent riboswitch as a novel genetic tool for strict regulation of protein expression in Cyanobacterium Synechococcus elongatus PCC 7942.

    Science.gov (United States)

    Nakahira, Yoichi; Ogawa, Atsushi; Asano, Hiroyuki; Oyama, Tokitaka; Tozawa, Yuzuru

    2013-10-01

    The cyanobacterium Synechococcus elongatus PCC 7942 is a major model species for studies of photosynthesis. It is are also a potential cell factory for the production of renewable biofuels and valuable chemicals. We employed engineered riboswitches to control translational initiation of target genes in this cyanobacterium. A firefly luciferase reporter assay revealed that three theophylline riboswitches performed as expected in the cyanobacterium. Riboswitch-E* exhibited very low leaky expression of luciferase and superior and dose-dependent on/off regulation of protein expression by theophylline. The maximum magnitude of the induction vs. basal level was ∼190-fold. Furthermore, the induction level was responsive to a wide range of theophylline concentrations in the medium, from 0 to 2 mM, facilitating the fine-tuning of luciferase expression. We adapted this riboswitch to another gene regulation system, in which expression of the circadian clock kaiC gene product is controlled by the theophylline concentration in the culture medium. The results demonstrated that the adequately adjusted expression level of KaiC restored complete circadian rhythm in the kaiC-deficient arrhythmic mutant. This theophylline-dependent riboswitch system has potential for various applications as a useful genetic tool in cyanobacteria.

  14. Experimental treatment of Staphylococcus aureus bovine intramammary infection using a guanine riboswitch ligand analog.

    Science.gov (United States)

    Ster, C; Allard, M; Boulanger, S; Lamontagne Boulet, M; Mulhbacher, J; Lafontaine, D A; Marsault, E; Lacasse, P; Malouin, F

    2013-02-01

    Staphylococcus aureus is a leading cause of intramammary infections (IMI). We recently demonstrated that Staph. aureus strains express the gene guaA during bovine IMI. This gene codes for a guanosine monophosphate synthetase and its expression is regulated by a guanine riboswitch. The guanine analog 2,5,6-triaminopyrimidine-4-one (PC1) is a ligand of the guanine riboswitch. Interactions between PC1 and its target result in inhibition of guanosine monophosphate synthesis and subsequent death of the bacterium. The present study describes the investigational use of PC1 for therapy of Staph. aureus IMI in lactating cows. The in vitro minimal inhibitory concentration of PC1 ranged from 0.5 to 4 μg/mL for a variety of Staph. aureus and Staphylococcus epidermidis strains and required a reducing agent for stability and full potency. A safety assessment study was performed, whereby the healthy quarters of 4 cows were infused with increasing doses of PC1 (0, 150, 250, and 500 mg). Over the 44 h following infusions, no obvious adverse effect was observed. Ten Holstein multiparous cows in mid lactation were then experimentally infused into 3 of the quarters with approximately 50 cfu of Staph. aureus strain SHY97-3906 and infection was allowed to progress for 2 wk before starting PC1 treatment. Bacterial counts reached then about 10(3) to 10(4) cfu/mL of milk. Infected quarters were treated with 1 of 3 doses of PC1 (0, 250, or 500 mg) after each morning and evening milking for 7d (i.e., 14 intramammary infusions of PC1). During the treatment period, milk from PC1-treated quarters showed a significant reduction in bacterial concentrations. However, this reduction of Staph. aureus count in milk was not maintained during the 4 wk following the end of the treatment and only 15% of the PC1-treated quarters underwent bacteriological cure. The somatic cell count and the quarter milk production were not affected by treatments. Although bacterial clearance was not achieved following

  15. Osteomyelitis diagnosis by {sup 99m}Tc radiolabeled aptamers

    Energy Technology Data Exchange (ETDEWEB)

    Santos, S.R.; Ferreira, I.M.; Andrade, A.S.R., E-mail: sararoberta7@hotmail.com, E-mail: imendesf@yahoo.com.br, E-mail: antero@cdtn.br [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil); Barros, A.L.B.; Cardoso, V.N.; Diniz, O.F., E-mail: brancodebarros@yahoo.com.br, E-mail: valbertcardoso@yahoo.com.br, E-mail: simoneodilia@yahoo.com.br [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Faculdade de Farmacia. Departamento de Analises Clinicas e Toxicologicas

    2015-07-01

    Osteomyelitis, which is characterized by progressive inflammatory destruction and new opposition of bone, is still a difficult infection to treat. The clinical diagnosis in late stages is achieved easily, but an early diagnosis is more challenging. Staphylococcus aureus is a common agent found in osteomyelitis and bone prostheses infection. Diagnosis by scintigraphy has advantages because it is a non-invasive procedure and is able to perform an early diagnosis even before anatomic changes. Thus, nuclear medicine could contribute to an accurate diagnosis since specific radiopharmaceuticals were developed. In this study, aptamers selected to Staphylococcus aureus were labeled with {sup 99m}Tc and used for bacteria identification in an osteomyelitis experimental model. The aptamers selected to S. aureus were directly labelled with {sup 99m}Tc and were evaluated by biodistribution studies. Wistar rats with intraosseous infection in the right paw were used. A random aptamer labelled with {sup 99m}Tc was as control. Six animals were used in each group. The aptamers labeled with {sup 99m}Tc were able to identify the infection foci caused by S. aureus displaying a target/non-target ratio of 2,23 ± 0,20, after 3 h. The control group presented a target/non-target ratio 1,08 ± 0.23. The results indicated that the radiolabeled aptamers were able to identify specifically the infection foci and they should be further explored for infection diagnosis by scintigraphy. (author)

  16. MIPs and Aptamers for Recognition of Proteins in Biomimetic Sensing.

    Science.gov (United States)

    Menger, Marcus; Yarman, Aysu; Erdőssy, Júlia; Yildiz, Huseyin Bekir; Gyurcsányi, Róbert E; Scheller, Frieder W

    2016-07-18

    Biomimetic binders and catalysts have been generated in order to substitute the biological pendants in separation techniques and bioanalysis. The two major approaches use either "evolution in the test tube" of nucleotides for the preparation of aptamers or total chemical synthesis for molecularly imprinted polymers (MIPs). The reproducible production of aptamers is a clear advantage, whilst the preparation of MIPs typically leads to a population of polymers with different binding sites. The realization of binding sites in the total bulk of the MIPs results in a higher binding capacity, however, on the expense of the accessibility and exchange rate. Furthermore, the readout of the bound analyte is easier for aptamers since the integration of signal generating labels is well established. On the other hand, the overall negative charge of the nucleotides makes aptamers prone to non-specific adsorption of positively charged constituents of the sample and the "biological" degradation of non-modified aptamers and ionic strength-dependent changes of conformation may be challenging in some application.

  17. Aptamers in Diagnostics and Treatment of Viral Infections

    Directory of Open Access Journals (Sweden)

    Tomasz Wandtke

    2015-02-01

    Full Text Available Aptamers are in vitro selected DNA or RNA molecules that are capable of binding a wide range of nucleic and non-nucleic acid molecules with high affinity and specificity. They have been conducted through the process known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment. It serves to reach specificity and considerable affinity to target molecules, including those of viral origin, both proteins and nucleic acids. Properties of aptamers allow detecting virus infected cells or viruses themselves and make them competitive to monoclonal antibodies. Specific aptamers can be used to interfere in each stage of the viral replication cycle and also inhibit its penetration into cells. Many current studies have reported possible application of aptamers as a treatment or diagnostic tool in viral infections, e.g., HIV (Human Immunodeficiency Virus, HBV (Hepatitis B Virus, HCV (Hepatitis C Virus, SARS (Severe Acute Respiratory Syndrome, H5N1 avian influenza and recently spread Ebola. This review presents current developments of using aptamers in the diagnostics and treatment of viral diseases.

  18. Label-free selection of RNA aptamers for metabolic engineering.

    Science.gov (United States)

    Hwang, Chuhern; Carothers, James M

    2016-08-15

    RNA aptamers can be assembled into genetic regulatory devices that sense and respond to levels of specific cellular metabolites and thus serve an integral part of designing dynamic control into engineered metabolic pathways. Here, we describe a practical method for generating specific and high affinity aptamers to enable the wider use of in vitro selection and a broader application of aptamers for metabolic engineering. Conventional selection methods involving either radioactive labeling of RNA or the use of label-free methods such as SPR to track aptamer enrichment require resources that are not widely accessible to research groups. We present a label-free selection method that uses small volume spectrophotometers to track RNA enrichment paired with previously characterized affinity chromatography methods. Borrowing techniques used in solid phase peptide synthesis, we present an approach for immobilizing a wide range of metabolites to an amino PEGA matrix. As an illustration, we detail laboratory techniques employed to generate aptamers that bind p-aminophenylalanine, a metabolic precursor for bio-based production of plastics and the pristinamycin family of antibiotics. We focused on the development of methods for ligand immobilization, selection via affinity chromatography, and nucleic acid quantification that can be performed with common laboratory equipment.

  19. Reagentless, Structure-Switching, Electrochemical Aptamer-Based Sensors

    Science.gov (United States)

    Schoukroun-Barnes, Lauren R.; Macazo, Florika C.; Gutierrez, Brenda; Lottermoser, Justine; Liu, Juan; White, Ryan J.

    2016-06-01

    The development of structure-switching, electrochemical, aptamer-based sensors over the past ˜10 years has led to a variety of reagentless sensors capable of analytical detection in a range of sample matrices. The crux of this methodology is the coupling of target-induced conformation changes of a redox-labeled aptamer with electrochemical detection of the resulting altered charge transfer rate between the redox molecule and electrode surface. Using aptamer recognition expands the highly sensitive detection ability of electrochemistry to a range of previously inaccessible analytes. In this review, we focus on the methods of sensor fabrication and how sensor signaling is affected by fabrication parameters. We then discuss recent studies addressing the fundamentals of sensor signaling as well as quantitative characterization of the analytical performance of electrochemical aptamer-based sensors. Although the limits of detection of reported electrochemical aptamer-based sensors do not often reach that of gold-standard methods such as enzyme-linked immunosorbent assays, the operational convenience of the sensor platform enables exciting analytical applications that we address. Using illustrative examples, we highlight recent advances in the field that impact important areas of analytical chemistry. Finally, we discuss the challenges and prospects for this class of sensors.

  20. Aptamer-Based Technologies in Foodborne Pathogen Detection

    Directory of Open Access Journals (Sweden)

    Jun Teng

    2016-09-01

    Full Text Available Aptamers are single stranded DNA or RNA ligands, which can be selected by a method called systematic evolution of ligands by exponential enrichment (SELEX; and they can specifically recognize and bind to their targets. These unique characteristics of aptamers offer great potentials in applications such as pathogen detection and biomolecular screening. Pathogen detection is the first and critical means in detecting and identifying the problems related to public health and food safety; and only the rapid, sensitive and efficient detection technologies can enable the users to make to accurate assessments on the risk of infections (humans and animals or contaminations (foods and other commodities caused by various pathogens. This article reviews the developments in the field of the aptamer-based approaches for pathogen detection, including whole-cell SELEX and Genomic SELEX. Nowadays, a variety of aptamer-based biosensors have been developed for pathogen detection. Thus, in this review, we also cover the development of aptamer-based biosensors including optical biosensors for multiple pathogen detection in multiple-labeling or label-free models such as fluorescence detection and surface plasmon resonance, electrochemical biosensors, and lateral chromatography test strips, and their applications in the pathogen detection and biomolecular screening. While notable progress has been made in the field in the last decade, challenges or drawbacks in their applications such as pathogen detection and biomolecular screening, remain to be overcome.

  1. Reagentless, Structure-Switching, Electrochemical Aptamer-Based Sensors.

    Science.gov (United States)

    Schoukroun-Barnes, Lauren R; Macazo, Florika C; Gutierrez, Brenda; Lottermoser, Justine; Liu, Juan; White, Ryan J

    2016-06-12

    The development of structure-switching, electrochemical, aptamer-based sensors over the past ∼10 years has led to a variety of reagentless sensors capable of analytical detection in a range of sample matrices. The crux of this methodology is the coupling of target-induced conformation changes of a redox-labeled aptamer with electrochemical detection of the resulting altered charge transfer rate between the redox molecule and electrode surface. Using aptamer recognition expands the highly sensitive detection ability of electrochemistry to a range of previously inaccessible analytes. In this review, we focus on the methods of sensor fabrication and how sensor signaling is affected by fabrication parameters. We then discuss recent studies addressing the fundamentals of sensor signaling as well as quantitative characterization of the analytical performance of electrochemical aptamer-based sensors. Although the limits of detection of reported electrochemical aptamer-based sensors do not often reach that of gold-standard methods such as enzyme-linked immunosorbent assays, the operational convenience of the sensor platform enables exciting analytical applications that we address. Using illustrative examples, we highlight recent advances in the field that impact important areas of analytical chemistry. Finally, we discuss the challenges and prospects for this class of sensors.

  2. DNA aptamers as molecular probes for colorectal cancer study.

    Directory of Open Access Journals (Sweden)

    Kwame Sefah

    Full Text Available BACKGROUND: Understanding the molecular features of specific tumors can increase our knowledge about the mechanism(s underlying disease development and progression. This is particularly significant for colorectal cancer, which is a heterogeneous complex of diseases developed in a sequential manner through a multistep carcinogenic process. As such, it is likely that tumors with similar characteristics might originate in the same manner and have a similar molecular behavior. Therefore, specific mapping of the molecular features can be potentially useful for both tumor classification and the development of appropriate therapeutic regimens. However, this can only be accomplished by developing high-affinity molecular probes with the ability to recognize specific markers associated with different tumors. Aptamers can most easily meet this challenge based on their target diversity, flexible manipulation and ease of development. METHODOLOGY AND RESULTS: Using a method known as cell-based Systematic Evolution of Ligands by Exponential enrichment (cell-SELEX and colorectal cancer cultured cell lines DLD-1 and HCT 116, we selected a panel of target-specific aptamers. Binding studies by flow cytometry and confocal microscopy showed that these aptamers have high affinity and selectivity. Our data further show that these aptamers neither recognize normal colon cells (cultured and fresh, nor do they recognize most other cancer cell lines tested. CONCLUSION/SIGNIFICANCE: The selected aptamers can identify specific biomarkers associated with colorectal cancers. We believe that these probes could be further developed for early disease detection, as well as prognostic markers, of colorectal cancers.

  3. Engineered Aptamers to Probe Molecular Interactions on the Cell Surface.

    Science.gov (United States)

    Batool, Sana; Bhandari, Sanam; George, Shanell; Okeoma, Precious; Van, Nabeela; Zümrüt, Hazan E; Mallikaratchy, Prabodhika

    2017-08-29

    Significant progress has been made in understanding the nature of molecular interactions on the cell membrane. To decipher such interactions, molecular scaffolds can be engineered as a tool to modulate these events as they occur on the cell membrane. To guarantee reliability, scaffolds that function as modulators of cell membrane events must be coupled to a targeting moiety with superior chemical versatility. In this regard, nucleic acid aptamers are a suitable class of targeting moieties. Aptamers are inherently chemical in nature, allowing extensive site-specific chemical modification to engineer sensing molecules. Aptamers can be easily selected using a simple laboratory-based in vitro evolution method enabling the design and development of aptamer-based functional molecular scaffolds against wide range of cell surface molecules. This article reviews the application of aptamers as monitors and modulators of molecular interactions on the mammalian cell surface with the aim of increasing our understanding of cell-surface receptor response to external stimuli. The information gained from these types of studies could eventually prove useful in engineering improved medical diagnostics and therapeutics.

  4. Capture-SELEX: Selection of DNA Aptamers for Aminoglycoside Antibiotics

    Science.gov (United States)

    2012-01-01

    Small organic molecules are challenging targets for an aptamer selection using the SELEX technology (SELEX—Systematic Evolution of Ligans by EXponential enrichment). Often they are not suitable for immobilization on solid surfaces, which is a common procedure in known aptamer selection methods. The Capture-SELEX procedure allows the selection of DNA aptamers for solute targets. A special SELEX library was constructed with the aim to immobilize this library on magnetic beads or other surfaces. For this purpose a docking sequence was incorporated into the random region of the library enabling hybridization to a complementary oligo fixed on magnetic beads. Oligonucleotides of the library which exhibit high affinity to the target and a secondary structure fitting to the target are released from the beads for binding to the target during the aptamer selection process. The oligonucleotides of these binding complexes were amplified, purified, and immobilized via the docking sequence to the magnetic beads as the starting point of the following selection round. Based on this Capture-SELEX procedure, the successful DNA aptamer selection for the aminoglycoside antibiotic kanamycin A as a small molecule target is described. PMID:23326761

  5. Targeting Insulin Receptor with a Novel Internalizing Aptamer

    Directory of Open Access Journals (Sweden)

    Margherita Iaboni

    2016-01-01

    Full Text Available Nucleic acid-based aptamers are emerging as therapeutic antagonists of disease-associated proteins such as receptor tyrosine kinases. They are selected by an in vitro combinatorial chemistry approach, named Systematic Evolution of Ligands by Exponential enrichment (SELEX, and thanks to their small size and unique chemical characteristics, they possess several advantages over antibodies as diagnostics and therapeutics. In addition, aptamers that rapidly internalize into target cells hold as well great potential for their in vivo use as delivery tools of secondary therapeutic agents. Here, we describe a nuclease resistant RNA aptamer, named GL56, which specifically recognizes the insulin receptor (IR. Isolated by a cell-based SELEX method that allows enrichment for internalizing aptamers, GL56 rapidly internalizes into target cells and is able to discriminate IR from the highly homologous insulin-like growth factor receptor 1. Notably, when applied to IR expressing cancer cells, the aptamer inhibits IR dependent signaling. Given the growing interest in the insulin receptor as target for cancer treatment, GL56 reveals a novel molecule with great translational potential as inhibitor and delivery tool for IR-dependent cancers.

  6. Aptamer-Based Technologies in Foodborne Pathogen Detection

    Science.gov (United States)

    Teng, Jun; Yuan, Fang; Ye, Yingwang; Zheng, Lei; Yao, Li; Xue, Feng; Chen, Wei; Li, Baoguang

    2016-01-01

    Aptamers are single stranded DNA or RNA ligands, which can be selected by a method called systematic evolution of ligands by exponential enrichment (SELEX); and they can specifically recognize and bind to their targets. These unique characteristics of aptamers offer great potentials in applications such as pathogen detection and biomolecular screening. Pathogen detection is the critical means in detecting and identifying the problems related to public health and food safety; and only the rapid, sensitive and efficient detection technologies can enable the users to make the accurate assessments on the risks of infections (humans and animals) or contaminations (foods and other commodities) caused by various pathogens. This article reviews the development in the field of the aptamer-based approaches for pathogen detection, including whole-cell SELEX and Genomic SELEX. Nowadays, a variety of aptamer-based biosensors have been developed for pathogen detection. Thus, in this review, we also cover the development in aptamer-based biosensors including optical biosensors for multiple pathogen detection by multiple-labeling or label-free models such as fluorescence detection and surface plasmon resonance, electrochemical biosensors and lateral chromatography test strips, and their applications in pathogen detection and biomolecular screening. While notable progress has been made in the field in the last decade, challenges or drawbacks in their applications such as pathogen detection and biomolecular screening remain to be overcome. PMID:27672383

  7. Development of a Sphingosylphosphorylcholine Detection System Using RNA Aptamers

    Directory of Open Access Journals (Sweden)

    Iwao Waga

    2010-08-01

    Full Text Available Sphingosylphosphorylcholine (SPC is a lysosphingolipid that exerts multiple functions, including acting as a spasmogen, as a mitogenic factor for various types of cells, and sometimes as an inflammatory mediator. Currently, liquid chromatography/tandem mass spectrometry (LC/MS/MS is used for the quantitation of SPC. However, because of the complicated procedures required it may not be cost effective, hampering its regular usage in a routine practical SPC monitoring. In this report, we have generated RNA aptamers that bind to SPC with high affinity using an in vitro selection procedure and developed an enzyme-linked aptamer assay system using the minimized SPC aptamer that can successfully distinguish SPC from the structurally related sphingosine 1-phosphate (S1P. This is the first case of the Systematic Evolution of Ligands by EXponential enrichment (SELEX process being performed with a lysosphingolipid. The SPC aptamers would be valuable tools for the development of aptamer-based medical diagnosis and for elucidating the biological role of SPC.

  8. Aptamers: An in vitro Evolution of Therapeutic and Diagnostic Applications in Medicine

    Directory of Open Access Journals (Sweden)

    Basak KAYHAN

    2013-06-01

    Full Text Available Aptamers are nucleic acid oligomers with distinct conformational shapes that allow binding targets with high affinity and specificity. Selective Evolution of Ligands by Exponential Enrichment (SELEX; an in vitro selection process to develop aptamers, has been invented in 1990. Despite more than 20 years have passed after its discovery, products of SELEX technology are in use in medicine. In this review we discuss why we need aptamers not only in therapeutic but also in diagnostic applications; and also critical points in SELEX technology. Finally; we present the aptamers in use and some patented aptamers awaiting approval. [Dis Mol Med 2013; 1(3.000: 54-60

  9. Aptamer-Based Therapeutics: New Approaches to Combat Human Viral Diseases

    Directory of Open Access Journals (Sweden)

    Ka-To Shum

    2013-11-01

    Full Text Available Viruses replicate inside the cells of an organism and continuously evolve to contend with an ever-changing environment. Many life-threatening diseases, such as AIDS, SARS, hepatitis and some cancers, are caused by viruses. Because viruses have small genome sizes and high mutability, there is currently a lack of and an urgent need for effective treatment for many viral pathogens. One approach that has recently received much attention is aptamer-based therapeutics. Aptamer technology has high target specificity and versatility, i.e., any viral proteins could potentially be targeted. Consequently, new aptamer-based therapeutics have the potential to lead a revolution in the development of anti-infective drugs. Additionally, aptamers can potentially bind any targets and any pathogen that is theoretically amenable to rapid targeting, making aptamers invaluable tools for treating a wide range of diseases. This review will provide a broad, comprehensive overview of viral therapies that use aptamers. The aptamer selection process will be described, followed by an explanation of the potential for treating virus infection by aptamers. Recent progress and prospective use of aptamers against a large variety of human viruses, such as HIV-1, HCV, HBV, SCoV, Rabies virus, HPV, HSV and influenza virus, with particular focus on clinical development of aptamers will also be described. Finally, we will discuss the challenges of advancing antiviral aptamer therapeutics and prospects for future success.

  10. Aptamer-based therapeutics: new approaches to combat human viral diseases.

    Science.gov (United States)

    Shum, Ka-To; Zhou, Jiehua; Rossi, John J

    2013-11-25

    Viruses replicate inside the cells of an organism and continuously evolve to contend with an ever-changing environment. Many life-threatening diseases, such as AIDS, SARS, hepatitis and some cancers, are caused by viruses. Because viruses have small genome sizes and high mutability, there is currently a lack of and an urgent need for effective treatment for many viral pathogens. One approach that has recently received much attention is aptamer-based therapeutics. Aptamer technology has high target specificity and versatility, i.e., any viral proteins could potentially be targeted. Consequently, new aptamer-based therapeutics have the potential to lead a revolution in the development of anti-infective drugs. Additionally, aptamers can potentially bind any targets and any pathogen that is theoretically amenable to rapid targeting, making aptamers invaluable tools for treating a wide range of diseases. This review will provide a broad, comprehensive overview of viral therapies that use aptamers. The aptamer selection process will be described, followed by an explanation of the potential for treating virus infection by aptamers. Recent progress and prospective use of aptamers against a large variety of human viruses, such as HIV-1, HCV, HBV, SCoV, Rabies virus, HPV, HSV and influenza virus, with particular focus on clinical development of aptamers will also be described. Finally, we will discuss the challenges of advancing antiviral aptamer therapeutics and prospects for future success.

  11. In vitro Selection of DNA Aptamers and Fluorescence-Based Recognition for Rapid Detection Listeria monocytogenes

    Institute of Scientific and Technical Information of China (English)

    LIU Guo-qing; LIAN Ying-qi; GAO Chao; YU Xiao-feng; ZHU Ming; ZONG Kai; CHEN Xue-jiao; YAN Yi

    2014-01-01

    Aptamers are speciifc nucleic acid sequences that can bind to a wide range of nucleic acid and non-nucleic acid targets with high afifnity and speciifcity. Nucleic acid aptamers are selected in vitro from single stranded DNA or RNA ligands containing random sequences of up to a few hundred nucleotides. Systematic evolution of ligands by exponential enrichment (SELEX) was used to select and PCR amplify DNA sequences (aptamers) capable of binding to and detecting Listeria monocytogenes, one of the major food-borne pathogens. A simpliifed afifnity separation approach was employed, in which L. monocytogenes in exponential (log) phase of growth was used as the separation target. A lfuorescently-labeled aptamer assay scheme was devised for detecting L. monocytogenes. This report described a novel approach to the detection of L. monocytogenes using DNA aptamers. Aptamers were developed by nine rounds of SELEX. A high afifnity aptamer was successfully selected from the initial random DNA pool, and its secondary structure was also investigated. One of aptamers named e01 with the highest afifnity was further tested in aptamer-peroxidase and aptamer-lfuorescence staining protocols. This study has proved the principle that the whole-cell SELEX could be a promising technique to design aptamer-based molecular probes for dectection of pathogenic microorganisms without tedious isolation and puriifcation of complex markers or targets.

  12. A colorimetric detection method of pesticide acetamiprid by fine-tuning aptamer length.

    Science.gov (United States)

    Tian, Yu; Wang, Yuan; Sheng, Zhi; Li, Tingting; Li, Xu

    2016-11-15

    This work investigates the effect of shortening aptamer sequences on the colorimetric detection of acetamiprid using aptamer-wrapped gold nanoparticles (AuNPs). Truncated 37-mer and 25-mer aptamers were generated by deleting excess flanking nucleotides from parental 49-mer acetamiprid-target aptamer. In comparing the responses of the three sequences, truncated aptamers did not improve the ability to discriminate against other tested pesticides. However, comparison between 49-mer and other shorter aptamers showed that shortening aptamer sequences through removing excess flanking nucleotides outsides of binding region improved colorimetric sensitivity for acetamiprid by 3.3 fold. Due to excess bases, the target-bound aptamer might still adhere to AuNPs, resulting in incomplete dissociation of aptamer from AuNPs and therefore the suppression of aggregation responses. This work provides further insight to the effects of aptamer structure on detection of the target, as well as a method by fine-tuning aptamer length for rapid detection of pesticide residues in environments or food.

  13. Progress and Challenges in Developing Aptamer-Functionalized Targeted Drug Delivery Systems

    Directory of Open Access Journals (Sweden)

    Feng Jiang

    2015-10-01

    Full Text Available Aptamers, which can be screened via systematic evolution of ligands by exponential enrichment (SELEX, are superior ligands for molecular recognition due to their high selectivity and affinity. The interest in the use of aptamers as ligands for targeted drug delivery has been increasing due to their unique advantages. Based on their different compositions and preparation methods, aptamer-functionalized targeted drug delivery systems can be divided into two main categories: aptamer-small molecule conjugated systems and aptamer-nanomaterial conjugated systems. In this review, we not only summarize recent progress in aptamer selection and the application of aptamers in these targeted drug delivery systems but also discuss the advantages, challenges and new perspectives associated with these delivery systems.

  14. Development of an aptamer-based affinity purification method for vascular endothelial growth factor

    Directory of Open Access Journals (Sweden)

    Maren Lönne

    2015-12-01

    Full Text Available Since aptamers bind their targets with high affinity and specificity, they are promising alternative ligands in protein affinity purification. As aptamers are chemically synthesized oligonucleotides, they can be easily produced in large quantities regarding GMP conditions allowing their application in protein production for therapeutic purposes. Several advantages of aptamers compared to antibodies are described in general within this paper. Here, an aptamer directed against the human Vascular Endothelial Growth Factor (VEGF was used as affinity ligand for establishing a purification platform for VEGF in small scale. The aptamer was covalently immobilized on magnetic beads in a controlled orientation resulting in a functional active affinity matrix. Target binding was optimized by introduction of spacer molecules and variation of aptamer density. Further, salt-induced target elution was demonstrated as well as VEGF purification from a complex protein mixture proving the specificity of protein-aptamer binding.

  15. Progress and Challenges in Developing Aptamer-Functionalized Targeted Drug Delivery Systems.

    Science.gov (United States)

    Jiang, Feng; Liu, Biao; Lu, Jun; Li, Fangfei; Li, Defang; Liang, Chao; Dang, Lei; Liu, Jin; He, Bing; Badshah, Shaikh Atik; Lu, Cheng; He, Xiaojuan; Guo, Baosheng; Zhang, Xiao-Bing; Tan, Weihong; Lu, Aiping; Zhang, Ge

    2015-01-01

    Aptamers, which can be screened via systematic evolution of ligands by exponential enrichment (SELEX), are superior ligands for molecular recognition due to their high selectivity and affinity. The interest in the use of aptamers as ligands for targeted drug delivery has been increasing due to their unique advantages. Based on their different compositions and preparation methods, aptamer-functionalized targeted drug delivery systems can be divided into two main categories: aptamer-small molecule conjugated systems and aptamer-nanomaterial conjugated systems. In this review, we not only summarize recent progress in aptamer selection and the application of aptamers in these targeted drug delivery systems but also discuss the advantages, challenges and new perspectives associated with these delivery systems.

  16. Affinity hydrogels for controlled protein release using nucleic acid aptamers and complementary oligonucleotides.

    Science.gov (United States)

    Soontornworajit, Boonchoy; Zhou, Jing; Snipes, Matthew P; Battig, Mark R; Wang, Yong

    2011-10-01

    Biomaterials for the precise control of protein release are important to the development of new strategies for treating human diseases. This study aimed to fundamentally understand aptamer--protein dissociation triggered by complementary oligonucleotides, and to apply this understanding to develop affinity hydrogels for controlled protein release. The results showed that the oligonucleotide tails of the aptamers played a critical role in inducing intermolecular hybridization and triggering aptamer--protein dissociation. In addition, the attachment of the oligonucleotide tails to the aptamers and the increase of hybridizing length could produce a synergistic effect on the dissociation of bound proteins from their aptamers. More importantly, pegylated complementary oligonucleotides could successfully trigger protein release from the aptamer-functionalized hydrogels at multiple time points. Based on these results, it is believed that aptamer-functionalized hydrogels and complementary oligonucleotides hold great potential of controlling the release of protein drugs to treat human diseases.

  17. Probing the coagulation pathway with aptamers identifies combinations that synergistically inhibit blood clot formation.

    Science.gov (United States)

    Bompiani, Kristin M; Lohrmann, Jens L; Pitoc, George A; Frederiksen, James W; Mackensen, George B; Sullenger, Bruce A

    2014-08-14

    Coordinated enzymatic reactions regulate blood clot generation. To explore the contributions of various coagulation enzymes in this process, we utilized a panel of aptamers against factors VIIa, IXa, Xa, and prothrombin. Each aptamer dose-dependently inhibited clot formation, yet none was able to completely impede this process in highly procoagulant settings. However, several combinations of two aptamers synergistically impaired clot formation. One extremely potent aptamer combination was able to maintain human blood fluidity even during extracorporeal circulation, a highly procoagulant setting encountered during cardiopulmonary bypass surgery. Moreover, this aptamer cocktail could be rapidly reversed with antidotes to restore normal hemostasis, indicating that even highly potent aptamer combinations can be rapidly controlled. These studies highlight the potential utility of using sets of aptamers to probe the functions of proteins in molecular pathways for research and therapeutic ends. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Improved Aptamers for the Diagnosis and Potential Treatment of HER2-Positive Cancer

    Directory of Open Access Journals (Sweden)

    Marlies Gijs

    2016-05-01

    Full Text Available Aptamers provide a potential source of alternative targeting molecules for existing antibody diagnostics and therapeutics. In this work, we selected novel DNA aptamers targeting the HER2 receptor by an adherent whole-cell SELEX approach. Individual aptamers were identified by next generation sequencing and bioinformatics analysis. Two aptamers, HeA2_1 and HeA2_3, were shown to bind the HER2 protein with affinities in the nanomolar range. In addition, both aptamers were able to bind with high specificity to HER2-overexpressing cells and HER2-positive tumor tissue samples. Furthermore, we demonstrated that aptamer HeA2_3 is being internalized into cancer cells and has an inhibitory effect on cancer cell growth and viability. In the end, we selected novel DNA aptamers with great potential for the diagnosis and possible treatment of HER2-positive cancer.

  19. Kissing loop interaction in adenine riboswitch: insights from umbrella sampling simulations.

    Science.gov (United States)

    Di Palma, Francesco; Bottaro, Sandro; Bussi, Giovanni

    2015-01-01

    Riboswitches are cis-acting regulatory RNA elements prevalently located in the leader sequences of bacterial mRNA. An adenine sensing riboswitch cis-regulates adeninosine deaminase gene (add) in Vibrio vulnificus. The structural mechanism regulating its conformational changes upon ligand binding mostly remains to be elucidated. In this open framework it has been suggested that the ligand stabilizes the interaction of the distal "kissing loop" complex. Using accurate full-atom molecular dynamics with explicit solvent in combination with enhanced sampling techniques and advanced analysis methods it could be possible to provide a more detailed perspective on the formation of these tertiary contacts. In this work, we used umbrella sampling simulations to study the thermodynamics of the kissing loop complex in the presence and in the absence of the cognate ligand. We enforced the breaking/formation of the loop-loop interaction restraining the distance between the two loops. We also assessed the convergence of the results by using two alternative initialization protocols. A structural analysis was performed using a novel approach to analyze base contacts. Contacts between the two loops were progressively lost when larger inter-loop distances were enforced. Inter-loop Watson-Crick contacts survived at larger separation when compared with non-canonical pairing and stacking interactions. Intra-loop stacking contacts remained formed upon loop undocking. Our simulations qualitatively indicated that the ligand could stabilize the kissing loop complex. We also compared with previously published simulation studies. Kissing complex stabilization given by the ligand was compatible with available experimental data. However, the dependence of its value on the initialization protocol of the umbrella sampling simulations posed some questions on the quantitative interpretation of the results and called for better converged enhanced sampling simulations.

  20. Massively Parallel Interrogation of Aptamer Sequence, Structure and Function

    Energy Technology Data Exchange (ETDEWEB)

    Fischer, N O; Tok, J B; Tarasow, T M

    2008-02-08

    Optimization of high affinity reagents is a significant bottleneck in medicine and the life sciences. The ability to synthetically create thousands of permutations of a lead high-affinity reagent and survey the properties of individual permutations in parallel could potentially relieve this bottleneck. Aptamers are single stranded oligonucleotides affinity reagents isolated by in vitro selection processes and as a class have been shown to bind a wide variety of target molecules. Methodology/Principal Findings. High density DNA microarray technology was used to synthesize, in situ, arrays of approximately 3,900 aptamer sequence permutations in triplicate. These sequences were interrogated on-chip for their ability to bind the fluorescently-labeled cognate target, immunoglobulin E, resulting in the parallel execution of thousands of experiments. Fluorescence intensity at each array feature was well resolved and shown to be a function of the sequence present. The data demonstrated high intra- and interchip correlation between the same features as well as among the sequence triplicates within a single array. Consistent with aptamer mediated IgE binding, fluorescence intensity correlated strongly with specific aptamer sequences and the concentration of IgE applied to the array. The massively parallel sequence-function analyses provided by this approach confirmed the importance of a consensus sequence found in all 21 of the original IgE aptamer sequences and support a common stem:loop structure as being the secondary structure underlying IgE binding. The microarray application, data and results presented illustrate an efficient, high information content approach to optimizing aptamer function. It also provides a foundation from which to better understand and manipulate this important class of high affinity biomolecules.

  1. Selective Targeting to Glioma with Nucleic Acid Aptamers.

    Directory of Open Access Journals (Sweden)

    Shraddha Aptekar

    Full Text Available Malignant glioma is characterised by a rapid growth rate and high capacity for invasive infiltration to surrounding brain tissue; hence, diagnosis and treatment is difficult and patient survival is poor. Aptamers contribute a promising and unique technology for the in vitro imaging of live cells and tissues, with a potentially bright future in clinical diagnostics and therapeutics for malignant glioma. The binding selectivity, uptake capacity and binding target of two DNA aptamers, SA43 and SA44, were investigated in glioma cells and patient tissues. The binding assay showed that SA43 and SA44 bound with strong affinity (Kd, 21.56 ± 4.60 nM and Kd, 21.11 ± 3.30 nM respectively to the target U87MG cells. Quantitative analysis by flow cytometry showed that the aptamers were able to actively internalise in U87MG and 1321N1 glioma cells compared to the non-cancerous and non-glioma cell types. Confocal microscopy confirmed staining in the cytoplasm, and co-localisation studies with endoplasmic reticulum, Golgi apparatus and lysosomal markers suggested internalisation and compartmentalisation within the endomembrane system. Both aptamers selectively bound to Ku 70 and Ku 80 DNA repair proteins as determined by aptoprecipitation (AP followed by mass spectrometry analysis and confirmation by Western blot. In addition, aptohistochemical (AHC staining on paraffin embedded, formalin fixed patient tissues revealed that the binding selectivity was significantly higher for SA43 aptamer in glioma tissues (grade I, II, III and IV compared to the non-cancerous tissues, whereas SA44 did not show selectivity towards glioma tissues. The results indicate that SA43 aptamer can differentiate between glioma and non-cancerous cells and tissues and therefore, shows promise for histological diagnosis of glioma.

  2. Massively parallel interrogation of aptamer sequence, structure and function.

    Directory of Open Access Journals (Sweden)

    Nicholas O Fischer

    Full Text Available BACKGROUND: Optimization of high affinity reagents is a significant bottleneck in medicine and the life sciences. The ability to synthetically create thousands of permutations of a lead high-affinity reagent and survey the properties of individual permutations in parallel could potentially relieve this bottleneck. Aptamers are single stranded oligonucleotides affinity reagents isolated by in vitro selection processes and as a class have been shown to bind a wide variety of target molecules. METHODOLOGY/PRINCIPAL FINDINGS: High density DNA microarray technology was used to synthesize, in situ, arrays of approximately 3,900 aptamer sequence permutations in triplicate. These sequences were interrogated on-chip for their ability to bind the fluorescently-labeled cognate target, immunoglobulin E, resulting in the parallel execution of thousands of experiments. Fluorescence intensity at each array feature was well resolved and shown to be a function of the sequence present. The data demonstrated high intra- and inter-chip correlation between the same features as well as among the sequence triplicates within a single array. Consistent with aptamer mediated IgE binding, fluorescence intensity correlated strongly with specific aptamer sequences and the concentration of IgE applied to the array. CONCLUSION AND SIGNIFICANCE: The massively parallel sequence-function analyses provided by this approach confirmed the importance of a consensus sequence found in all 21 of the original IgE aptamer sequences and support a common stem:loop structure as being the secondary structure underlying IgE binding. The microarray application, data and results presented illustrate an efficient, high information content approach to optimizing aptamer function. It also provides a foundation from which to better understand and manipulate this important class of high affinity biomolecules.

  3. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Deng-Liang [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China); Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan [State Key Laboratory for Physical Chemistry of Solid Surfaces, Key Laboratory for Chemical Biology of Fujian Province, Key Laboratory of Analytical Chemistry, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China); Yang, Hai-Tao; Wang, Jiang-Jie [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Yao, Pei-Sen [Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China); Pan, Ru-Jun [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Yang, Chaoyong James, E-mail: cyyang@xmu.edu.cn [State Key Laboratory for Physical Chemistry of Solid Surfaces, Key Laboratory for Chemical Biology of Fujian Province, Key Laboratory of Analytical Chemistry, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China); Kang, De-Zhi, E-mail: kdzy99988@163.com [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China)

    2014-10-31

    Highlights: • This is the first report of DNA aptamer against EGFR in vitro. • Aptamer can bind targets with high affinity and selectivity. • DNA aptamers are more stable, cheap and efficient than RNA aptamers. • Our selected DNA aptamer against EGFR has high affinity with K{sub d} 56 ± 7.3 nM. • Our selected DNA aptamer against EGFR has high selectivity. - Abstract: Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K{sub d} 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy.

  4. Aptamer conjugated magnetic nanoparticles as nanosurgeons

    Science.gov (United States)

    Nair, Baiju G.; Nagaoka, Yutaka; Morimoto, Hisao; Yoshida, Yasuhiko; Maekawa, Toru; Sakthi Kumar, D.

    2010-11-01

    Magnetic nanoparticles have shown promise in the fields of targeted drug delivery, hyperthermia and magnetic resonance imaging (MRI) in cancer therapy. The ability of magnetic nanoparticles to undergo surface modification and the effect of external magnetic field in the dynamics of their movement make them an excellent nanoplatform for cancer destruction. Surgical removal of cancerous or unwanted cells selectively from the interior of an organ or tissue without any collateral damage is a serious problem due to the highly infiltrative nature of cancer. To address this problem in surgery, we have developed a nanosurgeon for the selective removal of target cells using aptamer conjugated magnetic nanoparticles controlled by an externally applied three-dimensional rotational magnetic field. With the help of the nanosurgeon, we were able to perform surgical actions on target cells in in vitro studies. LDH and intracellular calcium release assay confirmed the death of cancer cells due to the action of the nanosurgeon which in turn nullifies the possibility of proliferation by the removed cells. The nanosurgeon will be a useful tool in the medical field for selective surgery and cell manipulation studies. Additionally, this system could be upgraded for the selective removal of complex cancers from diverse tissues by incorporating various target specific ligands on magnetic nanoparticles.

  5. A Tyrosine-Dependent Riboswitch Controls the Expression of a Tyrosyl-tRNA Synthetase from Acidithiobacillus ferrooxidans

    Directory of Open Access Journals (Sweden)

    Paula Bustamante

    2016-06-01

    Full Text Available Expression of aminoacyl-tRNA synthetases is regulated by a variety of mechanisms at the level of transcription or translation. A T-box dependent transcription termination / antitermination riboswitch system that responds to charged / uncharged tRNA regulates expression of aminoacyl tRNA synthetase genes in Gram-positive bacteria. TyrZ, the gene encoding tyrosyl-tRNA synthetase from Acidithiobacillus ferrooxidans, a Gram-negative acidophilic bacterium that participates in bioleaching of minerals, resembles the gene from Bacillus subtilis including the 5´-untranslated region encoding the riboswitch. Transcription of A. ferrooxidans tyrZ is induced by the presence of tyrosine by a mechanism involving antitermination of transcription. This mechanism is probably adapted to the low supply of amino acids of acidic environments of autotrophic bioleaching microorganisms. This work is licensed under a Creative Commons Attribution 4.0 International License.

  6. Bioactivity of 2′-deoxyinosine-incorporated aptamer AS1411

    OpenAIRE

    Xinmeng Fan; Lidan Sun; Yun Wu; Lihe Zhang; Zhenjun Yang

    2016-01-01

    Aptamers can be chemically modified to enhance nuclease resistance and increase target affinity. In this study, we performed chemical modification of 2′-deoxyinosine in AS1411, an anti-proliferative G-rich oligodeoxynucleotide aptamer, which binds selectively to the nucleolin protein. Its function was augmented when 2′-deoxyinosine was incorporated at positions 12, 13, 15, and 24 of AS1411, respectively. In addition, double incorporation of 2′-deoxyinosine at positions 12 and 24 (FAN-1224dI),...

  7. Aptamer-Binding Directed DNA Origami Pattern for Logic Gates.

    Science.gov (United States)

    Yang, Jing; Jiang, Shuoxing; Liu, Xiangrong; Pan, Linqiang; Zhang, Cheng

    2016-12-14

    In this study, an aptamer-substrate strategy is introduced to control programmable DNA origami pattern. Combined with DNA aptamer-substrate binding and DNAzyme-cutting, small DNA tiles were specifically controlled to fill into the predesigned DNA origami frame. Here, a set of DNA logic gates (OR, YES, and AND) are performed in response to the stimuli of adenosine triphosphate (ATP) and cocaine. The experimental results are confirmed by AFM imaging and time-dependent fluorescence changes, demonstrating that the geometric patterns are regulated in a controllable and programmable manner. Our approach provides a new platform for engineering programmable origami nanopatterns and constructing complex DNA nanodevices.

  8. A Highly Coupled Network of Tertiary Interactions in the SAM-I Riboswitch and Their Role in Regulatory Tuning.

    Science.gov (United States)

    Wostenberg, Christopher; Ceres, Pablo; Polaski, Jacob T; Batey, Robert T

    2015-11-06

    RNA folding in vivo is significantly influenced by transcription, which is not necessarily recapitulated by Mg(2+)-induced folding of the corresponding full-length RNA in vitro. Riboswitches that regulate gene expression at the transcriptional level are an ideal system for investigating this aspect of RNA folding as ligand-dependent termination is obligatorily co-transcriptional, providing a clear readout of the folding outcome. The folding of representative members of the SAM-I family of riboswitches has been extensively analyzed using approaches focusing almost exclusively upon Mg(2+) and/or S-adenosylmethionine (SAM)-induced folding of full-length transcripts of the ligand binding domain. To relate these findings to co-transcriptional regulatory activity, we have investigated a set of structure-guided mutations of conserved tertiary architectural elements of the ligand binding domain using an in vitro single-turnover transcriptional termination assay, complemented with phylogenetic analysis and isothermal titration calorimetry data. This analysis revealed a conserved internal loop adjacent to the SAM binding site that significantly affects ligand binding and regulatory activity. Conversely, most single point mutations throughout key conserved features in peripheral tertiary architecture supporting the SAM binding pocket have relatively little impact on riboswitch activity. Instead, a secondary structural element in the peripheral subdomain appears to be the key determinant in observed differences in regulatory properties across the SAM-I family. These data reveal a highly coupled network of tertiary interactions that promote high-fidelity co-transcriptional folding of the riboswitch but are only indirectly linked to regulatory tuning.

  9. Structural and Dynamic Basis for Low-Affinity, High-Selectivity Binding of L-Glutamine by the Glutamine Riboswitch

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    Aiming Ren

    2015-12-01

    Full Text Available Naturally occurring L-glutamine riboswitches occur in cyanobacteria and marine metagenomes, where they reside upstream of genes involved in nitrogen metabolism. By combining X-ray, NMR, and MD, we characterized an L-glutamine-dependent conformational transition in the Synechococcus elongatus glutamine riboswitch from tuning fork to L-shaped alignment of stem segments. This transition generates an open ligand-binding pocket with L-glutamine selectivity enforced by Mg2+-mediated intermolecular interactions. The transition also stabilizes the P1 helix through a long-range “linchpin” Watson-Crick G-C pair-capping interaction, while melting a short helix below P1 potentially capable of modulating downstream readout. NMR data establish that the ligand-free glutamine riboswitch in Mg2+ solution exists in a slow equilibrium between flexible tuning fork and a minor conformation, similar, but not identical, to the L-shaped bound conformation. We propose that an open ligand-binding pocket combined with a high conformational penalty for forming the ligand-bound state provide mechanisms for reducing binding affinity while retaining high selectivity.

  10. Structural and Dynamic Basis for Low-Affinity, High-Selectivity Binding of L-Glutamine by the Glutamine Riboswitch.

    Science.gov (United States)

    Ren, Aiming; Xue, Yi; Peselis, Alla; Serganov, Alexander; Al-Hashimi, Hashim M; Patel, Dinshaw J

    2015-12-01

    Naturally occurring L-glutamine riboswitches occur in cyanobacteria and marine metagenomes, where they reside upstream of genes involved in nitrogen metabolism. By combining X-ray, NMR, and MD, we characterized an L-glutamine-dependent conformational transition in the Synechococcus elongatus glutamine riboswitch from tuning fork to L-shaped alignment of stem segments. This transition generates an open ligand-binding pocket with L-glutamine selectivity enforced by Mg(2+)-mediated intermolecular interactions. The transition also stabilizes the P1 helix through a long-range "linchpin" Watson-Crick G-C pair-capping interaction, while melting a short helix below P1 potentially capable of modulating downstream readout. NMR data establish that the ligand-free glutamine riboswitch in Mg(2+) solution exists in a slow equilibrium between flexible tuning fork and a minor conformation, similar, but not identical, to the L-shaped bound conformation. We propose that an open ligand-binding pocket combined with a high conformational penalty for forming the ligand-bound state provide mechanisms for reducing binding affinity while retaining high selectivity.

  11. RNA aptamers inhibit the growth of the fish pathogen viral hemorrhagic septicemia virus (VHSV).

    Science.gov (United States)

    Punnarak, Porntep; Santos, Mudjekeewis D; Hwang, Seong Don; Kondo, Hidehiro; Hirono, Ikuo; Kikuchi, Yo; Aoki, Takashi

    2012-12-01

    Viral hemorrhagic septicemia virus (VHSV) is a serious disease impacting wild and cultured fish worldwide. Hence, an effective therapeutic method against VHSV infection needs to be developed. Aptamer technology is a new and promising method for diagnostics and therapeutics. It revolves around the use of an aptamer molecule, an artificial ligand (nucleic acid or protein), which has the capacity to recognize target molecules with high affinity and specificity. Here, we aimed at selecting RNA aptamers that can specifically bind to and inhibit the growth of a strain of fish VHSV both in vitro and in vivo. Three VHSV-specific RNA aptamers (F1, F2, and C6) were selected from a pool of artificially and randomly produced oligonucleotides using systematic evolution of ligands by exponential enrichment. The three RNA aptamers showed obvious binding to VHSV in an electrophoretic mobility shift assay but not to other tested viruses. The RNA aptamers were tested for their ability to inhibit VHSV in vitro using hirame natural embryo (HINAE) cells. Cytopathic effect and plaque assays showed that all aptamers inhibited the growth of VHSV in HINAE cells. In vivo tests using RNA aptamers produced by Rhodovulum sulfidophilum showed that extracellular RNA aptamers inhibited VHSV infection in Japanese flounder. These results suggest that the RNA aptamers are a useful tool for protection against VHSV infection in Japanese flounder.

  12. Modified AS1411 Aptamer Suppresses Hepatocellular Carcinoma by Up-Regulating Galectin-14.

    Science.gov (United States)

    Cho, Yuri; Lee, Yun Bin; Lee, Jeong-Hoon; Lee, Dong Hyeon; Cho, Eun Ju; Yu, Su Jong; Kim, Yoon Jun; Kim, Jong In; Im, Jong Hun; Lee, Jung Hwan; Oh, Eun Ju; Yoon, Jung-Hwan

    2016-01-01

    Aptamers are small synthetic oligonucleotides that bind to target proteins with high specificity and affinity. AS1411 is an aptamer that binds to nucleolin, which is overexpressed in the cytoplasm and occurs on the surface of cancer cells. We investigated the therapeutic potential of aptamers in hepatocellular carcinoma (HCC) by evaluating anti-tumor effects and confirming the affinity and specificity of AS1411- and modified AS1411-aptamers in HCC cells. Cell growth was assessed using the MTS assay, and cell death signaling was explored by immunoblot analysis. Fluorescence-activated cell sorting was performed to evaluate the affinity and specificity of AS1411-aptamers in SNU-761 HCC cells. We investigated the in vivo effects of the AS1411-aptamer using BALB/c nude mice in a subcutaneous xenograft model with SNU-761 cells. Treatment with a modified AS1411-aptamer significantly decreased in vitro (under normoxic [P = 0.035] and hypoxic [P = 0.018] conditions) and in vivo (under normoxic conditions, P = 0.041) HCC cell proliferation compared to control aptamers. AS1411- and control aptamers failed to control HCC cell proliferation. However, AS1411- and the modified AS1411-aptamer did not induce caspase activation. Decrease in cell growth by AS1411 or modified AS1411 was not prevented by caspase or necrosis inhibitors. In a microarray, AS1411 significantly enhanced galectin-14 expression. Suppression of HCC cell proliferation by the modified AS1411-aptamer was attenuated by galectin-14 siRNA transfection. Modified AS1411-aptamer suppressed HCC cell growth in vitro and in vivo by up-regulating galectin-14 expressions. Modified AS1411-aptamers may have therapeutic potential as a novel targeted therapy for HCC.

  13. Approach to the unfolding and folding dynamics of add A-riboswitch upon adenine dissociation using a coarse-grained elastic network model

    Science.gov (United States)

    Li, Chunhua; Lv, Dashuai; Zhang, Lei; Yang, Feng; Wang, Cunxin; Su, Jiguo; Zhang, Yang

    2016-07-01

    Riboswitches are noncoding mRNA segments that can regulate the gene expression via altering their structures in response to specific metabolite binding. We proposed a coarse-grained Gaussian network model (GNM) to examine the unfolding and folding dynamics of adenosine deaminase (add) A-riboswitch upon the adenine dissociation, in which the RNA is modeled by a nucleotide chain with interaction networks formed by connecting adjoining atomic contacts. It was shown that the adenine binding is critical to the folding of the add A-riboswitch while the removal of the ligand can result in drastic increase of the thermodynamic fluctuations especially in the junction regions between helix domains. Under the assumption that the native contacts with the highest thermodynamic fluctuations break first, the iterative GNM simulations showed that the unfolding process of the adenine-free add A-riboswitch starts with the denature of the terminal helix stem, followed by the loops and junctions involving ligand binding pocket, and then the central helix domains. Despite the simplified coarse-grained modeling, the unfolding dynamics and pathways are shown in close agreement with the results from atomic-level MD simulations and the NMR and single-molecule force spectroscopy experiments. Overall, the study demonstrates a new avenue to investigate the binding and folding dynamics of add A-riboswitch molecule which can be readily extended for other RNA molecules.

  14. Orchestration of thiamin biosynthesis and central metabolism by combined action of the thiamin pyrophosphate riboswitch and the circadian clock in Arabidopsis.

    Science.gov (United States)

    Bocobza, Samuel E; Malitsky, Sergey; Araújo, Wagner L; Nunes-Nesi, Adriano; Meir, Sagit; Shapira, Michal; Fernie, Alisdair R; Aharoni, Asaph

    2013-01-01

    Riboswitches are natural RNA elements that posttranscriptionally regulate gene expression by binding small molecules and thereby autonomously control intracellular levels of these metabolites. Although riboswitch-based mechanisms have been examined extensively, the integration of their activity with global physiology and metabolism has been largely overlooked. Here, we explored the regulation of thiamin biosynthesis and the consequences of thiamin pyrophosphate riboswitch deficiency on metabolism in Arabidopsis thaliana. Our results show that thiamin biosynthesis is largely regulated by the circadian clock via the activity of the THIAMIN C SYNTHASE (THIC) promoter, while the riboswitch located at the 3' untranslated region of this gene controls overall thiamin biosynthesis. Surprisingly, the results also indicate that the rate of thiamin biosynthesis directs the activity of thiamin-requiring enzymes and consecutively determines the rate of carbohydrate oxidation via the tricarboxylic acid cycle and pentose-phosphate pathway. Our model suggests that in Arabidopsis, the THIC promoter and the thiamin-pyrophosphate riboswitch act simultaneously to tightly regulate thiamin biosynthesis in a circadian manner and consequently sense and control vital points of core cellular metabolism.

  15. Electrochemical biosensors and logic devices based on aptamers

    Institute of Scientific and Technical Information of China (English)

    Zuo Xiaolei; Lin Meihua; Fan Chunhai

    2013-01-01

    Aptamers are molecular recognition elements with high specificity that are selected from deoxyribonucleic acid/ribonucleic acid (DNA/RNA) library.Compared with the traditional protein recognition elements,aptamers have excellent properties such as cost-effective,stable,easy for synthesis and modification.In recent years,electrochemistry plays an important role in biosensor field because of its high sensitivity,high stability,fast response and easy miniaturization.Through the combination of these two technologies and our rational design,we constructed a series of biosensors and biochips that are simple,fast,cheap and miniaturized.Firstly,we designed an adenosine triphosphate (ATP) electrochemical biosensor based on the strand displacement strategy.We can detect as low as 10 nmol/L of ATP both in pure solution and complicated cell lysates.Secondly,we creatively split the aptamers into two fragments and constructed the sandwich assay platform only based on single aptamer sequence.We successfully transferred this design on biochips with multiple micro electrodes (6×6) and accomplished multiplex detection.In the fields of biochips and biocomputers,we designed several DNA logic gates with electric (electrochemical) signal as output which paves a new way for the development of DNA computer.

  16. Immobilized aptamer paper spray ionization source for ion mobility spectrometry.

    Science.gov (United States)

    Zargar, Tahereh; Khayamian, Taghi; Jafari, Mohammad T

    2017-01-05

    A selective thin-film microextraction based on aptamer immobilized on cellulose paper was used as a paper spray ionization source for ion mobility spectrometry (PSI-IMS), for the first time. In this method, the paper is not only used as an ionization source but also it is utilized for the selective extraction of analyte, based on immobilized aptamer. This combination integrates both sample preparation and analyte ionization in a Whatman paper. To that end, an appropriate sample introduction system with a novel design was constructed for the paper spray ionization source. Using this system, a continuous solvent flow works as an elution and spray solvent simultaneously. In this method, analyte is adsorbed on a triangular paper with immobilized aptamer and then it is desorbed and ionized by elution solvent and applied high voltage on paper, respectively. The effects of different experimental parameters such as applied voltage, angle of paper tip, distance between paper tip and counter electrode, elution solvent type, and solvent flow rate were optimized. The proposed method was exhaustively validated in terms of sensitivity and reproducibility by analyzing the standard solutions of codeine and acetamiprid. The analytical results obtained are promising enough to ensure the use of immobilized aptamer paper-spray as both the extraction and ionization techniques in IMS for direct analysis of biomedicine. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Regulation of photosensitisation processes by an RNA aptamer

    Science.gov (United States)

    Thoa, Tran Thi Thanh; Minagawa, Noriko; Aigaki, Toshiro; Ito, Yoshihiro; Uzawa, Takanori

    2017-02-01

    One of the most powerful attributes of proteins is their ability to bind to and modulate the chemistry of cofactors and prosthetic groups. Here, we demonstrated the ability of an artificial nucleic acid (an aptamer) to similarly control the functionality of a non-biological element. Specifically, we selected an RNA aptamer that binds tris(bipyridine) ruthenium (II), Ru(bpy)32+, an inorganic complex that has attracted intense interest due to its photoredox chemistry, including its ability to split water by visible light. We found that a newly discovered aptamer strongly and enantioselectively binds Λ-Ru(bpy)32+ (Kd = 65 nM) and, in doing so, selectively suppresses deactivation via energy transfer, thereby elongating the lifetime of its photo-excited state by four-fold. The ability of the aptamer to enhance this important aspect of Ru(bpy)32+ chemistry illustrates a broader point concerning the potential power of combining in vitro-created biomolecules with non-biological reactants to perform enhanced chemical reactions.

  18. Single-Round Patterned DNA Library Microarray Aptamer Lead Identification

    Directory of Open Access Journals (Sweden)

    Jennifer A. Martin

    2015-01-01

    Full Text Available A method for identifying an aptamer in a single round was developed using custom DNA microarrays containing computationally derived patterned libraries incorporating no information on the sequences of previously reported thrombin binding aptamers. The DNA library was specifically designed to increase the probability of binding by enhancing structural complexity in a sequence-space confined environment, much like generating lead compounds in a combinatorial drug screening library. The sequence demonstrating the highest fluorescence intensity upon target addition was confirmed to bind the target molecule thrombin with specificity by surface plasmon resonance, and a novel imino proton NMR/2D NOESY combination was used to screen the structure for G-quartet formation. We propose that the lack of G-quartet structure in microarray-derived aptamers may highlight differences in binding mechanisms between surface-immobilized and solution based strategies. This proof-of-principle study highlights the use of a computational driven methodology to create a DNA library rather than a SELEX based approach. This work is beneficial to the biosensor field where aptamers selected by solution based evolution have proven challenging to retain binding function when immobilized on a surface.

  19. Aptamer-based Field-Effect Biosensor for Tenofovir Detection

    Science.gov (United States)

    Aliakbarinodehi, N.; Jolly, P.; Bhalla, N.; Miodek, A.; De Micheli, G.; Estrela, P.; Carrara, S.

    2017-01-01

    During medical treatment it is critical to maintain the circulatory concentration of drugs within their therapeutic range. A novel biosensor is presented in this work to address the lack of a reliable point-of-care drug monitoring system in the market. The biosensor incorporates high selectivity and sensitivity by integrating aptamers as the recognition element and field-effect transistors as the signal transducer. The drug tenofovir was used as a model small molecule. The biointerface of the sensor is a binary self-assembled monolayer of specific thiolated aptamer and 6-mercapto-1-hexanol (MCH), whose ratio was optimized by electrochemical impedance spectroscopy measurements to enhance the sensitivity towards the specific target. Surface plasmon resonance, performed under different buffer conditions, shows optimum specific and little non-specific binding in phosphate buffered saline. The dose-response behavior of the field-effect biosensor presents a linear range between 1 nM and 100 nM of tenofovir and a limit of detection of 1.2 nM. Two non-specific drugs and one non-specific aptamer, tested as stringent control candidates, caused negligible responses. The applications were successfully extended to the detection of the drug in human serum. As demonstrated by impedance measurements, the aptamer-based sensors can be used for real-time drug monitoring. PMID:28294122

  20. Long Shelf Life of a Lyophilized DNA Aptamer Beacon Assay.

    Science.gov (United States)

    Bruno, John G

    2017-03-01

    An aptamer beacon previously developed to detect C-telopeptide (CTx) from human bone collagen breakdown was lyophilized and shown to give a "lights on" concentration-dependent spectral fluorescence response essentially identical to that of the fresh reagent despite storage in a dark dry environment for the past 5.5 years.

  1. Aptamer conjugated silver nanoparticles for the detection of interleukin 6

    Science.gov (United States)

    Locke, Andrea K.; Norwood, Nicole; Marks, Haley L.; Schechinger, Monika; Jackson, George W.; Graham, Duncan; Coté, Gerard L.

    2016-03-01

    The controlled assembly of plasmonic nanoparticles by a molecular binding event has emerged as a simple yet sensitive methodology for protein detection. Metallic nanoparticles (NPs) coated with functionalized aptamers can be utilized as biosensors by monitoring changes in particle optical properties, such as the LSPR shift and enhancement of the SERS spectra, in the presence of a target protein. Herein we test this method using two modified aptamers selected for the protein biomarker interleukin 6, an indicator of the dengue fever virus and other diseases including certain types of cancers, diabetes, and even arthritis. IL6 works by inducing an immunological response within the body that can be either anti-inflammatory or pro-inflammatory. The results show that the average hydrodynamic diameter of the NPs as measured by Dynamic Light Scattering was ~42 nm. After conjugation of the aptamers, the peak absorbance of the AgNPs shifted from 404 to 408 nm indicating a surface modification of the NPs due to the presence of the aptamer. Lastly, preliminary results were obtained showing an increase in SERS intensity occurs when the IL-6 protein was introduced to the conjugate solution but the assay will still need to be optimized in order for it to be able to monitor varying concentration changes within and across the desired range.

  2. Development of aptamer based HIV-1 entry inhibitor prophylactic drugs

    CSIR Research Space (South Africa)

    London, G

    2013-08-01

    Full Text Available AIDS remains a major public health problem globally, especially in Southern Africa where over 6.4 million people are infected by the most prevalent HIV-1 subtype C. To help stop the spread of HIV-1 subtype C, we isolated 2ʹ-F-RNA aptamers against gp...

  3. Analytical bioconjugates, aptamers, enable specific quantitative detection of Listeria monocytogenes.

    Science.gov (United States)

    Lee, Sang-Hee; Ahn, Ji-Young; Lee, Kyeong-Ah; Um, Hyun-Ju; Sekhon, Simranjeet Singh; Sun Park, Tae; Min, Jiho; Kim, Yang-Hoon

    2015-06-15

    As a major human pathogen in the Listeria genus, Listeria monocytogenes causes the bacterial disease listeriosis, which is a serious infection caused by eating food contaminated with the bacteria. We have developed an aptamer-based sandwich assay (ABSA) platform that demonstrates a promising potential for use in pathogen detection using aptamers as analytical bioconjugates. The whole-bacteria SELEX (WB-SELEX) strategy was adopted to generate aptamers with high affinity and specificity against live L. monocytogenes. Of the 35 aptamer candidates tested, LMCA2 and LMCA26 reacted to L. monocytogenes with high binding, and were consequently chosen as sensing probes. The ABSA platform can significantly enhance the sensitivity by employing a very specific aptamer pair for the sandwich complex. The ABSA platform exhibited a linear response over a wide concentration range of L. monocytogenes from 20 to 2×10(6) CFU per mL and was closely correlated with the following relationship: y=9533.3x+1542.3 (R(2)=0.99). Our proposed ABSA platform also provided excellent specificity for the tests to distinguish L. monocytogenes from other Listeria species and other bacterial genera (3 Listeria spp., 4 Salmonella spp., 2 Vibrio spp., 3 Escherichia coli and 3 Shigella spp.). Improvements in the sensitivity and specificity have not only facilitated the reliable detection of L. monocytogenes at extremely low concentrations, but also allowed for the development of a 96-well plate-based routine assay platform for multivalent diagnostics. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Aptamer-based multiplexed proteomic technology for biomarker discovery.

    Directory of Open Access Journals (Sweden)

    Larry Gold

    Full Text Available BACKGROUND: The interrogation of proteomes ("proteomics" in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology and medicine. METHODOLOGY/PRINCIPAL FINDINGS: We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 µL of serum or plasma. Our current assay measures 813 proteins with low limits of detection (1 pM median, 7 logs of overall dynamic range (~100 fM-1 µM, and 5% median coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding signature of DNA aptamer concentrations, which is quantified on a DNA microarray. Our assay takes advantage of the dual nature of aptamers as both folded protein-binding entities with defined shapes and unique nucleotide sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD. We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to rapidly discover unique protein signatures characteristic of various disease states. CONCLUSIONS/SIGNIFICANCE: We describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next

  5. Novel protein detection method based on proximity-dependent polymerase reaction and aptamers

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    In recent years, specific detection of proteins is one of the hot issues about aptamers in proteomics.Here we reported a simple, sensitive and specific proximity-dependent protein assay with dual DNA aptamers. Thrombin was used as the model protein, and two aptamer probes with complementary sequence at 3'-end were designed for the two distinct epitopes of the protein. Association of the two aptamers with thrombin resulted in stable hybrids due to the proximity of 3'-end, then polymerase reaction was induced. The amount of obtained dsDNA was indicated using the fluorescence dye Sybr Green 1. The results showed that the initial velocity of polymerase reaction had a positive correlation with concentration of thrombin. The advantages of this dual-aptamer-based approach included simple and flexible design of aptamer probes, high selectivity and high sensitivity. The detection limit was 6.9pmol/L.

  6. Modeling the microscopic electrical properties of thrombin binding aptamer (TBA) for label-free biosensors

    Science.gov (United States)

    Alfinito, Eleonora; Reggiani, Lino; Cataldo, Rosella; De Nunzio, Giorgio; Giotta, Livia; Guascito, Maria Rachele

    2017-02-01

    Aptamers are chemically produced oligonucleotides, able to bind a variety of targets such as drugs, proteins and pathogens with high sensitivity and selectivity. Therefore, aptamers are largely employed for producing label-free biosensors (aptasensors), with significant applications in diagnostics and drug delivery. In particular, the anti-thrombin aptamers are biomolecules of high interest for clinical use, because of their ability to recognize and bind the thrombin enzyme. Among them, the DNA 15-mer aptamer (TBA), has been widely explored around the possibility of using it in aptasensors. This paper proposes a microscopic model of the electrical properties of TBA and of the aptamer-thrombin complex, combining information from both structure and function, following the issues addressed in an emerging branch of electronics known as proteotronics. The theoretical results are compared and validated with measurements reported in the literature. Finally, the model suggests resistance measurements as a novel tool for testing aptamer-target affinity.

  7. Comparison of In-Solution Biorecognition Properties of Aptamers against Ochratoxin A

    Science.gov (United States)

    McKeague, Maureen; Velu, Ranganathan; De Girolamo, Annalisa; Valenzano, Stefania; Pascale, Michelangelo; Smith, McKenzie; DeRosa, Maria C.

    2016-01-01

    Ochratoxin A (OTA) is a mycotoxin produced as a secondary metabolite by several species of Aspergillus and Penicillium and frequently found as a natural contaminant in a wide range of food commodities. Novel and robust biorecognition agents for detecting this molecule are required. Aptamers are artificial nucleic acid ligands able to bind with high affinity and specificity to a given target molecule. In the last few years, three separate research groups have selected aptamers for ochratoxin A. While each of these three families of aptamers have been incorporated into various methods for detecting OTA, it is unclear if each aptamer candidate is better suited for a particular application. Here, we perform the first head-to-head comparison of solution-based binding parameters for these groups of aptamers. Based on our results, we provide recommendations for the appropriate choice of aptamer for incorporation into solution-based biorecognition assays and applications. PMID:27854269

  8. Comparison of the 'chemical' and 'structural' approaches to the optimization of the thrombin-binding aptamer.

    Directory of Open Access Journals (Sweden)

    Olga Tatarinova

    Full Text Available Noncanonically structured DNA aptamers to thrombin were examined. Two different approaches were used to improve stability, binding affinity and biological activity of a known thrombin-binding aptamer. These approaches are chemical modification and the addition of a duplex module to the aptamer core structure. Several chemically modified aptamers and the duplex-bearing ones were all studied under the same conditions by a set of widely known and some relatively new methods. A number of the thrombin-binding aptamer analogs have demonstrated improved characteristics. Most importantly, the study allowed us to compare directly the two approaches to aptamer optimization and to analyze their relative advantages and disadvantages as well as their potential in drug design and fundamental studies.

  9. Modeling the microscopic electrical properties of thrombin binding aptamer (TBA) for label-free biosensors

    CERN Document Server

    Alfinito, Eleonora; Cataldo, Rosella; De Nunzio, Giorgio; Giotta, Livia; Guascito, Maria Rachele

    2016-01-01

    Aptamers are chemically produced oligonucleotides, able to bind a variety of targets such as drugs, proteins and pathogens with high sensitivity and selectivity. Therefore, aptamers are largely employed for producing label-free biosensors, with significant applications in diagnostics and drug delivery. In particular, the anti-thrombin aptamers are biomolecules of high interest for clinical use, because of their ability to recognize and bind the thrombin enzyme. Among them, the DNA 15-mer thrombin-binding aptamer (TBA), has been widely explored concerning both its structure, which was resolved with different techniques, and its function, especially about the possibility of using it as the active part of biosensors. This paper proposes a microscopic model of the electrical properties of TBA and the aptamer-thrombin complex, combining information from both structure and function. The novelty consists in describing both the aptamer alone and the complex as an impedance network, thus going deeper inside the issues...

  10. SELEX Modifications and Bioanalytical Techniques for Aptamer-Target Binding Characterization.

    Science.gov (United States)

    Tan, Sze Y; Acquah, Caleb; Sidhu, Amandeep; Ongkudon, Clarence M; Yon, L S; Danquah, Michael K

    2016-11-01

    The quest to improve the detection of biomolecules and cells in health and life sciences has led to the discovery and characterization of various affinity bioprobes. Libraries of synthetic oligonucleotides (ssDNA/ssRNA) with randomized sequences are employed during Systematic Evolution of Ligands by Exponential Enrichment (SELEX) to select highly specific affinity probes called aptamers. With much focus on the generation of aptamers for a variety of target molecules, conventional SELEX protocols have been modified to develop new and improved SELEX protocols yielding highly specific and stable aptamers. Various techniques have been used to analyze the binding interactions between aptamers and their cognate molecules with associated merits and limitations. This article comprehensively reviews research advancements in the generation of aptamers, analyses physicochemical conditions affecting their binding characteristics to cellular and biomolecular targets, and discusses various field applications of aptameric binding. Biophysical techniques employed in the characterization of the molecular and binding features of aptamers to their cognate targets are also discussed.

  11. A general double library SELEX strategy for aptamer selection using unmodified nonimmobilized targets.

    Science.gov (United States)

    Lee, Kyung Hyun; Zeng, Huaqiang

    2017-08-01

    Aptamer discovery for unmodified nonimmobilized targets has been constantly presenting itself as a significant challenge to the research community. We demonstrate here a novel double library (DL) SELEX strategy and its usefulness and generality toward discovering both ssDNA- and RNA-based aptamers with nanomolar binding affinities toward unmodified targets of both small (e.g., doxycycline) and large (e.g., VEGF165) sizes. The same selection strategy further allows for concurrent selection of an aptamer pair, recognizing discrete epitopes on the same protein, from the same selection cycles for the sandwich aptamer pair-based biosensor development (e.g., one aptamer for the recognition and the other for the signal transduction). These results establish the DL-SELEX method developed here as a valuable and highly accessible selection strategy for aptamer discovery, especially when chemical modifications of target molecules are not preferred or simply impossible.

  12. In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A.

    Directory of Open Access Journals (Sweden)

    Regina Stoltenburg

    Full Text Available A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for monitoring the aptamer selection progress. Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5'-end including the 5'-primer binding site is essential for aptamer-target binding. Extensive interaction analyses between aptamer and Protein A were performed by methods like surface plasmon resonance, MicroScale Thermophoresis and bead-based binding assays using fluorescence measurements. The binding of the aptamer to its target was thus investigated in assays with immobilization of one of the binding partners each, and with both binding partners in solution. Affinity constants were determined in the low micromolar to submicromolar range, increasing to the nanomolar range under the assumption of avidity. Protein A provides more than one binding site for the aptamer, which may overlap with the known binding sites for immunoglobulins. The aptamer binds specifically to both native and recombinant Protein A, but not to other immunoglobulin-binding proteins like Protein G and L. Cross specificity to other proteins was not found. The application of the aptamer is directed to Protein A detection or affinity purification. Moreover, whole cells of Staphylococcus aureus, presenting Protein A on the cell surface, could also be bound by the aptamer.

  13. Affinity Probe Capillary Electrophoresis Evaluation of Aptamer Binding to Campylobacter jejuni Bacteria

    Science.gov (United States)

    2009-11-01

    Affinity Probe Capillary Electrophoresis Evaluation of Aptamer Binding to Campylobacter jejuni Bacteria by Dimitra N. Stratis-Cullum, Sun...Aptamer Binding to Campylobacter jejuni Bacteria Dimitra N. Stratis-Cullum, Sun McMasters, and Paul M. Pellegrino Sensors and Electron Devices...To) 2007–2008 4. TITLE AND SUBTITLE Affinity Probe Capillary Electrophoresis Evaluation of Aptamer Binding to Campylobacter jejuni Bacteria 5a

  14. Aptamer-gelatin composite for a trigger release system mediated by oligonucleotide hybridization.

    Science.gov (United States)

    Soontornworajit, Boonchoy; Srakaew, Prangkamol; Naramitpanich, Pajaree

    2014-01-01

    Nucleic acid aptamers not only specifically bind to their target proteins with high affinity but also form intermolecular hybridization with their complementary oligonucleotides (CO). The hybridization can interrupt aptamer/protein interaction due to the changes of aptamer secondary structure which rely on hybridization length and base-pairing positions. Herein we aim to use this unique property of the aptamers, when combined with gelatin to develop a novel composite with desirable protein release profiles. Platelet-derived growth factor-BB (PDGF-BB) and its aptamer were used as target molecules. Prior to performing the release study, the effects of CO on aptamer-protein interaction were observed by surface plasmon resonance (SPR). The SPR sensorgram indicated that the aptamer dissociated from the bounded proteins when it hybridized with the CO. The aptamer was then immobilized onto streptavidin coated polystyrene particles via biotin/streptavidin interaction. Then, PDGF-BB and aptamer functionalized particles were mixed with gelatin solution and cast as small pieces of composite. The success of the composite preparation was confirmed by flow cytometry and microscopy. PDGF-BB release at several time points was quantified by ELISA. The results showed that the aptamer-gelatin composite could slow the release rate of the proteins from the composite due to strong binding of proteins and aptamers. Once the CO was added to the system, the release rate was significantly enhanced because the aptamer hybridized with the CO and lost its active secondary structure. Therefore, the proteins were triggered to release out from the composite. This work suggests a promising strategy for controlling the release of bioactive molecules in medical treatments.

  15. Trends in the Design and Development of Specific Aptamers Against Peptides and Proteins.

    Science.gov (United States)

    Tabarzad, Maryam; Jafari, Marzieh

    2016-04-01

    Aptamers are single stranded oligonucleotides, comparable to monoclonal antibodies (mAbs) in selectivity and affinity and have significant strategic properties in design, development and applications more than mAbs. Ease of design and development, simple chemical modification and the attachment of functional groups, easily handling and more adaptability with analytical methods, small size and adaptation with nanostructures are the valuable characteristics of aptamers in comparison to large protein based ligands. Among a broad range of targets that their specific aptamers developed, proteins and peptides have significant position according to the number of related studies performed so far. Since proteins control many of important physiological and pathological incidents in the living organisms, particularly human beings and because of the benefits of aptamers in clinical and analytical applications, aptamer related technologies in the field of proteins and peptides are under progress, exclusively. Currently, there is only one FDA approved therapeutic aptamer in the pharmaceutical market, which is specific to vascular endothelial growth factor and is prescribed for age related macular degenerative disease. Additionally, there are several aptamers in the different phases of clinical trials. Almost all of these aptamers are specific to clinically important peptide or protein targets. In addition, the application of protein specific aptamers in the design and development of targeted drug delivery systems and diagnostic biosensors is another interesting field of aptamer technology. In this review, significant efforts related to development and applications of aptamer technologies in proteins and peptides sciences were considered to emphasis on the importance of aptamers in medicinal and clinical applications.

  16. Development of a Quartz Crystal Microbalance Biosensor with Aptamers as Bio-recognition Element

    Directory of Open Access Journals (Sweden)

    Chunyan Yao

    2010-06-01

    Full Text Available The ultimate goal in any biosensor development project is its use for actual sample detection. Recently, there has been an interest in biosensors with aptamers as bio-recognition elements, but reported examples all deal with standards, not human serum. In order to verify the differences of aptamer-based biosensor and antibody-based biosensor in clinical detection, a comparison of the performance of aptamer-based and antibody-based quartz crystal microbalance (QCM biosensors for the detection of immunoglobulin E (IgE in human serum was carried out. Aptamers (or antibodies specific to IgE were immobilized on the gold surface of a quartz crystal. The frequency shifts of the QCM were measured. The linear range with the antibody (10–240 μg/L compared to that of the aptamer (2.5–200 μg/L, but a lower detection limit could be observed in the aptamer-based biosensor. The reproducibility of the two biosensors was comparable. The aptamers were equivalent or superior to antibodies in terms of specificity and sensitivity. In addition, the aptamer receptors could tolerate repeated affine layer regeneration after ligand binding and recycling of the biosensor with little loss of sensitivity. When stored for three weeks, the frequency shifts of the aptamer-coated crystals were all greater than 90% of those on the response at the first day.

  17. Robust aptamer sol-gel solid phase microextraction of very polar adenosine from human plasma.

    Science.gov (United States)

    Mu, Li; Hu, Xiangang; Wen, Jianping; Zhou, Qixing

    2013-03-01

    Conventional solid phase microextraction (SPME) has a limited capacity to extract very polar analytes, such as adenosine. To solve this problem, aptamer conjugating sol-gel methodology was coupled with an SPME fiber. According to the authors' knowledge, this is the first reported use of aptamer SPME. The fiber of aptamer sol-gel SPME with a mesoporous structure has high porosity, large surface area, and small water contact angle. Rather than employing direct entrapment, covalent immobilization was the dominant method of aptamer loading in sol-gel. Aptamer sol-gel fiber captured a specified analyte from among the analog molecules, thereby, exhibiting an excellent selective property. Compared with commercial SPME fibers, this aptamer fiber was suitable for extracting adenosine, presenting an extraction efficiency higher than 20-fold. The values of repeatability and reproducibility expressed by relative standard deviation were low (9.4%). Interestingly, the sol-gel network enhanced the resistance of aptamer SPME to both nuclease and nonspecific proteins. Furthermore, the aptamer sol-gel fiber was applied in human plasma with LOQ 1.5 μg/L, which is an acceptable level. This fiber also demonstrates durability and regeneration over 20-cycles without significant loss of efficiency. Given the various targets (from metal ions to biomacromolecules and cells) of aptamers, this methodology will extend the multi-domain applications of SPME.

  18. Inhibition of HCV NS3 protease by RNA aptamers in cells.

    Science.gov (United States)

    Nishikawa, Fumiko; Kakiuchi, Nobuko; Funaji, Kohei; Fukuda, Kotaro; Sekiya, Satoru; Nishikawa, Satoshi

    2003-04-01

    Non-structural protein 3 (NS3) of hepatitis C virus (HCV) has two distinct activities, protease and helicase, which are essential for HCV proliferation. In previous work, we obtained RNA aptamers (G9-I, II and III) which specifically bound the NS3 protease domain (DeltaNS3), efficiently inhibiting protease activity in vitro. To utilize these aptamers in vivo, we constructed a G9 aptamer expression system in cultured cells, using the cytomegarovirus enhancer + chicken beta-actin globin (CAG) promoter. By conjugating the cis-acting genomic human hepatitis delta virus (HDV) ribozyme and G9-II aptamer, a chimeric HDV ribozyme-G9-II aptamer (HA) was constructed, which was used to produce stable RNA in vivo and to create tandem repeats of the functional unit. To target the transcribed RNA aptamers to the cytoplasm, the minimal mutant of constitutive transport element (CTE), derived from type D retroviruses, was conjugated at the 3' end of HA (HAC). Transcript RNAs from (HA)(n) and (HAC)(n) were processed into the G9-II aptamer unit by the cis-acting HDV ribozyme, both in vitro and in vivo. Efficient protease inhibition activity of HDV ribozyme-G9-II aptamer expression plasmid was demonstrated in HeLa cells. Protease inhibition activity level of tandem chimeric aptamers, (HA)(n) and (HAC)(n), rose with the increase of n from 1 to 4.

  19. A Small Aptamer with Strong and Specific Recognition of the Triphosphate of ATP

    Science.gov (United States)

    Sazani, Peter L.; Larralde, Rosa

    2004-01-01

    We report the in vitro selection of an RNA-based ATP aptamer with the ability to discriminate between adenosine ligands based on their 5‘ phosphorylation state. Previous selection of ATP aptamers yielded molecules that do not significantly discriminate between ligands at the 5‘ position. By applying a selective pressure that demands recognition of the 5‘ triphosphate, we obtained an aptamer that binds to ATP with a Kd of approximately 5 μM, and to AMP with a Kd of approximately 5.5 mM, a difference of 1100-fold. This aptamer demonstrates the ability of small RNAs to interact with negatively charged moieties. PMID:15237981

  20. One-step selection of Vaccinia virus-binding DNA aptamers by MonoLEX

    Directory of Open Access Journals (Sweden)

    Stöcklein Walter

    2007-08-01

    Full Text Available Abstract Background As a new class of therapeutic and diagnostic reagents, more than fifteen years ago RNA and DNA aptamers were identified as binding molecules to numerous small compounds, proteins and rarely even to complete pathogen particles. Most aptamers were isolated from complex libraries of synthetic nucleic acids by a process termed SELEX based on several selection and amplification steps. Here we report the application of a new one-step selection method (MonoLEX to acquire high-affinity DNA aptamers binding Vaccinia virus used as a model organism for complex target structures. Results The selection against complete Vaccinia virus particles resulted in a 64-base DNA aptamer specifically binding to orthopoxviruses as validated by dot blot analysis, Surface Plasmon Resonance, Fluorescence Correlation Spectroscopy and real-time PCR, following an aptamer blotting assay. The same oligonucleotide showed the ability to inhibit in vitro infection of Vaccinia virus and other orthopoxviruses in a concentration-dependent manner. Conclusion The MonoLEX method is a straightforward procedure as demonstrated here for the identification of a high-affinity DNA aptamer binding Vaccinia virus. MonoLEX comprises a single affinity chromatography step, followed by subsequent physical segmentation of the affinity resin and a single final PCR amplification step of bound aptamers. Therefore, this procedure improves the selection of high affinity aptamers by reducing the competition between aptamers of different affinities during the PCR step, indicating an advantage for the single-round MonoLEX method.

  1. Selection and Identification of Chloramphenicol-Specific DNA Aptamers by Mag-SELEX.

    Science.gov (United States)

    Duan, Ye; Gao, Zhiqiang; Wang, Lihui; Wang, Huishan; Zhang, Hexiao; Li, Hao

    2016-12-01

    Chloramphenicol (CAP) has been widely used to treat bacterial infections in livestock and aquatic animals. To reduce the risk of CAP residues, an efficient technology to rapidly detect CAP residues in animal-sourced food is expressly needed. In this study, magnetic bead-based systematic evolution of ligands by exponential enrichment (Mag-SELEX) strategy was performed to select and identify CAP-specific single-stranded DNA (ssDNA) aptamers from a random oligonucleotide library. After nine rounds of selection, five potential ssDNA aptamers were selected. Low homology indicated that they might belong to different families. To identify an aptamer with the highest affinity for CAP, the dissociation constant (K d) values of these selected aptamers were determined. The lowest K d values of two potential aptamers (i.e., No. 4 and No. 5) were, respectively, 0.10162 ± 0.0111 and 0.03224 ± 0.00819 μM, which were much lower than previously reported lowest K d value (i.e., 0.766 μM) of CAP aptamer. Moreover, compared with No. 4, aptamer No. 5 had higher binding rate, which is quite different among those with CAP and with CAP's structural analogs (i.e., thiamphenicol (TAP) and florfenicol (FF)). These results indicated that the potential aptamer No. 5 with highest specificity and affinity for CAP would be an ideal aptamer for future detection of residual CAP in animal-sourced food.

  2. Galaxy Workflows for Web-based Bioinformatics Analysis of Aptamer High-throughput Sequencing Data

    Directory of Open Access Journals (Sweden)

    William H Thiel

    2016-01-01

    Full Text Available Development of RNA and DNA aptamers for diagnostic and therapeutic applications is a rapidly growing field. Aptamers are identified through iterative rounds of selection in a process termed SELEX (Systematic Evolution of Ligands by EXponential enrichment. High-throughput sequencing (HTS revolutionized the modern SELEX process by identifying millions of aptamer sequences across multiple rounds of aptamer selection. However, these vast aptamer HTS datasets necessitated bioinformatics techniques. Herein, we describe a semiautomated approach to analyze aptamer HTS datasets using the Galaxy Project, a web-based open source collection of bioinformatics tools that were originally developed to analyze genome, exome, and transcriptome HTS data. Using a series of Workflows created in the Galaxy webserver, we demonstrate efficient processing of aptamer HTS data and compilation of a database of unique aptamer sequences. Additional Workflows were created to characterize the abundance and persistence of aptamer sequences within a selection and to filter sequences based on these parameters. A key advantage of this approach is that the online nature of the Galaxy webserver and its graphical interface allow for the analysis of HTS data without the need to compile code or install multiple programs.

  3. Aptamer-Functionalized Fluorescent Silica Nanoparticles for Highly Sensitive Detection of Leukemia Cells

    Science.gov (United States)

    Tan, Juntao; Yang, Nuo; Hu, Zixi; Su, Jing; Zhong, Jianhong; Yang, Yang; Yu, Yating; Zhu, Jianmeng; Xue, Dabin; Huang, Yingying; Lai, Zongqiang; Huang, Yong; Lu, Xiaoling; Zhao, Yongxiang

    2016-06-01

    A simple, highly sensitive method to detect leukemia cells has been developed based on aptamer-modified fluorescent silica nanoparticles (FSNPs). In this strategy, the amine-labeled Sgc8 aptamer was conjugated to carboxyl-modified FSNPs via amide coupling between amino and carboxyl groups. Sensitivity and specificity of Sgc8-FSNPs were assessed using flow cytometry and fluorescence microscopy. These results showed that Sgc8-FSNPs detected leukemia cells with high sensitivity and specificity. Aptamer-modified FSNPs hold promise for sensitive and specific detection of leukemia cells. Changing the aptamer may allow the FSNPs to detect other types of cancer cells.

  4. Discrimination between Closely Related Cellular Metabolites by the SAM-I Riboswitch

    Energy Technology Data Exchange (ETDEWEB)

    Montange, R.; Mondragon, E; van Tyne, D; Garst, A; Ceres, P; Batey, R

    2010-01-01

    The SAM-I riboswitch is a cis-acting element of genetic control found in bacterial mRNAs that specifically binds S-adenosylmethionine (SAM). We previously determined the 2.9-{angstrom} X-ray crystal structure of the effector-binding domain of this RNA element, revealing details of RNA-ligand recognition. To improve this structure, variations were made to the RNA sequence to alter lattice contacts, resulting in a 0.5-{angstrom} improvement in crystallographic resolution and allowing for a more accurate refinement of the crystallographic model. The basis for SAM specificity was addressed by a structural analysis of the RNA complexed to S-adenosylhomocysteine (SAH) and sinefungin and by measuring the affinity of SAM and SAH for a series of mutants using isothermal titration calorimetry. These data illustrate the importance of two universally conserved base pairs in the RNA that form electrostatic interactions with the positively charged sulfonium group of SAM, thereby providing a basis for discrimination between SAM and SAH.

  5. Controlling expression of genes in the unicellular alga Chlamydomonas reinhardtii with a vitamin-repressible riboswitch.

    Science.gov (United States)

    Ramundo, Silvia; Rochaix, Jean-David

    2015-01-01

    Chloroplast genomes of land plants and algae contain generally between 100 and 150 genes. These genes are involved in plastid gene expression and photosynthesis and in various other tasks. The function of some chloroplast genes is still unknown and some of them appear to be essential for growth and survival. Repressible and reversible expression systems are highly desirable for functional and biochemical characterization of these genes. We have developed a genetic tool that allows one to regulate the expression of any coding sequence in the chloroplast genome of the unicellular alga Chlamydomonas reinhardtii. Our system is based on vitamin-regulated expression of the nucleus-encoded chloroplast Nac2 protein, which is specifically required for the expression of any plastid gene fused to the psbD 5'UTR. With this approach, expression of the Nac2 gene in the nucleus and, in turn, that of the chosen chloroplast gene artificially driven by the psbD 5'UTR, is controlled by the MetE promoter and Thi4 riboswitch, which can be inactivated in a reversible way by supplying vitamin B12 and thiamine to the growth medium, respectively. This system opens interesting possibilities for studying the assembly and turnover of chloroplast multiprotein complexes such as the photosystems, the ribosome, and the RNA polymerase. It also provides a way to overcome the toxicity often associated with the expression of proteins of biotechnological interest in the chloroplast.

  6. High-resolution structures of two complexes between thrombin and thrombin-binding aptamer shed light on the role of cations in the aptamer inhibitory activity

    Science.gov (United States)

    Russo Krauss, Irene; Merlino, Antonello; Randazzo, Antonio; Novellino, Ettore; Mazzarella, Lelio; Sica, Filomena

    2012-01-01

    The G-quadruplex architecture is a peculiar structure adopted by guanine-rich oligonucleotidic sequences, and, in particular, by several aptamers, including the thrombin-binding aptamer (TBA) that has the highest inhibitory activity against human α-thrombin. A crucial role in determining structure, stability and biological properties of G-quadruplexes is played by ions. In the case of TBA, K+ ions cause an enhancement of the aptamer clotting inhibitory activity. A detailed picture of the interactions of TBA with the protein and with the ions is still lacking, despite the importance of this aptamer in biomedical field for detection and inhibition of α-thrombin. Here, we fill this gap by presenting a high-resolution crystallographic structural characterization of the thrombin–TBA complex formed in the presence of Na+ or K+ and a circular dichroism study of the structural stability of the aptamer both free and complexed with α-thrombin, in the presence of the two ionic species. The results indicate that the different effects exerted by Na+ and K+ on the inhibitory activity of TBA are related to a subtle perturbation of a few key interactions at the protein–aptamer interface. The present data, in combination with those previously obtained on the complex between α-thrombin and a modified aptamer, may allow the design of new TBA variants with a pharmacological performance enhancement. PMID:22669903

  7. Selective Aptamers for Detection of Estradiol and Ethynylestradiol in Natural Waters

    KAUST Repository

    Akki, Spurti U.

    2015-08-18

    © 2015 American Chemical Society. We used in vitro selection to identify new DNA aptamers for two endocrine-disrupting compounds often found in treated and natural waters, 17β-estradiol (E2) and 17α-ethynylestradiol (EE). We used equilibrium filtration to determine aptamer sensitivity/selectivity and dimethyl sulfate (DMS) probing to explore aptamer binding sites. The new E2 aptamers are at least 74-fold more sensitive for E2 than is a previously reported DNA aptamer, with dissociation constants (Kd values) of 0.6 μM. Similarly, the EE aptamers are highly sensitive for EE, with Kd of 0.5-1.0 μM. Selectivity values indicate that the E2 aptamers bind E2 and a structural analogue, estrone (E1), equally well and are up to 74-fold selective over EE. One EE aptamer is 53-fold more selective for EE over E2 or E1, but the other binds EE, E2, and E1 with similar affinity. The new aptamers do not lose sensitivity or selectivity in natural water from a local lake, despite the presence of natural organic matter (∼4 mg/L TOC). DMS probing suggests that E2 binding occurs in relatively flexible single-stranded DNA regions, an important finding for rational redesign of aptamers and their incorporation into sensing platforms. This is the first report of aptamers with strong selectivity for E2 and E1 over EE, or with strong selectivity for EE over E2 and E1. Such selectivity is important for achieving the goal of creating practically useful DNA-based sensors that can distinguish structurally similar estrogenic compounds in natural waters.

  8. Rupture of DNA aptamer: New insights from simulations

    Energy Technology Data Exchange (ETDEWEB)

    Mishra, Rakesh Kumar; Nath, Shesh; Kumar, Sanjay [Department of Physics, Banaras Hindu University, Varanasi 221 005 (India)

    2015-10-28

    Base-pockets (non-complementary base-pairs) in a double-stranded DNA play a crucial role in biological processes. Because of thermal fluctuations, it can lower the stability of DNA, whereas, in case of DNA aptamer, small molecules, e.g., adenosinemonophosphate and adenosinetriphosphate, form additional hydrogen bonds with base-pockets termed as “binding-pockets,” which enhance the stability. Using the Langevin dynamics simulations of coarse grained model of DNA followed by atomistic simulations, we investigated the influence of base-pocket and binding-pocket on the stability of DNA aptamer. Striking differences have been reported here for the separation induced by temperature and force, which require further investigation by single molecule experiments.

  9. Current approaches in SELEX: An update to aptamer selection technology.

    Science.gov (United States)

    Darmostuk, Mariia; Rimpelova, Silvie; Gbelcova, Helena; Ruml, Tomas

    2015-11-01

    Systematic evolution of ligands by exponential enrichment (SELEX) is a well-established and efficient technology for the generation of oligonucleotides with a high target affinity. These SELEX-derived single stranded DNA and RNA molecules, called aptamers, were selected against various targets, such as proteins, cells, microorganisms, chemical compounds etc. They have a great potential in the use as novel antibodies, in cancer theragnostics and in biomedical research. Vast interest in aptamers stimulated continuous development of SELEX, which underwent numerous modifications since its first application in 1990. Novel modifications made the selection process more efficient, cost-effective and significantly less time-consuming. This article brings a comprehensive and up-to-date review of recent advances in SELEX methods and pinpoints advantages, main obstacles and limitations. The post-SELEX strategies and examples of application are also briefly outlined in this review. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Smart ligand: aptamer-mediated targeted delivery of chemotherapeutic drugs and siRNA for cancer therapy.

    Science.gov (United States)

    Li, Xin; Zhao, Qinghe; Qiu, Liyan

    2013-10-28

    Aptamers are a class of oligonucleotides that can specifically bind to different targets with high affinity. Since their discovery in 1980s, aptamers have attracted considerable interests in medical applications. So far, initial research using aptamers as delivery systems has produced exciting results. In this review, we summarize recent progress in aptamer-mediated chemotherapeutic drug and siRNA delivery systems in tumor treatment. With regard to chemotherapeutic drugs, the 2 main methods for targeted delivery using aptamers are as follows: aptamer-drug systems (in which aptamers directly deliver the drug both as a carrier and as a ligand) and aptamer-nanoparticles systems (in which nanoparticles function together with aptamers for targeted delivery of drugs). For delivery of siRNA, aptamers can be utilized by the following ways to facilitate targeting: (1) linked by a connector; (2) form a chimera; and (3) combined with nanoparticles. In co-delivery system, the advantages associated with the use of aptamers are beginning to become apparent also. Here, the challenges and new perspectives in the field of aptamer-mediated delivery have been discussed.

  11. Using atomic force microscopy and surface plasmon resonance to detect specific interactions between ricin and anti-ricin aptamers

    Science.gov (United States)

    Nucleic acid aptamers have been widely used as binding reagents for the label free detections of biomolecules. Compare to antibodies, aptamers have demonstrated advantages such as easy synthesis, low cost, and better stability. Therefore, aptamers can be integrated into various detection platforms ...

  12. Selection of an aptamer antidote to the anticoagulant drug bivalirudin.

    Science.gov (United States)

    Martin, Jennifer A; Parekh, Parag; Kim, Youngmi; Morey, Timothy E; Sefah, Kwame; Gravenstein, Nikolaus; Dennis, Donn M; Tan, Weihong

    2013-01-01

    Adverse drug reactions, including severe patient bleeding, may occur following the administration of anticoagulant drugs. Bivalirudin is a synthetic anticoagulant drug sometimes employed as a substitute for heparin, a commonly used anticoagulant that can cause a condition called heparin-induced thrombocytopenia (HIT). Although bivalrudin has the advantage of not causing HIT, a major concern is lack of an antidote for this drug. In contrast, medical professionals can quickly reverse the effects of heparin using protamine. This report details the selection of an aptamer to bivalirudin that functions as an antidote in buffer. This was accomplished by immobilizing the drug on a monolithic column to partition binding sequences from nonbinding sequences using a low-pressure chromatography system and salt gradient elution. The elution profile of binding sequences was compared to that of a blank column (no drug), and fractions with a chromatographic difference were analyzed via real-time PCR (polymerase chain reaction) and used for further selection. Sequences were identified by 454 sequencing and demonstrated low micromolar dissociation constants through fluorescence anisotropy after only two rounds of selection. One aptamer, JPB5, displayed a dose-dependent reduction of the clotting time in buffer, with a 20 µM aptamer achieving a nearly complete antidote effect. This work is expected to result in a superior safety profile for bivalirudin, resulting in enhanced patient care.

  13. Selection of an aptamer antidote to the anticoagulant drug bivalirudin.

    Directory of Open Access Journals (Sweden)

    Jennifer A Martin

    Full Text Available Adverse drug reactions, including severe patient bleeding, may occur following the administration of anticoagulant drugs. Bivalirudin is a synthetic anticoagulant drug sometimes employed as a substitute for heparin, a commonly used anticoagulant that can cause a condition called heparin-induced thrombocytopenia (HIT. Although bivalrudin has the advantage of not causing HIT, a major concern is lack of an antidote for this drug. In contrast, medical professionals can quickly reverse the effects of heparin using protamine. This report details the selection of an aptamer to bivalirudin that functions as an antidote in buffer. This was accomplished by immobilizing the drug on a monolithic column to partition binding sequences from nonbinding sequences using a low-pressure chromatography system and salt gradient elution. The elution profile of binding sequences was compared to that of a blank column (no drug, and fractions with a chromatographic difference were analyzed via real-time PCR (polymerase chain reaction and used for further selection. Sequences were identified by 454 sequencing and demonstrated low micromolar dissociation constants through fluorescence anisotropy after only two rounds of selection. One aptamer, JPB5, displayed a dose-dependent reduction of the clotting time in buffer, with a 20 µM aptamer achieving a nearly complete antidote effect. This work is expected to result in a superior safety profile for bivalirudin, resulting in enhanced patient care.

  14. Bioactivity of 2′-deoxyinosine-incorporated aptamer AS1411

    Science.gov (United States)

    Fan, Xinmeng; Sun, Lidan; Wu, Yun; Zhang, Lihe; Yang, Zhenjun

    2016-01-01

    Aptamers can be chemically modified to enhance nuclease resistance and increase target affinity. In this study, we performed chemical modification of 2′-deoxyinosine in AS1411, an anti-proliferative G-rich oligodeoxynucleotide aptamer, which binds selectively to the nucleolin protein. Its function was augmented when 2′-deoxyinosine was incorporated at positions 12, 13, 15, and 24 of AS1411, respectively. In addition, double incorporation of 2′-deoxyinosine at positions 12 and 24 (FAN-1224dI), 13 and 24 (FAN-1324dI), and 15 and 24 (FAN-1524dI) promoted G-quartet formation, as well as inhibition of DNA replication and tumor cell growth, and induced S-phase cell cycle arrest. In further animal experiments, FAN-1224dI, FAN-1324dI and FAN-1524dI resulted in enhanced treatment effects than AS1411 alone. These results suggested that the position and number of modification substituents in AS1411 are critical parameters to improve the diagnostic and therapeutic function of the aptamer. Structural investigations of the FAN-1524dI/nucleolin complex structure, using molecular dynamics simulation, revealed the critical interactions involving nucleolin and 2′-dI incorporated AS1411 compared with AS1411 alone. These findings augment understanding of the role of 2′-deoxyinosine moieties in interactive binding processes. PMID:27194215

  15. Bioactivity of 2'-deoxyinosine-incorporated aptamer AS1411.

    Science.gov (United States)

    Fan, Xinmeng; Sun, Lidan; Wu, Yun; Zhang, Lihe; Yang, Zhenjun

    2016-05-19

    Aptamers can be chemically modified to enhance nuclease resistance and increase target affinity. In this study, we performed chemical modification of 2'-deoxyinosine in AS1411, an anti-proliferative G-rich oligodeoxynucleotide aptamer, which binds selectively to the nucleolin protein. Its function was augmented when 2'-deoxyinosine was incorporated at positions 12, 13, 15, and 24 of AS1411, respectively. In addition, double incorporation of 2'-deoxyinosine at positions 12 and 24 (FAN-1224dI), 13 and 24 (FAN-1324dI), and 15 and 24 (FAN-1524dI) promoted G-quartet formation, as well as inhibition of DNA replication and tumor cell growth, and induced S-phase cell cycle arrest. In further animal experiments, FAN-1224dI, FAN-1324dI and FAN-1524dI resulted in enhanced treatment effects than AS1411 alone. These results suggested that the position and number of modification substituents in AS1411 are critical parameters to improve the diagnostic and therapeutic function of the aptamer. Structural investigations of the FAN-1524dI/nucleolin complex structure, using molecular dynamics simulation, revealed the critical interactions involving nucleolin and 2'-dI incorporated AS1411 compared with AS1411 alone. These findings augment understanding of the role of 2'-deoxyinosine moieties in interactive binding processes.

  16. Development of a thermal-stable structure-switching cocaine-binding aptamer.

    Science.gov (United States)

    Shoara, Aron A; Reinstein, Oren; Borhani, Okty Abbasi; Martin, Taylor R; Slavkovic, Sladjana; Churcher, Zachary R; Johnson, Philip E

    2017-08-21

    We have developed a new cocaine-binding aptamer variant that has a significantly higher melt temperature when bound to a ligand than the currently used sequence. Retained in this new construct is the ligand-induced structure-switching binding mechanism that is important in biosensing applications of the cocaine-binding aptamer. Isothermal titration calorimetry methods show that the binding affinity of this new sequence is slightly tighter than the existing cocaine-binding aptamer. The improved thermal performance, a Tm increase of 4 °C for the cocaine-bound aptamer and 9 °C for the quinine-bound aptamer, was achieved by optimizing the DNA sequence in stem 2 of the aptamer to have the highest stability based on the nearest neighbor thermodynamic parameters and confirmed by UV and fluorescence spectroscopy. The sequences in stem 1 and stem 3 were unchanged in order to retain the structure switching and ligand binding functions. The more favorable thermal stability characteristics of the OR3 aptamer should make it a useful construct for sensing applications employing the cocaine-binding aptamer system. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  17. Thermal Stability of siRNA Modulates Aptamer- conjugated siRNA Inhibition

    Directory of Open Access Journals (Sweden)

    Alexey Berezhnoy

    2012-01-01

    Full Text Available Oligonucleotide aptamer-mediated in vivo cell targeting of small interfering RNAs (siRNAs is emerging as a useful approach to enhance the efficacy and reduce the adverse effects resulting from siRNA-mediated genetic interference. A current main impediment in aptamer-mediated siRNA targeting is that the activity of the siRNA is often compromised when conjugated to an aptamer, often requiring labor intensive and time consuming design and testing of multiple configurations to identify a conjugate in which the siRNA activity has not been significantly reduced. Here, we show that the thermal stability of the siRNA is an important parameter of siRNA activity in its conjugated form, and that siRNAs with lower melting temperature (Tm are not or are minimally affected when conjugated to the 3′ end of 2′F-pyrimidine-modified aptamers. In addition, the configuration of the aptamer-siRNA conjugate retains activity comparable with the free siRNA duplex when the passenger strand is co-transcribed with the aptamer and 3′ overhangs on the passenger strand are removed. The approach described in this paper significantly reduces the time and effort necessary to screening siRNA sequences that retain biological activity upon aptamer conjugation, facilitating the process of identifying candidate aptamer-siRNA conjugates suitable for in vivo testing.

  18. Aptamer-functionalized porous phospholipid nanoshells for direct measurement of Hg(2+) in urine.

    Science.gov (United States)

    Li, Zhen; Muhandiramlage, Thusitha P; Keogh, John P; Hall, Henry K; Aspinwall, Craig A

    2015-01-01

    A porous phospholipid nanoshell (PPN) sensor functionalized with a specific aptamer sensor agent was prepared for rapid detection of Hg(2+) in human urine with minimal sample preparation. Aptamer sensors provide an important class of optical transducers that can be readily and reproducibly synthesized. A key limitation of aptamer sensors, and many other optical sensors, is the potential of biofouling or biodegradation when used in complex biological matrices such as serum or urine, particularly when high levels of nucleases are present. We prepared Hg(2+)-responsive, PPN-encapsulated aptamer sensors that overcome these limitations. PPNs provide a protective barrier to encapsulate the aptamer sensor in an aqueous environment free of diffusional restrictions encountered with many polymer nanomaterials. The unique porous properties of the PPN membrane enable ready and rapid transfer of small molecular weight ions and molecules into the sensor interior while minimizing the macromolecular interactions between the transducer and degradants or interferents in the exterior milieu. Using Hg(2+)-responsive, PPN-encapsulated aptamer sensors, we were able to detect sub-100 ppb (chronic threshold limit from urine test) Hg(2+) in human urine with no sample preparation, whereas free aptamer sensors yielded inaccurate results due to interferences from the matrix. The PPN architecture provides a new platform for construction of aptamer-functionalized sensors that target low molecular weight species in complex matrices, beyond the Hg(2+) demonstrated here.

  19. Potent Inhibition of HIV-1 Reverse Transcriptase and Replication by Nonpseudoknot, "UCAA-motif" RNA Aptamers.

    Science.gov (United States)

    Whatley, Angela S; Ditzler, Mark A; Lange, Margaret J; Biondi, Elisa; Sawyer, Andrew W; Chang, Jonathan L; Franken, Joshua D; Burke, Donald H

    2013-02-05

    RNA aptamers that bind the reverse transcriptase (RT) of human immunodeficiency virus (HIV) compete with nucleic acid primer/template for access to RT, inhibit RT enzymatic activity in vitro, and suppress viral replication when expressed in human cells. Numerous pseudoknot aptamers have been identified by sequence analysis, but relatively few have been confirmed experimentally. In this work, a screen of nearly 100 full-length and >60 truncated aptamer transcripts established the predictive value of the F1Pk and F2Pk pseudoknot signature motifs. The screen also identified a new, nonpseudoknot motif with a conserved unpaired UCAA element. High-throughput sequence (HTS) analysis identified 181 clusters capable of forming this novel element. Comparative sequence analysis, enzymatic probing and RT inhibition by aptamer variants established the essential requirements of the motif, which include two conserved base pairs (AC/GU) on the 5' side of the unpaired UCAA. Aptamers in this family inhibit RT in primer extension assays with IC(50) values in the low nmol/l range, and they suppress viral replication with a potency that is comparable with that of previously studied aptamers. All three known anti-RT aptamer families (pseudoknots, the UCAA element, and the recently described "(6/5)AL" motif) are therefore suitable for developing aptamer-based antiviral gene therapies.Molecular Therapy - Nucleic Acids (2013) 2, e71; doi:10.1038/mtna.2012.62; published online 5 February 2013.

  20. Intracellular Expression of PAI-1 Specific Aptamers Alters Breast Cancer Cell Migration, Invasion and Angiogenesis

    Science.gov (United States)

    Fortenberry, Yolanda M.; Brandal, Stephanie M.; Carpentier, Gilles; Hemani, Malvi; Pathak, Arvind P.

    2016-01-01

    Plasminogen activator inhibitor-1 (PAI-1) is elevated in various cancers, where it has been shown to effect cell migration and invasion and angiogenesis. While, PAI-1 is a secreted protein, its intercellular levels are increased in cancer cells. Consequently, intracellular PAI-1 could contribute to cancer progression. While various small molecule inhibitors of PAI-1 are currently being investigated, none specifically target intracellular PAI-1. A class of inhibitors, termed aptamers, has been used effectively in several clinical applications. We previously generated RNA aptamers that target PAI-1 and demonstrated their ability to inhibit extracellular PAI-1. In the current study we explored the effect of these aptamers on intracellular PAI-1. We transiently transfected the PAI-1 specific aptamers into both MDA-MB-231 human breast cancer cells, and human umbilical vein endothelial cells (HUVECs) and studied their effects on cell migration, invasion and angiogenesis. Aptamer expressing MDA-MB-231 cells exhibited a decrease in cell migration and invasion. Additionally, intracellular PAI-1 and urokinase plasminogen activator (uPA) protein levels decreased, while the PAI-1/uPA complex increased. Moreover, a significant decrease in endothelial tube formation in HUVECs transfected with the aptamers was observed. In contrast, conditioned media from aptamer transfected MDA-MB-231 cells displayed a slight pro-angiogenic effect. Collectively, our study shows that expressing functional aptamers inside breast and endothelial cells is feasible and may exhibit therapeutic potential. PMID:27755560

  1. Modular Assembly of Cell-targeting Devices Based on an Uncommon G-quadruplex Aptamer

    DEFF Research Database (Denmark)

    Opazo, Felipe; Eiden, Laura; Hansen, Line

    2015-01-01

    Aptamers are valuable tools that provide great potential to develop cost-effective diagnostics and therapies in the biomedical field. Here, we report a novel DNA aptamer that folds into an unconventional G-quadruplex structure able to recognize and enter specifically into human Burkitt's lymphoma...

  2. A replaceable liposomal aptamer for the ultrasensitive and rapid detection of biotin

    Science.gov (United States)

    Sung, Tzu-Cheng; Chen, Wen-Yih; Shah, Pramod; Chen, Chien-Sheng

    2016-02-01

    Biotin is an essential vitamin which plays an important role for maintaining normal physiological function. A rapid, sensitive, and simple method is necessary to monitor the biotin level. Here, we reported a replacement assay for the detection of biotin using a replaceable liposomal aptamer. Replacement assay is a competitive assay where a sample analyte replaces the labeled competitor of analyte out of its biorecognition element on a surface. It is user friendly and time-saving because of washing free. We used aptamer as a competitor, not a biorecognition element as tradition. To label aptamers, we used cholesterol-conjugated aptamers to tag signal-amplifying-liposomes. Without the need of conjugation procedure, aptamers can be easily incorporated into the surface of dye-encapsulating liposomes. Two aptamers as competitors of biotin, ST-21 and ST-21M with different affinities to streptavidin, were studied in parallel for the detection of biotin using replacement assays. ST-21 and ST-21M aptamers reached to limits of detection of 1.32 pg/80 μl and 0.47 pg/80 μl, respectively. The dynamic ranges of our assays using ST-21 and ST-21M aptamers were seven and four orders of magnitude, respectively. This assay can be completed in 20 minutes without washing steps. These results were overall better than previous reported assays.

  3. DNA aptamer beacon assay for C-telopeptide and handheld fluorometer to monitor bone resorption.

    Science.gov (United States)

    Bruno, John Gordon; Carrillo, Maria P; Phillips, Taylor; Hanson, Douglas; Bohmann, Jonathan A

    2011-09-01

    A novel DNA aptamer beacon is described for quantification of a 26-amino acid C-telopeptide (CTx) of human type I bone collagen. One aptamer sequence and its reverse complement dominated the aptamer pool (31.6% of sequenced clones). Secondary structures of these aptamers were examined for potential binding pockets. Three-dimensional computer models which analyzed docking topologies and binding energies were in agreement with empirical fluorescence experiments used to select one candidate loop for beacon assay development. All loop structures from the aptamer finalists were end-labeled with TYE 665 and Iowa Black quencher for comparison of beacon fluorescence levels as a function of CTx concentration. The optimal beacon, designated CTx 2R-2h yielded a low ng/ml limit of detection using a commercially available handheld fluorometer. The CTx aptamer beacon bound full-length 26-amino acid CTx peptide, but not a shorter 8-amino acid segment of CTx peptide which is a common target for commercial CTx ELISA kits. The prototype assay was shown to detect CTx peptide from human urine after creatinine and urea were removed by size-exclusion chromatography to prevent nonspecific denaturing of the aptamer beacon. This work demonstrates the potential of aptamer beacons to be utilized for rapid and sensitive bone health monitoring in a handheld or point-of-care format.

  4. Aptamers as a promising approach for the control of parasitic diseases

    Directory of Open Access Journals (Sweden)

    Juan David Ospina-Villa

    Full Text Available ABSTRACT Aptamers are short single-stranded RNA or DNA oligonucleotides that are capable of binding various biological targets with high affinity and specificity. Their identification initially relies on a molecular process named SELEX (Systematic Evolution of Ligands by EXponential enrichment that has been later modified in order to improve aptamer sensitivity, minimize duration and cost of the assay, as well as increase target types. Several biochemical modifications can help to enhance aptamer stability without affecting significantly target interaction. As a result, aptamers have generated a large interest as promising tools to compete with monoclonal antibodies for detection and inhibition of specific markers of human diseases. One aptamer-based drug is currently authorized and several others are being clinically evaluated. Despite advances in the knowledge of parasite biology and host-parasite interactions from "omics" data, protozoan parasites still affect millions of people around the world and there is an urgent need for drug target discovery and novel therapeutic concepts. In this context, aptamers represent promising tools for pathogen identification and control. Recent studies have reported the identification of "aptasensors" for parasite diagnosis, and "intramers" targeting intracellular proteins. Here we discuss various strategies that have been employed for intracellular expression of aptamers and expansion of their possible application, and propose that they may be suitable for the clinical use of aptamers in parasitic infections.

  5. DNA aptamer release from the DNA-SWNT hybrid by protein recognition.

    Science.gov (United States)

    Yoo, Chang-Hyuk; Jung, Seungwon; Bae, Jaehyun; Kim, Gunn; Ihm, Jisoon; Lee, Junghoon

    2016-02-14

    Here we show the formation of the complex between a DNA aptamer and a single-walled carbon nanotube (SWNT) and its reaction with its target protein. The aptamer, which is specifically bound with thrombin, the target protein in this study, easily wraps and disperses the SWNT by noncovalent π-π stacking.

  6. RNA aptamer-based electrochemical biosensor for selective and label-free analysis of dopamine

    DEFF Research Database (Denmark)

    Farjami, Elahe; Campos, Rui; Nielsen, Jesper Sejrup

    2013-01-01

    , including dopamine precursors and metabolites and other neurotransmitters (NT). Here we report an electrochemical RNA aptamer-based biosensor for analysis of dopamine in the presence of other NT. The biosensor exploits a specific binding of dopamine by the RNA aptamer, immobilized at a cysteamine...

  7. Nucleic Acid Aptamers Against Biotoxins: A New Paradigm Toward the Treatment and Diagnostic Approach

    DEFF Research Database (Denmark)

    Lauridsen, Lasse Holm; Veedu, Rakesh N.

    2012-01-01

    to combat these problems. Fully sequestered in vitro, aptamers eliminate the need for a living host. Furthermore, one of the key advantages of using aptamers instead of antibodies is that they can be selected against very weakly immunogenic and cytotoxic substances. In this review, we focus on nucleic acid...

  8. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity.

    Science.gov (United States)

    Wang, Deng-Liang; Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan; Yang, Hai-Tao; Wang, Jiang-Jie; Yao, Pei-Sen; Pan, Ru-Jun; Yang, Chaoyong James; Kang, De-Zhi

    2014-10-31

    Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher's attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with Kd 56±7.3nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy.

  9. Intracellular Expression of PAI-1 Specific Aptamers Alters Breast Cancer Cell Migration, Invasion and Angiogenesis.

    Science.gov (United States)

    Fortenberry, Yolanda M; Brandal, Stephanie M; Carpentier, Gilles; Hemani, Malvi; Pathak, Arvind P

    2016-01-01

    Plasminogen activator inhibitor-1 (PAI-1) is elevated in various cancers, where it has been shown to effect cell migration and invasion and angiogenesis. While, PAI-1 is a secreted protein, its intercellular levels are increased in cancer cells. Consequently, intracellular PAI-1 could contribute to cancer progression. While various small molecule inhibitors of PAI-1 are currently being investigated, none specifically target intracellular PAI-1. A class of inhibitors, termed aptamers, has been used effectively in several clinical applications. We previously generated RNA aptamers that target PAI-1 and demonstrated their ability to inhibit extracellular PAI-1. In the current study we explored the effect of these aptamers on intracellular PAI-1. We transiently transfected the PAI-1 specific aptamers into both MDA-MB-231 human breast cancer cells, and human umbilical vein endothelial cells (HUVECs) and studied their effects on cell migration, invasion and angiogenesis. Aptamer expressing MDA-MB-231 cells exhibited a decrease in cell migration and invasion. Additionally, intracellular PAI-1 and urokinase plasminogen activator (uPA) protein levels decreased, while the PAI-1/uPA complex increased. Moreover, a significant decrease in endothelial tube formation in HUVECs transfected with the aptamers was observed. In contrast, conditioned media from aptamer transfected MDA-MB-231 cells displayed a slight pro-angiogenic effect. Collectively, our study shows that expressing functional aptamers inside breast and endothelial cells is feasible and may exhibit therapeutic potential.

  10. Shortening full-length aptamer by crawling base deletion – Assisted by Mfold web server application

    Directory of Open Access Journals (Sweden)

    Subash C.B. Gopinath

    2017-06-01

    Full Text Available Systematic Evolution of Ligands by EXponential enrichment (SELEX is the method to select the specific aptamer against a wide range of targets. For this process, the initial library usually has a length of random sequences from ∼25 and it reaches over 100 bases. The lengthy sequences have disadvantages such as difficult to prepare, less stable and expensive. It is wise to prefer shorter version of aptamer for a wide range of applications including drug delivery process. It is a common practice to shorten the full-length aptamer by mapping analyses and it is tedious. Here, we used a crawling method to shorten the aptamer by different sequential deletion of bases from both 5′ and 3′ ends, assisted by Mfold web server application. Two different kinds of aptamer with varied lengths (randomized region of 30 and 74 bases were desired for this study, generated against Influenza A/Panama/2007/1999 (H3N2 and gD protein of Herpes Simplex Virus-1. It was found that shortening the aptamer length by crawling pattern is possible with the assistance of Mfold web server application. The obtained results resemble the shortened aptamer derived by mapping analyses. The proposed strategy is recommended to predict the shorter aptamer without involving any wet experimental section.

  11. Cell-SELEX-based selection of aptamers that recognize distinct targets on metastatic colorectal cancer cells.

    Science.gov (United States)

    Li, Wan-Ming; Bing, Tao; Wei, Jia-Yi; Chen, Zhe-Zhou; Shangguan, Di-Hua; Fang, Jin

    2014-08-01

    The development of diagnostic/therapeutic strategies against metastasis-related molecular targets is critical for improving the survival rate of cancer patients. Subtractive Cell-SELEX was performed using highly metastatic colorectal cancer (CRC) LoVo cells and non-metastatic HCT-8 cells as the target and negative cells, respectively, for the selection of metastatic-specific aptamers. This process generated seven aptamers that displayed highly specific binding to the target cells with Kds in the nanomolar range. Based on the distinct chemical/biological properties of their individual cell surface targets, the aptamers were separately functionalized: the receptor-targeting aptamer W14 was used as a carrier for doxorubicin, resulting in the specific delivery of the drug to the target cells and a significant reduction of its cytotoxicity to non-target cells, and the non-receptor-binding aptamer W3 was used as a molecular probe conjugated to quantum dots for the targeted imaging of metastatic cancer cell lines, spontaneous lung metastasis murine tissue, and metastatic CRC patient tissues. In addition, these aptamers can be used in combination due to their lack of detectable mutual-binding interference. The study demonstrates that a panel of aptamers that recognize distinct features of target molecules can be obtained through single Cell-SELEX selection, and the selected aptamers may be individually functionalized for specific applications and/or utilized in combination. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Selection and identification of DNA aptamers against okadaic acid for biosensing application.

    Science.gov (United States)

    Eissa, Shimaa; Ng, Andy; Siaj, Mohamed; Tavares, Ana C; Zourob, Mohammed

    2013-12-17

    This work describes the selection and identification of DNA aptamers that bind with high affinity and specificity to okadaic acid (OA), a lipophilic marine biotoxin that accumulates in shellfish. The aptamers selected using systematic evolution of ligands by exponential enrichment (SELEX) exhibited dissociation constants in the nanomolar range. The aptamer with the highest affinity was then used for the fabrication of a label-free electrochemical biosensor for okadaic acid detection. The aptamer was first immobilized on the gold electrode by a self-assembly approach through Au-S interaction. The binding of okadaic acid to the aptamer immobilized on the electrode surface induces an alteration of the aptamer conformation causing a significant decrease in the electron-transfer resistance monitored by electrochemical impedance spectroscopy. The aptasensor showed a linear range for the concentrations of OA between 100 pg/mL and 60 ng/mL with a detection limit of 70 pg/mL. The dissociation constant of okadaic acid with the aptamer immobilized on the electrode surface showed good agreement with that determined using fluorescence assay in solution. Moreover, the aptasensor did not show cross-reactivity toward toxins with structures similar to okadaic acid such as dinophysis toxin-1 and 2 (DTX-1, DTX-2). Further biosensing applications of the selected aptamers are expected to offer promising alternatives to the traditional analytical and immunological methods for OA detection.

  13. Selective Evolution of Ligands by Exponential Enrichment to Identify RNA Aptamers against Shiga Toxins.

    Science.gov (United States)

    Challa, Sreerupa; Tzipori, Saul; Sheoran, Abhineet

    2014-01-01

    Infection with Shiga toxin- (Stx-) producing E. coli causes life threatening hemolytic uremic syndrome (HUS), a leading cause of acute renal failure in children. Of the two antigenically distinct toxins, Stx1 and Stx2, Stx2 is more firmly linked with the development of HUS. In the present study, selective evolution of ligands by exponential enrichment (SELEX) was used in an attempt to identify RNA aptamers against Stx1 and Stx2. After 5 rounds of selection, significant enrichment of aptamer pool was obtained against Stx2, but not against Stx1, using a RNA aptamer library containing 56 random nucleotides (N56). Characterization of individual aptamer sequences revealed that six unique RNA aptamers (mA/pC, mB/pA, mC, mD, pB, and pD) recognized Stx2 in a filter binding assay. None of these aptamers bound Stx1. Aptamers mA/pC, mB/pA, mC, and mD, but not pB and pD, partially blocked binding of Alexa 488-labeled Stx2 with HeLa cells in a flow cytometry assay. However, none of the aptamers neutralized Stx2-mediated cytotoxicity and death of HeLa cells.

  14. Selective Evolution of Ligands by Exponential Enrichment to Identify RNA Aptamers against Shiga Toxins

    Directory of Open Access Journals (Sweden)

    Sreerupa Challa

    2014-01-01

    Full Text Available Infection with Shiga toxin- (Stx- producing E. coli causes life threatening hemolytic uremic syndrome (HUS, a leading cause of acute renal failure in children. Of the two antigenically distinct toxins, Stx1 and Stx2, Stx2 is more firmly linked with the development of HUS. In the present study, selective evolution of ligands by exponential enrichment (SELEX was used in an attempt to identify RNA aptamers against Stx1 and Stx2. After 5 rounds of selection, significant enrichment of aptamer pool was obtained against Stx2, but not against Stx1, using a RNA aptamer library containing 56 random nucleotides (N56. Characterization of individual aptamer sequences revealed that six unique RNA aptamers (mA/pC, mB/pA, mC, mD, pB, and pD recognized Stx2 in a filter binding assay. None of these aptamers bound Stx1. Aptamers mA/pC, mB/pA, mC, and mD, but not pB and pD, partially blocked binding of Alexa 488-labeled Stx2 with HeLa cells in a flow cytometry assay. However, none of the aptamers neutralized Stx2-mediated cytotoxicity and death of HeLa cells.

  15. Combining aptamers and in silico interaction studies to decipher the function of hypothetical proteins

    DEFF Research Database (Denmark)

    Suravajhala, Prashanth; Burri, Harsha Vardhan Reddy; Heiskanen, Arto

    2014-01-01

    We present the potential role of aptamers in elucidating the function of hypothetical proteins, as well as the possibilities provided by bioinformatics for establishing a benchmark for aptamer-protein prediction methods. With these future perspectives, the role of hypothetical proteins as target ...

  16. Identification of CRISPR and riboswitch related RNAs among novel noncoding RNAs of the euryarchaeon Pyrococcus abyssi

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    Carpousis Agamemnon J

    2011-06-01

    Full Text Available Abstract Background Noncoding RNA (ncRNA has been recognized as an important regulator of gene expression networks in Bacteria and Eucaryota. Little is known about ncRNA in thermococcal archaea except for the eukaryotic-like C/D and H/ACA modification guide RNAs. Results Using a combination of in silico and experimental approaches, we identified and characterized novel P. abyssi ncRNAs transcribed from 12 intergenic regions, ten of which are conserved throughout the Thermococcales. Several of them accumulate in the late-exponential phase of growth. Analysis of the genomic context and sequence conservation amongst related thermococcal species revealed two novel P. abyssi ncRNA families. The CRISPR family is comprised of crRNAs expressed from two of the four P. abyssi CRISPR cassettes. The 5'UTR derived family includes four conserved ncRNAs, two of which have features similar to known bacterial riboswitches. Several of the novel ncRNAs have sequence similarities to orphan OrfB transposase elements. Based on RNA secondary structure predictions and experimental results, we show that three of the twelve ncRNAs include Kink-turn RNA motifs, arguing for a biological role of these ncRNAs in the cell. Furthermore, our results show that several of the ncRNAs are subjected to processing events by enzymes that remain to be identified and characterized. Conclusions This work proposes a revised annotation of CRISPR loci in P. abyssi and expands our knowledge of ncRNAs in the Thermococcales, thus providing a starting point for studies needed to elucidate their biological function.

  17. Protein-binding RNA aptamers affect molecular interactions distantly from their binding sites

    DEFF Research Database (Denmark)

    Dupont, Daniel M; Thuesen, Cathrine K; Bøtkjær, Kenneth A;

    2015-01-01

    Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless...... potential, both binding to the serine protease urokinase-type plasminogen activator (uPA). We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A) controlling uPA activities. One of the aptamers (upanap-126) binds to the area...... around the C-terminal α-helix in pro-uPA, while the other aptamer (upanap-12) binds to both the β-hairpin of the growth factor domain and the kringle domain of uPA. Based on the mapping studies, combined with data from small-angle X-ray scattering analysis, we construct a model for the upanap-12:pro...

  18. Selection and characterization of DNA aptamers against Staphylococcus aureus enterotoxin C1.

    Science.gov (United States)

    Huang, Yukun; Chen, Xiujuan; Duan, Nuo; Wu, Shijia; Wang, Zhouping; Wei, Xinlin; Wang, Yuanfeng

    2015-01-01

    Enterotoxins from pathogenic bacteria are known as the main reason that can cause the bacterial foodborne diseases. In this study, aptamers that bound to Staphylococcus aureus enterotoxin C1 (SEC1) with high affinity and selectivity were generated in vitro by twelve rounds of selection based on magnetic separation technology, with a low-level dissociation constant (Kd) value of 65.14 ± 11.64 nmol/L of aptamer C10. Aptamer-based quantification of SEC1 in the food sample by a graphene oxide (GO)-based method was implemented to investigate the potential of the aptamer against SEC1 with a limit of detection of 6 ng/mL. On the basis of this work, biosensors using the selected SEC1 aptamers as new molecular recognition elements could be applied for innovative determinations of SEC1.

  19. IN VITRO SELECTION AND CHARACTERIZATION OF CELLULOSE-BINDING RNA APTAMERS USING ISOTHERMAL AMPLIFICATION

    Science.gov (United States)

    Boese, B. J.; Corbino, K.; Breaker, R. R.

    2017-01-01

    We sought to create new cellulose-binding RNA aptamers for use as modular components in the engineering of complex functional nucleic acids. We designed our in vitro selection strategy to incorporate self-sustained sequence replication (3SR), which is an isothermal nucleic acid amplification protocol that allows for the rapid amplification of RNAs with little manipulation. The best performing aptamer representative was chosen for reselection and further optimization. The aptamer exhibits robust affinity for cellulose in both the powdered and paper form, but did not show any significant affinity for closely related polysaccharides. The minimal cellulose-binding RNA aptamer also can be grafted onto other RNAs to permit the isolation of RNAs from complex biochemical mixtures via cellulose affinity chromatography. This was demonstrated by fusing the aptamer to a glmS ribozyme sequence, and selectively eluting ribozyme cleavage products from cellulose using the glucosamine 6-phosphate to activate glmS ribozyme function. PMID:18696364

  20. Antidote control of aptamer therapeutics: the road to a safer class of drug agents.

    Science.gov (United States)

    Bompiani, K M; Woodruff, R S; Becker, R C; Nimjee, S M; Sullenger, B A

    2012-08-01

    Aptamers, or nucleic acid ligands, have gained clinical interest over the past 20 years due to their unique characteristics, which are a combination of the best facets of small molecules and antibodies. The high binding affinity and specificity of aptamers allows for isolation of an artificial ligand for theoretically any therapeutic target of interest. Chemical manipulations of aptamers also allow for fine-tuning of their bioavailability, and antidote control greatly expands their clinical use. Here we review the various methods of antidote control of aptamer therapeutics--matched oligonucleotide antidotes and universal antidotes. We also describe the development, recent progress, and potential future therapeutic applications of these types of aptamer-antidote pairs.

  1. DNA nanostructure-decorated surfaces for enhanced aptamer-target binding and electrochemical cocaine sensors.

    Science.gov (United States)

    Wen, Yanli; Pei, Hao; Wan, Ying; Su, Yan; Huang, Qing; Song, Shiping; Fan, Chunhai

    2011-10-01

    The sensitivity of aptamer-based electrochemical sensors is often limited by restricted target accessibility and surface-induced perturbation of the aptamer structure, which arise from imperfect packing of probes on the heterogeneous and locally crowded surface. In this study, we have developed an ultrasensitive and highly selective electrochemical aptamer-based cocaine sensor (EACS), based on a DNA nanotechnology-based sensing platform. We have found that the electrode surface decorated with an aptamer probe-pendant tetrahedral DNA nanostructure greatly facilitates cocaine-induced fusion of the split anticocaine aptamer. This novel design leads to a sensitive cocaine sensor with a remarkably low detection limit of 33 nM. It is also important that the tetrahedra-decorated surface is protein-resistant, which not only suits the enzyme-based signal amplification scheme employed in this work, but ensures high selectivity of this sensor when deployed in sera or other adulterated samples.

  2. Selection of Aptamers Against Whole Living Cells: From Cell-SELEX to Identification of Biomarkers.

    Science.gov (United States)

    Quang, Nam Nguyen; Miodek, Anna; Cibiel, Agnes; Ducongé, Frédéric

    2017-01-01

    Aptamer selection protocols, named cell-SELEX, have been developed to target trans-membrane proteins using whole living cells as target. This technique presents several advantages. (1) It does not necessitate the use of purified proteins. (2) Aptamers are selected against membrane proteins in their native conformation. (3) Cell-SELEX can be performed to identify aptamers against biomarkers differentially expressed between different cell lines without prior knowledge of the targets. (4) Aptamers identified by cell-SELEX can be further used to purify their targets and to identify new biomarkers. Here, we provide a protocol of cell-SELEX including the preparation of an oligonucleotide library, next-generation sequencing and radioactive binding assays. Furthermore, we also provide a protocol to purify and identify the target of these aptamers. These protocols could be useful for the discovery of lead therapeutic compounds and diagnostic cell-surface biomarkers.

  3. Insights into Metalloregulation by M-box Riboswitch RNAs via Structural Analysis of Manganese-Bound Complexes

    Energy Technology Data Exchange (ETDEWEB)

    Ramesh, Arati; Wakeman, Catherine A.; Winkler, Wade C. (UTSMC)

    2011-12-09

    The M-box riboswitch couples intracellular magnesium levels to expression of bacterial metal transport genes. Structural analyses on other riboswitch RNA classes, which typically respond to a small organic metabolite, have revealed that ligand recognition occurs through a combination of base-stacking, electrostatic, and hydrogen-bonding interactions. In contrast, the M-box RNA triggers a change in gene expression upon association with an undefined population of metals, rather than responding to only a single ligand. Prior biophysical experimentation suggested that divalent ions associate with the M-box RNA to promote a compacted tertiary conformation, resulting in sequestration of a short sequence tract otherwise required for downstream gene expression. Electrostatic shielding from loosely associated metals is undoubtedly an important influence during this metal-mediated compaction pathway. However, it is also likely that a subset of divalent ions specifically occupies cation binding sites and promotes proper positioning of functional groups for tertiary structure stabilization. To better elucidate the role of these metal binding sites, we resolved a manganese-chelated M-box RNA complex to 1.86 {angstrom} by X-ray crystallography. These data support the presence of at least eight well-ordered cation binding pockets, including several sites that had been predicted by biochemical studies but were not observed in prior structural analysis. Overall, these data support the presence of three metal-binding cores within the M-box RNA that facilitate a network of long-range interactions within the metal-bound, compacted conformation.

  4. Cyclic di-GMP contributes to adaption and virulence of Bacillus thuringiensis through a riboswitch-regulated collagen adhesion protein.

    Science.gov (United States)

    Tang, Qing; Yin, Kang; Qian, Hongliang; Zhao, Youwen; Wang, Wen; Chou, Shan-Ho; Fu, Yang; He, Jin

    2016-07-06

    Cyclic di-GMP is a ubiquitous second messenger that regulates diverse cellular processes in bacteria by binding to various protein or riboswitch effectors. In Bacillus thuringiensis BMB171, a c-di-GMP riboswitch termed Bc2 RNA resides in the 5'-untranslated region (5'-UTR) of an mRNA that encodes a collagen adhesion protein (Cap). The expression of cap was strongly repressed in parent strain BMB171 because of the presence of Bc2 RNA but was significantly promoted in the Bc2 RNA markerless deletion mutant. Bc2 RNA acts as a genetic "on" switch, which forms an anti-terminator structure to promote cap read-through transcription upon c-di-GMP binding. As a result, cap transcription was de-repressed under high c-di-GMP levels. Therefore, Bc2 RNA regulates cap expression using a repression/de-repression model. Bc2 RNA-regulated Cap was also found to be tightly associated with motility, aggregation, exopolysaccharide secretion, biofilm formation, and virulence of B. thuringiensis BMB171 against its host insect Helicoverpa armigera.

  5. Aptamer conjugated paclitaxel and magnetic fluid loaded fluorescently tagged PLGA nanoparticles for targeted cancer therapy

    Energy Technology Data Exchange (ETDEWEB)

    Aravind, Athulya; Nair, Remya; Raveendran, Sreejith; Veeranarayanan, Srivani; Nagaoka, Yutaka; Fukuda, Takahiro; Hasumura, Takahashi; Morimoto, Hisao; Yoshida, Yasuhiko; Maekawa, Toru; Sakthi Kumar, D., E-mail: sakthi@toyo.jp

    2013-10-15

    Controlled and targeted drug delivery is an essential criterion in cancer therapy to reduce the side effects caused by non-specific drug release and toxicity. Targeted chemotherapy, sustained drug release and optical imaging have been achieved using a multifunctional nanocarrier constructed from poly (D, L-lactide-co-glycolide) nanoparticles (PLGA NPs), an anticancer drug paclitaxel (PTX), a fluorescent dye Nile red (NR), magnetic fluid (MF) and aptamers (Apt, AS1411, anti-nucleolin aptamer). The magnetic fluid and paclitaxel loaded fluorescently labeled PLGA NPs (MF-PTX-NR-PLGA NPs) were synthesized by a single-emulsion technique/solvent evaporation method using a chemical cross linker bis (sulfosuccinimidyl) suberate (BS3) to enable binding of aptamer on to the surface of the nanoparticles. Targeting aptamers were then introduced to the particles through the reaction with the cross linker to target the nucleolin receptors over expressed on the cancer cell surface. Specific binding and uptake of the aptamer conjugated magnetic fluid loaded fluorescently tagged PLGA NPs (Apt-MF-NR-PLGA NPs) to the target cancer cells induced by aptamers was observed using confocal microscopy. Cytotoxicity assay conducted in two cell lines (L929 and MCF-7) confirmed that targeted MCF-7 cancer cells were killed while control cells were unharmed. In addition, aptamer mediated delivery resulting in enhanced binding and uptake to the target cancer cells exhibited increased therapeutic effect of the drug. Moreover, these aptamer conjugated magnetic polymer vehicles apart from actively transporting drugs into specifically targeted tumor regions can also be used to induce hyperthermia or for facilitating magnetic guiding of particles to the tumor regions. - Highlights: • Aptamer escorted, theranostic biodegradable PLGA carriers were developed. • Can target cancer cells, control drug release, image and magnetically guide. • Highly specific to the targeted cancer cells thus delivering

  6. Surface biofunctionalization of β-TCP blocks using aptamer 74 for bone tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Ardjomandi, N.; Huth, J. [Department of Oral and Maxillofacial Surgery, University Hospital Tübingen (Germany); Stamov, D.R. [JPK Instruments AG, Berlin (Germany); Henrich, A. [Department of Oral and Maxillofacial Surgery, University Hospital Tübingen (Germany); Klein, C. [Dental Practice Zahngesundheit Waiblingen, Waiblingen (Germany); Wendel, H.-P. [Department of Thoracic, Cardiac and Vascular Surgery, University Hospital, Tübingen (Germany); Reinert, S. [Department of Oral and Maxillofacial Surgery, University Hospital Tübingen (Germany); Alexander, D., E-mail: dorothea.alexander@med.uni-tuebingen.de [Department of Oral and Maxillofacial Surgery, University Hospital Tübingen (Germany)

    2016-10-01

    Successful bone regeneration following oral and maxillofacial surgeries depends on efficient functionalization strategies that allow the recruitment of osteogenic progenitor cells at the tissue/implant interface. We have previously identified aptamer 74, which exhibited a binding affinity for osteogenically induced jaw periosteal cells (JPCs). In the present study, this aptamer was used for the surface biofunctionalization of β-tricalcium phosphate (β-TCP) blocks. Atomic force microscopy (AFM) measurements showed increased binding activity of aptamer 74 towards osteogenically induced JPCs compared to untreated controls. The immobilization efficiency of aptamer 74 was analyzed using the QuantiFluor ssDNA assay for 2D surfaces and by amino acid analysis for 3D β-TCP constructs. Following the successful immobilization of aptamer 74 in 2D culture wells and on 3D constructs, in vitro assays showed no significant differences in cell proliferation compared to unmodified surfaces. Interestingly, JPC mineralization was significantly higher on the 2D surfaces and higher cell adhesion was detected on the 3D constructs with immobilized aptamer. Herein, we report an established, biocompatible β-TCP matrix with surface immobilization of aptamer 74, which enhances properties such as cell adhesion on 3D constructs and mineralization on 2D surfaces. Further studies need to be performed to improve the immobilization efficiency and to develop a suitable approach for JPC mineralization growing within 3D β-TCP constructs. - Highlights: • Covalent binding of aptamer 74 on PLGA-coated β-tricalcium phosphate constructs. • AFM analysis of rupture forces between aptamer 74 and jaw periosteal cells. • Analysis of jaw periosteal cell functions on aptamer coated β-TCP constructs.

  7. Development of an aptamer beacon for detection of interferon-gamma.

    Science.gov (United States)

    Tuleuova, Nazgul; Jones, Caroline N; Yan, Jun; Ramanculov, Erlan; Yokobayashi, Yohei; Revzin, Alexander

    2010-03-01

    Traditional antibody-based affinity sensing strategies employ multiple reagents and washing steps and are unsuitable for real-time detection of analyte binding. Aptamers, on the other hand, may be designed to monitor binding events directly, in real-time, without the need for secondary labels. The goal of the present study was to design an aptamer beacon for fluorescence resonance energy transfer (FRET)-based detection of interferon-gamma (IFN-gamma)--an important inflammatory cytokine. Variants of DNA aptamer modified with biotin moieties and spacers were immobilized on avidin-coated surfaces and characterized by surface plasmon resonance (SPR). The SPR studies showed that immobilization of aptamer via the 3' end resulted in the best binding IFN-gamma (K(d) = 3.44 nM). This optimal aptamer variant was then used to construct a beacon by hybridizing fluorophore-labeled aptamer with an antisense oligonucleotide strand carrying a quencher. SPR studies revealed that IFN-gamma binding with an aptamer beacon occurred within 15 min of analyte introduction--suggesting dynamic replacement of the quencher-complementary strand by IFN-gamma molecules. To further highlight biosensing applications, aptamer beacon molecules were immobilized inside microfluidic channels and challenged with varying concentration of analyte. Fluorescence microscopy revealed low fluorescence in the absence of analyte and high fluorescence after introduction of IFN-gamma. Importantly, unlike traditional antibody-based immunoassays, the signal was observed directly upon binding of analyte without the need for multiple washing steps. The surface immobilized aptamer beacon had a linear range from 5 to 100 nM and a lower limit of detection of 5 nM IFN-gamma. In conclusion, we designed a FRET-based aptamer beacon for monitoring of an inflammatory cytokine-IFN-gamma. In the future, this biosensing strategy will be employed to monitor dynamics of cytokine production by the immune cells.

  8. Analysis and Identification of Aptamer-Compound Interactions with a Maximum Relevance Minimum Redundancy and Nearest Neighbor Algorithm

    Directory of Open Access Journals (Sweden)

    ShaoPeng Wang

    2016-01-01

    Full Text Available The development of biochemistry and molecular biology has revealed an increasingly important role of compounds in several biological processes. Like the aptamer-protein interaction, aptamer-compound interaction attracts increasing attention. However, it is time-consuming to select proper aptamers against compounds using traditional methods, such as exponential enrichment. Thus, there is an urgent need to design effective computational methods for searching effective aptamers against compounds. This study attempted to extract important features for aptamer-compound interactions using feature selection methods, such as Maximum Relevance Minimum Redundancy, as well as incremental feature selection. Each aptamer-compound pair was represented by properties derived from the aptamer and compound, including frequencies of single nucleotides and dinucleotides for the aptamer, as well as the constitutional, electrostatic, quantum-chemical, and space conformational descriptors of the compounds. As a result, some important features were obtained. To confirm the importance of the obtained features, we further discussed the associations between them and aptamer-compound interactions. Simultaneously, an optimal prediction model based on the nearest neighbor algorithm was built to identify aptamer-compound interactions, which has the potential to be a useful tool for the identification of novel aptamer-compound interactions. The program is available upon the request.

  9. Comparison of Flow Cytometry and ELASA for Screening of Proper Candidate Aptamer in Cell-SELEX Pool.

    Science.gov (United States)

    Nabavinia, Maryam Sadat; Charbgoo, Fahimeh; Alibolandi, Mona; Mosaffa, Fatemeh; Gholoobi, Aida; Ramezani, Mohammad; Abnous, Khalil

    2017-07-19

    Aptamers are single-stranded RNA or DNA, which bind to their target with high affinity and specificity. Method of isolating aptamers against cell surface protein is called cell-SELEX. Common approach for monitoring cell-SELEX developed aptamers is flow cytometry. Since flow cytometry is costly and requires sophisticated equipments, we suggested implementing easy access, high throughput enzyme-link apta-sorbent assay test (ELASA) to confirm the specificity of aptamers selected through cell-SELEX process. In this regard, we compared ELASA and flow cytometry techniques in order to screen potent candidate aptamers against A2780 Rcis cell line, which were selected by cell-SELEX. The obtained results demonstrated that both ELASA and flow cytometry are identical in terms of sensivity and precision for aptamers selection. Then it could be concluded that ELASA method could be used as a versatile, inexpensive procedure for in vito evaluation of isolated aptamers from cell-SELEX based process.

  10. Development, screening, and analysis of DNA aptamer libraries potentially useful for diagnosis and passive immunity of arboviruses

    Directory of Open Access Journals (Sweden)

    Bruno John G

    2012-11-01

    Full Text Available Abstract Background Nucleic acid aptamers have long demonstrated the capacity to bind viral envelope proteins and to inhibit the progression of pathogenic virus infections. Here we report on initial efforts to develop and screen DNA aptamers against recombinant envelope proteins or synthetic peptides and whole inactivated viruses from several virulent arboviruses including Chikungunya, Crimean-Congo hemorrhagic fever (CCHF, dengue, tickborne encephalitis and West Nile viruses. We also analyzed sequence data and secondary structures for commonalities that might reveal consensus binding sites among the various aptamers. Some of the highest affinity and most specific aptamers in the down-selected libraries were demonstrated to have diagnostic utility in lateral flow chromatographic assays and in a fluorescent aptamer-magnetic bead sandwich assay. Some of the reported aptamers may also be able to bind viral envelope proteins in vivo and therefore may have antiviral potential in passive immunity or prophylactic applications. Results Several arbovirus DNA aptamer sequences emerged multiple times in the various down selected aptamer libraries thereby suggesting some consensus sequences for binding arbovirus envelope proteins. Screening of aptamers by enzyme-linked aptamer sorbent assay (ELASA was useful for ranking relative aptamer affinities against their cognate viral targets. Additional study of the aptamer sequences and secondary structures of top-ranked anti-arboviral aptamers suggest potential virus binding motifs exist within some of the key aptamers and are highlighted in the supplemental figures for this article. One sequence segment (ACGGGTCCGGACA emerged 60 times in the anti-CCHF aptamer library, but nowhere else in the anti-arbovirus library and only a few other times in a larger library of aptamers known to bind bacteria and rickettsia or other targets. Diagnostic utility of some of the aptamers for arbovirus detection in lateral flow

  11. Development of an efficient targeted cell-SELEX procedure for DNA aptamer reagents.

    Directory of Open Access Journals (Sweden)

    Susanne Meyer

    Full Text Available BACKGROUND: DNA aptamers generated by cell-SELEX offer an attractive alternative to antibodies, but generating aptamers to specific, known membrane protein targets has proven challenging, and has severely limited the use of aptamers as affinity reagents for cell identification and purification. METHODOLOGY: We modified the BJAB lymphoblastoma cell line to over-express the murine c-kit cell surface receptor. After six rounds of cell-SELEX, high-throughput sequencing and bioinformatics analysis, we identified aptamers that bound BJAB cells expressing c-kit but not wild-type BJAB controls. One of these aptamers also recognizes c-kit endogenously expressed by a mast cell line or hematopoietic progenitor cells, and specifically blocks binding of the c-kit ligand stem cell factor (SCF. This aptamer enables better separation by fluorescence-activated cell sorting (FACS of c-kit(+ hematopoietic progenitor cells from mixed bone marrow populations than a commercially available antibody, suggesting that this approach may be broadly useful for rapid isolation of affinity reagents suitable for purification of other specific cell types. CONCLUSIONS/SIGNIFICANCE: Here we describe a novel procedure for the efficient generation of DNA aptamers that bind to specific cell membrane proteins and can be used as high affinity reagents. We have named the procedure STACS (Specific TArget Cell-SELEX.

  12. Surface biofunctionalization of β-TCP blocks using aptamer 74 for bone tissue engineering.

    Science.gov (United States)

    Ardjomandi, N; Huth, J; Stamov, D R; Henrich, A; Klein, C; Wendel, H-P; Reinert, S; Alexander, D

    2016-10-01

    Successful bone regeneration following oral and maxillofacial surgeries depends on efficient functionalization strategies that allow the recruitment of osteogenic progenitor cells at the tissue/implant interface. We have previously identified aptamer 74, which exhibited a binding affinity for osteogenically induced jaw periosteal cells (JPCs). In the present study, this aptamer was used for the surface biofunctionalization of β-tricalcium phosphate (β-TCP) blocks. Atomic force microscopy (AFM) measurements showed increased binding activity of aptamer 74 towards osteogenically induced JPCs compared to untreated controls. The immobilization efficiency of aptamer 74 was analyzed using the QuantiFluor ssDNA assay for 2D surfaces and by amino acid analysis for 3D β-TCP constructs. Following the successful immobilization of aptamer 74 in 2D culture wells and on 3D constructs, in vitro assays showed no significant differences in cell proliferation compared to unmodified surfaces. Interestingly, JPC mineralization was significantly higher on the 2D surfaces and higher cell adhesion was detected on the 3D constructs with immobilized aptamer. Herein, we report an established, biocompatible β-TCP matrix with surface immobilization of aptamer 74, which enhances properties such as cell adhesion on 3D constructs and mineralization on 2D surfaces. Further studies need to be performed to improve the immobilization efficiency and to develop a suitable approach for JPC mineralization growing within 3D β-TCP constructs. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Robust suppression of HIV replication by intracellularly expressed reverse transcriptase aptamers is independent of ribozyme processing.

    Science.gov (United States)

    Lange, Margaret J; Sharma, Tarun K; Whatley, Angela S; Landon, Linda A; Tempesta, Michael A; Johnson, Marc C; Burke, Donald H

    2012-12-01

    RNA aptamers that bind human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) also inhibit viral replication, making them attractive as therapeutic candidates and potential tools for dissecting viral pathogenesis. However, it is not well understood how aptamer-expression context and cellular RNA pathways govern aptamer accumulation and net antiviral bioactivity. Using a previously-described expression cassette in which aptamers were flanked by two "minimal core" hammerhead ribozymes, we observed only weak suppression of pseudotyped HIV. To evaluate the importance of the minimal ribozymes, we replaced them with extended, tertiary-stabilized hammerhead ribozymes with enhanced self-cleavage activity, in addition to noncleaving ribozymes with active site mutations. Both the active and inactive versions of the extended hammerhead ribozymes increased inhibition of pseudotyped virus, indicating that processing is not necessary for bioactivity. Clonal stable cell lines expressing aptamers from these modified constructs strongly suppressed infectious virus, and were more effective than minimal ribozymes at high viral multiplicity of infection (MOI). Tertiary stabilization greatly increased aptamer accumulation in viral and subcellular compartments, again regardless of self-cleavage capability. We therefore propose that the increased accumulation is responsible for increased suppression, that the bioactive form of the aptamer is one of the uncleaved or partially cleaved transcripts, and that tertiary stabilization increases transcript stability by reducing exonuclease degradation.

  14. Rationally Designing Aptamer Sequences with Reduced Affinity for Controlled Sensor Performance

    Directory of Open Access Journals (Sweden)

    Lauren R. Schoukroun-Barnes

    2015-03-01

    Full Text Available The relative ease of predicting the secondary structure of nucleic acid sequences lends itself to the design of sequences to perform desired functions. Here, we combine the utility of nucleic acid aptamers with predictable control over the secondary structure to rationally design sequences with controlled affinity towards a target analyte when employed as the recognition element in an electrochemical sensor. Specifically, we present a method to modify an existing high-gain aptamer sequence to create sequences that, when employed in an electrochemical, aptamer-based sensor, exhibit reduced affinity towards a small molecule analyte tobramycin. Sensors fabricated with the high-gain parent sequence saturate at concentrations much below the therapeutic window for tobramycin (7–18 µM. Accordingly, the rationale behind modifying this high-gain sequence to reduce binding affinity was to tune sensor performance for optimal sensitivity in the therapeutic window. Using secondary structure predictions and analysis of the NMR structure of an aminoglycoside RNA aptamer bound to tobramycin, we are able to successfully modify the aptamer sequence to tune the dissociation constants of electrochemical aptamer-based sensors between 0.17 and 3 µM. The guidelines we present represent a general strategy to lessening binding affinity of sensors employing aptamer-modified electrodes.

  15. Tumor targeting with a (99m)Tc-labeled AS1411 aptamer in prostate tumor cells.

    Science.gov (United States)

    Noaparast, Zohreh; Hosseinimehr, Seyed Jalal; Piramoon, Majid; Abedi, Seyed Mohammad

    2015-01-01

    AS1411, a 26-base guanine-rich oligonucleotide aptamer, has high affinity to nucleolin, mainly on tumor cell surfaces. In this study, a modified AS1411 was labeled with (99m)Tc and evaluated as a potential tumor-targeting agent for imaging. The AS1411 aptamer was conjugated with HYNIC and labeled with (99m)Tc in the presence a co-ligand. Radiochemical purity and stability testing of the (99m)Tc-HYNIC-AS1411 aptamer were carried out with thin layer chromatography and a size-exclusion column in normal saline and human serum. Cellular nucleolin-specific binding, cellular internalization in DU-145 cells, as high levels of nucleolin expression, were performed. Additionally, biodistribution in normal mice and DU-145 tumour-bearing mice was assessed. Radiolabeling of the aptamer resulted in a reasonable yield and radiochemical purity after purification. The aptamer was stable in normal saline and human serum, and cellular experiments demonstrated specific binding of the AS1411 aptamer to the nucleolin protein. Based on biodistribution assessment of (99m)Tc-HYNIC-AS1411, rapid blood clearance was seen after injection and it appears that the excretion route was via the urinary system at 1 h post-injection. Tumours also showed a higher accumulation of radioactivity with this labeled aptamer. (99m)Tc-AS1411 can be a potential tool for the molecular imaging of nucleolin-overexpressing cancers.

  16. Application of capillary electrophoresis to the development and evaluation of aptamer affinity probes

    Science.gov (United States)

    Sooter, Letha J.; McMasters, Sun; Stratis-Cullum, Dimitra N.

    2007-09-01

    Nucleic acid aptamers can exhibit high binding affinities for a wide variety of targets and have received much attention as molecular recognition elements for enhanced biosensor performance. These aptamers recognize target molecules through a combination of conformational dependent non-covalent interactions in aqueous media which can be investigated using capillary electrophoresis-based methods. In this paper we report on the results of our studies of the relative binding affinity of Campylobacter jejuni aptamers using a capillary electrophoretic immunoassay. Our results show preferential binding to C. jejuni over other common food pathogen bacteria. Capillary electrophoresis can also be used to develop new aptamer recognition elements using an in vitro selection process known as systematic evolution of ligand by exponential enrichment (SELEX). Recently, this process has been adapted to use capillary electrophoresis in an attempt to shorten the overall selection process. This smart selection of nucleic acid aptamers from a large diversity of a combinatorial DNA library is under optimization for the development of aptamers which bind to Army-relevant targets. This paper will include a discussion of the establishment of CE-SELEX methods for the future development of smart aptamer probes.

  17. Inhibition of PAI-1 antiproteolytic activity against tPA by RNA aptamers.

    Science.gov (United States)

    Damare, Jared; Brandal, Stephanie; Fortenberry, Yolanda M

    2014-08-01

    Plasminogen activator inhibitor-1 (PAI-1; SERPINE1) inhibits the plasminogen activators: tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). Elevated levels of PAI-1 have been correlated with an increased risk for cardiovascular disease. Pharmacologically suppressing PAI-1 might prevent, or successfully treat PAI-1 related vascular diseases. This can potentially be accomplished by using small RNA molecules (aptamers). This study's goal is to develop RNA aptamers to a region of PAI-1 that will prevent the ability of PAI-1 to interact with the plasminogen activators. The aptamers were generated through a systematic evolution of ligands via exponential enrichment approach that ensures the creation of RNA molecules that bind to our target protein, PAI-1. In vitro assays were used to determine the effect of these aptamers on PAI-1's inhibitory activity. Three aptamers that bind to PAI-1 with affinities in the nanomolar range were isolated. The aptamer clones R10-4 and R10-2 inhibited PAI-1's antiproteolytic activity against tPA and disrupted PAI-1's ability to form a stable covalent complex with tPA. Increasing aptamer concentrations correlated positively with an increase in cleaved PAI-1. To the best of our knowledge, this is the first report of RNA molecules that inhibit the antiproteolytic activity of PAI-1.

  18. Aptamer-based surface plasmon resonance sensing of glycated human blood proteins

    Science.gov (United States)

    Reaver, Nathan G. F.; Zheng, Rui; Kim, Dong-Shik; Cameron, Brent D.

    2013-02-01

    The concentration ratio of glycated to non-glycated forms of various blood proteins can be used as a diagnostic measure in diabetes to determine a history of glycemic compliance. Depending on a protein's half-life in blood, compliance can be assessed from a few days to several months in the past, which can then be used to provide additional therapeutic guidance. Current glycated protein detection methods are limited in their ability to measure multiple proteins, and are susceptible to interference from other blood pathologies. In this study, we developed and characterized DNA aptamers for use in Surface Plasmon Resonance (SPR) sensors to assess the blood protein hemoglobin. The aptamers were developed by way of a modified Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process which selects DNA sequences that have a high binding affinity to a specific protein. DNA products resulting from this process are sequenced and identified aptamers are then synthesized. The SELEX process was performed to produce aptamers for a glycated form of hemoglobin. Equilibrium dissociation constants for the binding of the identified aptamer to glycated hemoglobin, hemoglobin, and fibrinogen were calculated from fitted Langmuir isotherms obtained through SPR. These constants were determined to be 94 nM, 147 nM, and 244 nM respectively. This aptamer can potentially be used to create a SPR aptamer based biosensor for detection of glycated hemoglobin, a technology that has the potential to deliver low-cost and immediate glycemic compliance assessment in either a clinical or home setting.

  19. RAGE-Aptamer Blocks the Development and Progression of Experimental Diabetic Nephropathy.

    Science.gov (United States)

    Matsui, Takanori; Higashimoto, Yuichiro; Nishino, Yuri; Nakamura, Nobutaka; Fukami, Kei; Yamagishi, Sho-Ichi

    2017-06-01

    The interaction of advanced glycation end products (AGEs) and their receptor (RAGE) plays a central role in diabetic nephropathy. We screened DNA aptamers directed against RAGE (RAGE-aptamers) in vitro and examined the effects on the development and progression of diabetic nephropathy in streptozotocin-induced diabetic rats. RAGE-aptamer bound to RAGE with a Kd of 5.68 nmol/L and resultantly blocked the binding of AGEs to RAGE. When diabetic rats received continuous intraperitoneal injection of RAGE-aptamer from week 7 to 11 of diabetes, the increases in renal NADPH oxidase activity, oxidative stress generation, AGE, RAGE, inflammatory and fibrotic gene and protein levels, macrophage and extracellular matrix accumulation, and albuminuria were significantly suppressed, which were associated with improvement of podocyte damage. Two-week infusion of RAGE-aptamer just after the induction of diabetes also inhibited the AGE-RAGE-oxidative stress system and MCP-1 levels in the kidneys of 8-week-old diabetic rats and simultaneously ameliorated podocyte injury and albuminuria. Moreover, RAGE-aptamer significantly suppressed the AGE-induced oxidative stress generation and inflammatory and fibrotic reactions in human cultured mesangial cells. The findings suggest that continuous infusion of RAGE-aptamer could attenuate the development and progression of experimental diabetic nephropathy by blocking the AGE-RAGE axis. © 2017 by the American Diabetes Association.

  20. Computational Selection of RNA Aptamer against Angiopoietin-2 and Experimental Evaluation

    Directory of Open Access Journals (Sweden)

    Wen-Pin Hu

    2015-01-01

    Full Text Available Angiogenesis plays a decisive role in the growth and spread of cancer and angiopoietin-2 (Ang2 is in the spotlight of studies for its unique role in modulating angiogenesis. The aim of this study was to introduce a computational simulation approach to screen aptamers with high binding ability for Ang2. We carried out computational simulations of aptamer-protein interactions by using ZDOCK and ZRANK functions in Discovery Studio 3.5 starting from the available information of aptamers generated through the systematic evolution of ligands by exponential enrichment (SELEX in the literature. From the best of three aptamers on the basis of ZRANK scores, 189 sequences with two-point mutations were created and simulated with Ang2. Then, we used a surface plasmon resonance (SPR biosensor to test 3 mutant sequences of high ZRANK scores along with a high and a low affinity binding sequence as reported in the literature. We found a selected RNA aptamer has a higher binding affinity and SPR response than a reported sequence with the highest affinity. This is the first study of in silico selection of aptamers against Ang2 by using the ZRANK scoring function, which should help to increase the efficiency of selecting aptamers with high target-binding ability.

  1. Development of a fraction collection approach in capillary electrophoresis SELEX for aptamer selection.

    Science.gov (United States)

    Luo, Zhaofeng; Zhou, Hongmin; Jiang, Hao; Ou, Huichao; Li, Xin; Zhang, Liyun

    2015-04-21

    Aptamers have attracted much attention due to their ability to bind to target molecules with high affinity and specificity. The development of an approach capable of efficiently generating aptamers through systematic evolution of ligands by exponential enrichment (SELEX) is particularly challenging. Herein, a fraction collection approach in capillary electrophoresis SELEX (FCE-SELEX) for the partition of a bound DNA-target complex is developed. By integrating fraction collection with a facile oil seal method for avoiding contamination while amplifying the bound DNA-target complex, in a single round of selection, a streptavidin-binding aptamer (SBA) has been generated. The affinity of aptamer SBA-36 for streptavidin (SA) is determined as 30.8 nM by surface plasmon resonance (SPR). Selectivity and biotin competition experiments demonstrate that the SBA-36 aptamer selected by FCE-SELEX is as efficient as those from other methods. Based on the ability of fraction collection in partition and collection of the aptamer-target complex from the original DNA library, FCE-SELEX can be a universal tool for the development of aptamers.

  2. Development of an Efficient Targeted Cell-SELEX Procedure for DNA Aptamer Reagents

    Science.gov (United States)

    Nie, Jeff; Stewart, Ron; McIntosh, Brian E.; Conti, Lisa R.; Ahmad, Kareem M.; Soh, H. Tom; Thomson, James A.

    2013-01-01

    Background DNA aptamers generated by cell-SELEX offer an attractive alternative to antibodies, but generating aptamers to specific, known membrane protein targets has proven challenging, and has severely limited the use of aptamers as affinity reagents for cell identification and purification. Methodology We modified the BJAB lymphoblastoma cell line to over-express the murine c-kit cell surface receptor. After six rounds of cell-SELEX, high-throughput sequencing and bioinformatics analysis, we identified aptamers that bound BJAB cells expressing c-kit but not wild-type BJAB controls. One of these aptamers also recognizes c-kit endogenously expressed by a mast cell line or hematopoietic progenitor cells, and specifically blocks binding of the c-kit ligand stem cell factor (SCF). This aptamer enables better separation by fluorescence-activated cell sorting (FACS) of c-kit+ hematopoietic progenitor cells from mixed bone marrow populations than a commercially available antibody, suggesting that this approach may be broadly useful for rapid isolation of affinity reagents suitable for purification of other specific cell types. Conclusions/Significance Here we describe a novel procedure for the efficient generation of DNA aptamers that bind to specific cell membrane proteins and can be used as high affinity reagents. We have named the procedure STACS (Specific TArget Cell-SELEX). PMID:23967247

  3. Screening and identification of DNA aptamers against T-2 toxin assisted by graphene oxide.

    Science.gov (United States)

    Chen, Xiujuan; Huang, Yukun; Duan, Nuo; Wu, Shijia; Xia, Yu; Ma, Xiaoyuan; Zhu, Changqing; Jiang, Yuan; Wang, Zhouping

    2014-10-22

    A high-affinity ssDNA aptamer that specifically binds to T-2 toxin was generated by the systemic evolution of ligands by exponential enrichment (SELEX) procedure assisted by graphene oxide (GO). After 10 rounds of selection against T-2 toxin, a highly enriched ssDNA pool was sequenced and the representative aptamers were subjected to binding assays to evaluate their affinity and specificity. Circular dichroism spectroscopy was also used to study the inherent interaction of T-2 toxin and the preferred aptamer Seq.16, which demonstrated a low dissociation constant (Kd) of 20.8 ± 3.1 nM and excellent selectivity for T-2 toxin. Using the selected aptamer Seq.16 as the recognition element, an aptamer-based fluorescent bioassay was developed for the measurement of T-2 in beer samples with a linear range from 0.5 to 37.5 μM (R(2) = 0.988) and a limit of detection (LOD) of 0.4 μM. The results indicate that GO-SELEX technology is appropriate for the screening of aptamers against small-molecule toxins, offering a promising application for aptamer-based biosensors.

  4. A Review of Therapeutic Aptamer Conjugates with Emphasis on New Approaches

    Directory of Open Access Journals (Sweden)

    John G. Bruno

    2013-03-01

    Full Text Available The potential to emulate or enhance antibodies with nucleic acid aptamers while lowering costs has prompted development of new aptamer-protein, siRNA, drug, and nanoparticle conjugates. Specific focal points of this review discuss DNA aptamers covalently bound at their 3' ends to various proteins for enhanced stability and greater pharmacokinetic lifetimes in vivo. The proteins can include Fc tails of IgG for opsonization, and the first component of complement (C1q to trigger complement-mediated lysis of antibiotic-resistant Gram negative bacteria, cancer cells and possibly some parasites during vulnerable stages. In addition, the 3' protein adduct may be a biotoxin, enzyme, or may simply be human serum albumin (HSA or a drug known to bind HSA, thereby retarding kidney and other organ clearance and inhibiting serum exonucleases. In this review, the author summarizes existing therapeutic aptamer conjugate categories and describes his patented concept for PCR-based amplification of double-stranded aptamers followed by covalent attachment of proteins or other agents to the chemically vulnerable overhanging 3' adenine added by Taq polymerase. PCR amplification of aptamers could dramatically lower the current $2,000/gram cost of parallel chemical oligonucleotide synthesis, thereby enabling mass production of aptamer-3'-protein or drug conjugates to better compete against expensive humanized monoclonal antibodies.

  5. Isolation and Characterization of 2'-amino-modified RNA Aptamers for Human TNFα

    Institute of Scientific and Technical Information of China (English)

    Xinrui Yan; Xuwen Gao; Zhiqing Zhang

    2004-01-01

    Human tumor necrosis factor α (hTNFα), a pleiotropic cytokine with activities ranging from host defense mechanisms in infection and injury to severe toxicity in septic shock or other related diseases, is a promising target for drug screening. Using the SELEX (systematic evolution of ligands by exponential enrichment) process, we isolated oligonucleotide ligands (aptamers) with high affinities for hTNFα. Aptamers were selected from a starting pool of 40 randomized sequences composed of about 1015 RNA molecules. Representative aptamers were truncated to the minimal length with high affinity for hTNFα and were further modified by replacement of 2'-OH with 2'-F and 2'-NH2 at all ribopurine positions. These modified RNA aptamers were resistant to nuclease. The specificity of these aptamers for hTNFαwas confirmed, and their activity to inhibit the cytotoxicity of hTNFα on mouse L929 cells was determined. Results demonstrated that four 2'-NH2-modified aptamers bound to hTNFα with high affinity and blocked the binding of hTNFα to its receptor, thus protecting the L929 cells from the cytotoxicity of hTNFα. Oligonucleotide aptamers described here are potential therapeutics and diagnostics for hTNFc-related diseases.

  6. Fabrication of endothelial progenitor cell capture surface via DNA aptamer modifying dopamine/polyethyleneimine copolymer film

    Science.gov (United States)

    Li, Xin; Deng, Jinchuan; Yuan, Shuheng; Wang, Juan; Luo, Rifang; Chen, Si; Wang, Jin; Huang, Nan

    2016-11-01

    Endothelial progenitor cells (EPCs) are mainly located in bone marrow and circulate, and play a crucial role in repairmen of injury endothelium. One of the most promising strategies of stents designs were considered to make in-situ endothelialization in vivo via EPC-capture biomolecules on a vascular graft to capture EPCs directly from circulatory blood. In this work, an EPC specific aptamer with a 34 bases single strand DNA sequence was conjugated onto the stent surface via dopamine/polyethyleneimine copolymer film as a platform and linker. The assembled density of DNA aptamer could be regulated by controlling dopamine percentage in this copolymer film. X-ray photoelectron spectroscopy (XPS), water contact angle (WCA) and fluorescence test confirmed the successful immobilization of DNA aptamer. To confirm its biofunctionality and cytocompatibility, the capturing cells ability of the aptamer modified surface and the effects on the growth behavior of human umbilical vein endothelial cells (HUVECs), smooth muscle cells (SMCs) were investigated. The aptamer functionalized sample revealed a good EPC-capture ability, and had a cellular friendly feature for both EPC and EC growth, while not stimulated the hyperplasia of SMCs. And, the co-culture experiment of three types of cells confirmed the specificity capturing of EPCs to aptamer modified surface, rather than ECs and SMCs. These data suggested that this aptamer functionalized surface may have a large potentiality for the application of vascular grafts with targeted endothelialization.

  7. Imaging gene expression in real-time using aptamers

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Il Chung [Iowa State Univ., Ames, IA (United States)

    2011-01-01

    Signal transduction pathways are usually activated by external stimuli and are transient. The downstream changes such as transcription of the activated genes are also transient. Real-time detection of promoter activity is useful for understanding changes in gene expression, especially during cell differentiation and in development. A simple and reliable method for viewing gene expression in real time is not yet available. Reporter proteins such as fluorescent proteins and luciferase allow for non-invasive detection of the products of gene expression in living cells. However, current reporter systems do not provide for real-time imaging of promoter activity in living cells. This is because of the long time period after transcription required for fluorescent protein synthesis and maturation. We have developed an RNA reporter system for imaging in real-time to detect changes in promoter activity as they occur. The RNA reporter uses strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags), which can be expressed from a promoter of choice. The tobramycin, neomycin and PDC RNA aptamers have been utilized for this system and expressed in yeast from the GAL1 promoter. The IMAGEtag RNA kinetics were quantified by RT-qPCR. In yeast precultured in raffinose containing media the GAL1 promoter responded faster than in yeast precultured in glucose containing media. IMAGEtag RNA has relatively short half-life (5.5 min) in yeast. For imaging, the yeast cells are incubated with their ligands that are labeled with fluorescent dyes. To increase signal to noise, ligands have been separately conjugated with the FRET (Förster resonance energy transfer) pairs, Cy3 and Cy5. With these constructs, the transcribed aptamers can be imaged after activation of the promoter by galactose. FRET was confirmed with three different approaches, which were sensitized emission, acceptor photobleaching and donor lifetime by FLIM (fluorescence lifetime imaging

  8. Imaging gene expression in real-time using aptamers

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Ilchung [Iowa State Univ., Ames, IA (United States)

    2012-01-01

    Signal transduction pathways are usually activated by external stimuli and are transient. The downstream changes such as transcription of the activated genes are also transient. Real-time detection of promoter activity is useful for understanding changes in gene expression, especially during cell differentiation and in development. A simple and reliable method for viewing gene expression in real time is not yet available. Reporter proteins such as fluorescent proteins and luciferase allow for non-invasive detection of the products of gene expression in living cells. However, current reporter systems do not provide for real-time imaging of promoter activity in living cells. This is because of the long time period after transcription required for fluorescent protein synthesis and maturation. We have developed an RNA reporter system for imaging in real-time to detect changes in promoter activity as they occur. The RNA reporter uses strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags), which can be expressed from a promoter of choice. The tobramycin, neomycin and PDC RNA aptamers have been utilized for this system and expressed in yeast from the GAL1 promoter. The IMAGEtag RNA kinetics were quantified by RT-qPCR. In yeast precultured in raffinose containing media the GAL1 promoter responded faster than in yeast precultured in glucose containing media. IMAGEtag RNA has relatively short half-life (5.5 min) in yeast. For imaging, the yeast cells are incubated with their ligands that are labeled with fluorescent dyes. To increase signal to noise, ligands have been separately conjugated with the FRET (Förster resonance energy transfer) pairs, Cy3 and Cy5. With these constructs, the transcribed aptamers can be imaged after activation of the promoter by galactose. FRET was confirmed with three different approaches, which were sensitized emission, acceptor photobleaching and donor lifetime by FLIM (fluorescence lifetime imaging

  9. Recognition of Bungarus multicinctus venom by a DNA aptamer against β-bungarotoxin.

    Directory of Open Access Journals (Sweden)

    Fengping Ye

    Full Text Available Antibody-based technology is the main method for diagnosis and treatment of snake bite envenoming currently. However, the development of an antibody, polyclonal or monoclonal, is a complicated and costly procedure. Aptamers are single stranded oligonucleotides that recognize specific targets such as proteins and have shown great potential over the years as diagnostic and therapeutic agents. In contrast to antibodies, aptamers can be selected in vitro without immunization of animals, and synthesized chemically with extreme accuracy, low cost and high degree of purity. In this study we firstly report on the identification of DNA aptamers that bind to β-bungarotoxin (β-BuTx, a neurotoxin from the venom of Bungarus multicinctus. A plate-SELEX method was used for the selection of β-BuTx specific aptamers. After 10 rounds of selection, four aptamer candidates were obtained, with the dissociation constant ranged from 65.9 nM to 995 nM measured by fluorescence spectroscopy. Competitive binding assays using both the fluorescently labeled and unlabeled aptamers revealed that the four aptamers bound to the same binding site of β-BuTx. The best binder, βB-1, bound specifically to β-BuTx, but not to BSA, casein or α-Bungarotoxin. Moreover, electrophoretic mobility shift assay and enzyme-linked aptamer assay demonstrated that βB-1 could discriminate B. multicinctus venom from other snake venoms tested. The results suggest that aptamer βB-1 can serve as a useful tool for the design and development of drugs and diagnostic tests for β-BuTx poisoning and B. multicinctus bites.

  10. Evaluation of antithrombotic activity of thrombin DNA aptamers by a murine thrombosis model.

    Directory of Open Access Journals (Sweden)

    Elena Zavyalova

    Full Text Available Aptamers are nucleic acid based molecular recognition elements with a high potential for the theranostics. Some of the aptamers are under development for therapeutic applications as promising antithrombotic agents; and G-quadruplex DNA aptamers, which directly inhibit the thrombin activity, are among them. RA-36, the 31-meric DNA aptamer, consists of two thrombin binding pharmacophores joined with the thymine linker. It has been shown earlier that RA-36 directly inhibits thrombin in the reaction of fibrinogen hydrolysis, and also it inhibits plasma and blood coagulation. Studies of both inhibitory and anticoagulation effects had indicated rather high species specificity of the aptamer. Further R&D of RA-36 requires exploring its efficiency in vivo. Therefore the development of a robust and adequate animal model for effective physiological studies of aptamers is in high current demand. This work is devoted to in vivo study of the antithrombotic effect of RA-36 aptamer. A murine model of thrombosis has been applied to reveal a lag and even prevention of thrombus formation when RA-36 was intravenous bolus injected in high doses of 1.4-7.1 µmol/kg (14-70 mg/kg. A comparative study of RA-36 aptamer and bivalirudin reveals that both direct thrombin inhibitors have similar antithrombotic effects for the murine model of thrombosis; though in vitro bivalirudin has anticoagulation activity several times higher compared to RA-36. The results indicate that both RA-36 aptamer and bivalirudin are direct thrombin inhibitors of different potency, but possible interactions of the thrombin-inhibitor complex with other components of blood coagulation cascade level the physiological effects for both inhibitors.

  11. Evaluation of antithrombotic activity of thrombin DNA aptamers by a murine thrombosis model.

    Science.gov (United States)

    Zavyalova, Elena; Samoylenkova, Nadezhda; Revishchin, Alexander; Golovin, Andrey; Pavlova, Galina; Kopylov, Alexey

    2014-01-01

    Aptamers are nucleic acid based molecular recognition elements with a high potential for the theranostics. Some of the aptamers are under development for therapeutic applications as promising antithrombotic agents; and G-quadruplex DNA aptamers, which directly inhibit the thrombin activity, are among them. RA-36, the 31-meric DNA aptamer, consists of two thrombin binding pharmacophores joined with the thymine linker. It has been shown earlier that RA-36 directly inhibits thrombin in the reaction of fibrinogen hydrolysis, and also it inhibits plasma and blood coagulation. Studies of both inhibitory and anticoagulation effects had indicated rather high species specificity of the aptamer. Further R&D of RA-36 requires exploring its efficiency in vivo. Therefore the development of a robust and adequate animal model for effective physiological studies of aptamers is in high current demand. This work is devoted to in vivo study of the antithrombotic effect of RA-36 aptamer. A murine model of thrombosis has been applied to reveal a lag and even prevention of thrombus formation when RA-36 was intravenous bolus injected in high doses of 1.4-7.1 µmol/kg (14-70 mg/kg). A comparative study of RA-36 aptamer and bivalirudin reveals that both direct thrombin inhibitors have similar antithrombotic effects for the murine model of thrombosis; though in vitro bivalirudin has anticoagulation activity several times higher compared to RA-36. The results indicate that both RA-36 aptamer and bivalirudin are direct thrombin inhibitors of different potency, but possible interactions of the thrombin-inhibitor complex with other components of blood coagulation cascade level the physiological effects for both inhibitors.

  12. Studying small molecule-aptamer interactions using MicroScale Thermophoresis (MST).

    Science.gov (United States)

    Entzian, Clemens; Schubert, Thomas

    2016-03-15

    Aptamers are potent and versatile binding molecules recognizing various classes of target molecules. Even challenging targets such as small molecules can be identified and bound by aptamers. Studying the interaction between aptamers and drugs, antibiotics or metabolites in detail is however difficult due to the lack of sophisticated analysis methods. Basic binding parameters of these small molecule-aptamer interactions such as binding affinity, stoichiometry and thermodynamics are elaborately to access using the state of the art technologies. The innovative MicroScale Thermophoresis (MST) is a novel, rapid and precise method to characterize these small molecule-aptamer interactions in solution at microliter scale. The technology is based on the movement of molecules through temperature gradients, a physical effect referred to as thermophoresis. The thermophoretic movement of a molecule depends - besides on its size - on charge and hydration shell. Upon the interaction of a small molecule and an aptamer, at least one of these parameters is altered, leading to a change in the movement behavior, which can be used to quantify molecular interactions independent of the size of the target molecule. The MST offers free choice of buffers, even measurements in complex bioliquids are possible. The dynamic affinity range covers the pM to mM range and is therefore perfectly suited to analyze small molecule-aptamer interactions. This section describes a protocol how quantitative binding parameters for aptamer-small molecule interactions can be obtained by MST. This is demonstrated by mapping down the binding site of the well-known ATP aptamer DH25.42 to a specific region at the adenine of the ATP molecule.

  13. In vitro selection, characterization, and biosensing application of high-affinity cylindrospermopsin-targeting aptamers.

    Science.gov (United States)

    Elshafey, Reda; Siaj, Mohamed; Zourob, Mohammed

    2014-09-16

    Contamination of freshwater with cyanotoxin cylindrospermopsin (CYN) represents a significant global concern for public health. The sensitive detection of CYN is necessary to effectively manage and control the treatment of water resources. Here we report a novel, highly sensitive label-free aptasensor for CYN analysis, using aptamers as specific receptors. We have selected the DNA aptamers from a diverse random library using the in vitro screening SELEX approach. The aptamers exhibited high affinity for CYN with Kd of nanomolar range. One aptamer exhibited conformational change upon CYN recognition (CD analysis) and was used to fabricate the label-free impedimetric aptasensor for CYN. A self-assembled monolayer from a disulfide-derivatized aptamer was formed on a gold electrode to fabricate the aptasensor. Upon CYN capturing to the aptasensor surface, a marked drop in the electron transfer resistance was obtained, which was used as the principle of detection of CYN. This resulted from the aptamer's conformational change induced by CYN recognition. The present aptasensor could detect CYN with the limit of detection as low as 100 pM and a wide linear range of 0.1 to 80 nM. When mounted on the gold surface, the aptamer exhibited a lower dissociation constant for CYN than that observed in the fluorescence assay, implying that the anchoring of the aptamer on the Au surface improved its affinity to CYN. Moreover, the aptasensor showed high specificity toward other coexistent cyanobacterial toxins of microcystin-LR and Anatoxin-a. Further biosensor designs will be generated using those aptamers for simple and sensitive CYN monitoring.

  14. Detecting and Discriminating Shigella sonnei Using an Aptamer-Based Fluorescent Biosensor Platform

    Directory of Open Access Journals (Sweden)

    Myeong-Sub Song

    2017-05-01

    Full Text Available In this paper, a Whole-Bacteria SELEX (WB-SELEX strategy was adopted to isolate specific aptamers against Shigella sonnei. Real-time PCR amplification and post-SELEX experiment revealed that the selected aptmers possessed a high binding affinity and specificity for S. sonnei. Of the 21 aptamers tested, the C(t values of the SS-3 and SS-4 aptamers (Ct = 13.89 and Ct = 12.23, respectively had the lowest value compared to other aptamer candidates. The SS-3 and SS-4 aptamers also displayed a binding affinity (KD of 39.32 ± 5.02 nM and 15.89 ± 1.77 nM, respectively. An aptamer-based fluorescent biosensor assay was designed to detect and discriminate S. sonnei cells using a sandwich complex pair of SS-3 and SS-4. The detection of S. sonnei by the aptamer based fluorescent biosensor platform consisted of three elements: (1 5’amine-SS-4 modification in a 96-well type microtiter plate surface (N-oxysuccinimide, NOS as capture probes; (2 the incubation with S. sonnei and test microbes in functionalized 96 assay wells in parallel; (3 the readout of fluorescent activity using a Cy5-labeled SS-3 aptamer as the detector. Our platform showed a significant ability to detect and discriminate S. sonnei from other enteric species such as E. coli, Salmonella typhimurium and other Shigella species (S. flexneri, S. boydii. In this study, we demonstrated the feasibility of an aptamer sensor platform to detect S. sonnei in a variety of foods and pave the way for its use in diagnosing shigellosis through multiple, portable designs.

  15. Aptamer contained triple-helix molecular switch for rapid fluorescent sensing of acetamiprid.

    Science.gov (United States)

    Liu, Xin; Li, Ying; Liang, Jing; Zhu, Wenyue; Xu, Jingyue; Su, Ruifang; Yuan, Lei; Sun, Chunyan

    2016-11-01

    In this study, an aptamer-based fluorescent sensing platform using triple-helix molecular switch (THMS) was developed for the pesticide screening represented by acetamiprid. The THMS was composed of two tailored DNA probes: a label-free central target specific aptamer sequence flanked by two arm segments acting as a recognition probe; a hairpin-shaped structure oligonucleotide serving as a signal transduction probe (STP), labeled with a fluorophore and a quencher at the 3' and 5'-end, respectively. In the absence of acetamiprid, complementary bindings of two arm segments of the aptamers with the loop sequence of STP enforce the formation of THMS with the "open" configuration of STP, and the fluorescence of THMS is on. In the presence of target acetamiprid, the aptamer-target binding results in the formation of a structured aptamer/target complex, which disassembles the THMS and releases the STP. The free STP is folded to a stem loop structure, and the fluorescence is quenched. The quenched fluorescence intensity was proportional to the concentration of acetamiprid in the range from 100 to 1200nM, with the limit of detection (LOD) as low as 9.12nM. In addition, this THMS-based method has been successfully used to test and quantify acetamiprid in Chinese cabbage with satisfactory recoveries, and the results were in full agreement with those from LC-MS. The aptamer-based THMS presents distinct advantages, including high stability, remarkable sensitivity, and preservation of the affinity and specificity of the original aptamer. Most importantly, this strategy is convenient and generalizable by virtue of altering the aptamer sequence without changing the triple-helix structure. So, it is expected that this aptamer-based fluorescent assay could be extensively applied in the field of food safety inspection.

  16. DNA aptamers for the detection of Haemophilus influenzae type b by cell SELEX.

    Science.gov (United States)

    Bitaraf, F S; Rasooli, I; Mousavi Gargari, S L

    2016-03-01

    Haemophilus influenzae type b (Hib) causes acute bacterial meningitis (ABM) in children, with a mortality rate of about 3-6 % of the affected patients. ABM can lead to death during a period of hours to several days and, hence, rapid and early detection of the infection is crucial. Aptamers, the short single-stranded DNA or RNA with high affinity to target molecules, are selected by a high-flux screening technique known as in vitro screening and systematic evolution of ligands by exponential enrichment technology (SELEX). In this study, whole-cell SELEX was applied for the selection of target-specific aptamers with high affinity to Hib. ssDNA aptamers prepared by lambda exonuclease were incubated with the target cells (Hib). The aptameric binding rate to Hib was characterized for binding affinity after seven SELEX rounds by flow cytometry. The aptamers with higher binding affinity were cloned. Four of 68 aptamer clones were selected for sequencing. The dissociation constant (Kd) of the high-affinity aptamer clones 45 and 63 were 47.10 and 28.46 pM, respectively. These aptamers did not bind to other bacterial species, including the seven meningitis-causing bacteria. They showed distinct affinity to various H. influenzae strains only. These aptamers showed the highest affinity to Hib and the lowest affinity to H. influenzae type c and to other meningitis-causing bacteria. Clone 63 could detect Hib in patients' cerebrospinal fluid (CSF) samples at 60 colony-forming units (CFU)/mL. The results indicate applicability of the aptamers for rapid and early detection of infections brought about by Hib.

  17. Complementation of Cobalamin Auxotrophy in Synechococcus sp. Strain PCC 7002 and Validation of a Putative Cobalamin Riboswitch In Vivo.

    Science.gov (United States)

    Pérez, Adam A; Liu, Zhenfeng; Rodionov, Dmitry A; Li, Zhongkui; Bryant, Donald A

    2016-10-01

    The euryhaline cyanobacterium Synechococcus sp. strain PCC 7002 has an obligate requirement for exogenous vitamin B12 (cobalamin), but little is known about the roles of this compound in cyanobacteria. Bioinformatic analyses suggest that only the terminal enzyme in methionine biosynthesis, methionine synthase, requires cobalamin as a coenzyme in Synechococcus sp. strain PCC 7002. Methionine synthase (MetH) catalyzes the transfer of a methyl group from N(5)-methyl-5,6,7,8-tetrahydrofolate to l-homocysteine during l-methionine synthesis and uses methylcobalamin as an intermediate methyl donor. Numerous bacteria and plants alternatively employ a cobalamin-independent methionine synthase isozyme, MetE, that catalyzes the same methyl transfer reaction as MetH but uses N(5)-methyl-5,6,7,8-tetrahydrofolate directly as the methyl donor. The cobalamin auxotrophy of Synechococcus sp. strain PCC 7002 was complemented by using the metE gene from the closely related cyanobacterium Synechococcus sp. strain PCC 73109, which possesses genes for both methionine synthases. This result suggests that methionine biosynthesis is probably the sole use of cobalamin in Synechococcus sp. strain PCC 7002. Furthermore, a cobalamin-repressible gene expression system was developed in Synechococcus sp. strain PCC 7002 that was used to validate the presence of a cobalamin riboswitch in the promoter region of metE from Synechococcus sp. strain PCC 73109. This riboswitch acts as a cobalamin-dependent transcriptional attenuator for metE in that organism. Synechococcus sp. strain PCC 7002 is a cobalamin auxotroph because, like eukaryotic marine algae, it uses a cobalamin-dependent methionine synthase (MetH) for the final step of l-methionine biosynthesis but cannot synthesize cobalamin de novo Heterologous expression of metE, encoding cobalamin-independent methionine synthase, from Synechococcus sp. strain PCC 73109, relieved this auxotrophy and enabled the construction of a truly autotrophic

  18. Aptaligner: automated software for aligning pseudorandom DNA X-aptamers from next-generation sequencing data.

    Science.gov (United States)

    Lu, Emily; Elizondo-Riojas, Miguel-Angel; Chang, Jeffrey T; Volk, David E

    2014-06-10

    Next-generation sequencing results from bead-based aptamer libraries have demonstrated that traditional DNA/RNA alignment software is insufficient. This is particularly true for X-aptamers containing specialty bases (W, X, Y, Z, ...) that are identified by special encoding. Thus, we sought an automated program that uses the inherent design scheme of bead-based X-aptamers to create a hypothetical reference library and Markov modeling techniques to provide improved alignments. Aptaligner provides this feature as well as length error and noise level cutoff features, is parallelized to run on multiple central processing units (cores), and sorts sequences from a single chip into projects and subprojects.

  19. Molecular Diagnostic and Drug Delivery Agents based on Aptamer-Nanomaterial Conjugates

    Science.gov (United States)

    Lee, Jung Heon; Yigit, Mehmet V.; Mazumdar, Debapriya; Lu, Yi

    2010-01-01

    Recent progress in an emerging area of designing aptamer and nanomaterial conjugates as molecular diagnostic and drug delivery agents in biomedical applications is summarized. Aptamers specific for a wide range of targets are first introduced and compared to antibodies. Methods of integrating these aptamers with a variety of nanomaterials, such as gold nanoparticles, quantum dots, carbon nanotubes, and superparamagnetic iron oxide nanoparticles, each with unique optical, magnetic, and electrochemical properties, are reviewed. Applications of these systems as fluorescent, colorimetric, magnetic resonance imaging, and electrochemical sensors in medical diagnostics are given, along with new applications as smart drug delivery agents. PMID:20338204

  20. Targeting Two Coagulation Cascade Proteases with a Bivalent Aptamer Yields a Potent and Antidote-Controllable Anticoagulant.

    Science.gov (United States)

    Soule, Erin E; Bompiani, Kristin M; Woodruff, Rebecca S; Sullenger, Bruce A

    2016-02-01

    Potent and rapid-onset anticoagulation is required for several clinical settings, including cardiopulmonary bypass surgery. In addition, because anticoagulation is associated with increased bleeding following surgery, the ability to rapidly reverse such robust anticoagulation is also important. Previously, we observed that no single aptamer was as potent as heparin for anticoagulating blood. However, we discovered that combinations of two aptamers were as potent as heparin. Herein, we sought to combine two individual anticoagulant aptamers into a single bivalent RNA molecule in an effort to generate a single molecule that retained the potent anticoagulant activity of the combination of individual aptamers. We created four bivalent aptamers that can inhibit Factor X/Xa and prothrombin/thrombin and anticoagulate plasma, as well as the combination of individual aptamers. Detailed characterization of the shortest bivalent aptamer indicates that each aptamer retains full binding and functional activity when presented in the bivalent context. Finally, reversal of this bivalent aptamer with a single antidote was explored, and anticoagulant activity could be rapidly turned off in a dose-dependent manner. These studies demonstrate that bivalent anticoagulant aptamers represent a novel and potent approach to actively and reversibly control coagulation.

  1. RNA aptamers generated against oligomeric Abeta40 recognize common amyloid aptatopes with low specificity but high sensitivity.

    Directory of Open Access Journals (Sweden)

    Farid Rahimi

    Full Text Available Aptamers are useful molecular recognition tools in research, diagnostics, and therapy. Despite promising results in other fields, aptamer use has remained scarce in amyloid research, including Alzheimer's disease (AD. AD is a progressive neurodegenerative disease believed to be caused by neurotoxic amyloid beta-protein (Abeta oligomers. Abeta oligomers therefore are an attractive target for development of diagnostic and therapeutic reagents. We used covalently-stabilized oligomers of the 40-residue form of Abeta (Abeta40 for aptamer selection. Despite gradually increasing the stringency of selection conditions, the selected aptamers did not recognize Abeta40 oligomers but reacted with fibrils of Abeta40, Abeta42, and several other amyloidogenic proteins. Aptamer reactivity with amyloid fibrils showed some degree of protein-sequence dependency. Significant fibril binding also was found for the naïve library and could not be eliminated by counter-selection using Abeta40 fibrils, suggesting that aptamer binding to amyloid fibrils was RNA-sequence-independent. Aptamer binding depended on fibrillogenesis and showed a lag phase. Interestingly, aptamers detected fibril formation with > or =15-fold higher sensitivity than thioflavin T (ThT, revealing substantial beta-sheet and fibril formation undetected by ThT. The data suggest that under physiologic conditions, aptamers for oligomeric forms of amyloidogenic proteins cannot be selected due to high, non-specific affinity of oligonucleotides for amyloid fibrils. Nevertheless, the high sensitivity, whereby aptamers detect beta-sheet formation, suggests that they can serve as superior amyloid recognition tools.

  2. Electrochemical Impedance Spectroscopic Sensing of Methamphetamine by a Specific Aptamer

    Directory of Open Access Journals (Sweden)

    Omid Mashinchian

    2012-05-01

    Full Text Available Introduction: Electrochemical impedance spectroscopy (EIS is a simple and highly sensitive technique that can be used for evaluation of the aptamer-target interaction even in a label-free approach. Methods: To pursue the effectiveness of EIS, in the current study, the folding properties of specific aptamer for methamphetamine (METH (i.e., aptaMETH were evaluated in the presence of METH and amphetamine (Amph. Folded and unfolded aptaMETH was mounted on the gold electrode surface and the electron charge transfer was measured by EIS. Results: The Ret of methamphetamine-aptaMETH was significantly increased in comparison with other folding conditions, indicating specific detection of METH by aptaMETH. Conclusion: Based on these findings, methamphetamine-aptaMETH on the gold electrode surface displayed the most interfacial electrode resistance and thus the most folding situation. This clearly indicates that the aptaMETH can profoundly and specifically pinpoint METH; as a result we suggest utilization of this methodology for fast and cost-effective identification of METH.

  3. Harnessing Aptamers to Overcome Challenges in Gluten Detection.

    Science.gov (United States)

    Miranda-Castro, Rebeca; de-los-Santos-Álvarez, Noemí; Miranda-Ordieres, Arturo J; Lobo-Castañón, María Jesús

    2016-04-20

    Celiac disease is a lifelong autoimmune disorder triggered by foods containing gluten, the storage protein in wheat, rye, and barley. The rapidly escalating number of patients diagnosed with this disease poses a great challenge to both food industry and authorities to guarantee food safety for all. Therefore, intensive efforts are being made to establish minimal disease-eliciting doses of gluten and consequently to improve gluten-free labeling. These efforts depend to a high degree on the availability of methods capable of detecting the protein in food samples at levels as low as possible. Current analytical approaches rely on the use of antibodies as selective recognition elements. With limited sensitivity, these methods exhibit some deficiencies that compromise the accuracy of the obtained results. Aptamers provide an ideal alternative for designing biosensors for fast and selective measurement of gluten in foods. This article highlights the challenges in gluten detection, the current status of the use of aptamers for solving this problem, and what remains to be done to move these systems into commercial applications.

  4. Miniaturized Aptamer-Based Assays for Protein Detection

    Directory of Open Access Journals (Sweden)

    Alessandro Bosco

    2016-09-01

    Full Text Available The availability of devices for cancer biomarker detection at early stages of the disease is one of the most critical issues in biomedicine. Towards this goal, to increase the assay sensitivity, device miniaturization strategies empowered by the employment of high affinity protein binders constitute a valuable approach. In this work we propose two different surface-based miniaturized platforms for biomarker detection in body fluids: the first platform is an atomic force microscopy (AFM-based nanoarray, where AFM is used to generate functional nanoscale areas and to detect biorecognition through careful topographic measurements; the second platform consists of a miniaturized electrochemical cell to detect biomarkers through electrochemical impedance spectroscopy (EIS analysis. Both devices rely on robust and highly-specific protein binders as aptamers, and were tested for thrombin detection. An active layer of DNA-aptamer conjugates was immobilized via DNA directed immobilization on complementary single-stranded DNA self-assembled monolayers confined on a nano/micro area of a gold surface. Results obtained with these devices were compared with the output of surface plasmon resonance (SPR assays used as reference. We succeeded in capturing antigens in concentrations as low as a few nM. We put forward ideas to push the sensitivity further to the pM range, assuring low biosample volume (μL range assay conditions.

  5. Screening and Initial Binding Assessment of Fumonisin B1 Aptamers

    Directory of Open Access Journals (Sweden)

    Maria C. DeRosa

    2010-11-01

    Full Text Available Fumonisins are mycotoxins produced by Fusarium verticillioides and F. proliferatum, fungi that are ubiquitous in corn (maize. Insect damage and some other environmental conditions result in the accumulation of fumonisins in corn-based products worldwide. Current methods of fumonisin detection rely on the use of immunoaffinity columns and high-performance liquid chromatography (HPLC. The use of aptamers offers a good alternative to the use of antibodies in fumonisin cleanup and detection due to lower costs and improved stability. Aptamers are single-stranded oligonucleotides that are selected using Systematic Evolution of Ligands by EXponential enrichment (SELEX for their ability to bind to targets with high affinity and specificity. Sequences obtained after 18 rounds of SELEX were screened for their ability to bind to fumonisin B1. Six unique sequences were obtained, each showing improved binding to fumonisin B1 compared to controls. Sequence FB1 39 binds to fumonisin with a dissociation constant of 100 ± 30 nM and shows potential for use in fumonisin biosensors and solid phase extraction columns.

  6. Screening and initial binding assessment of fumonisin b(1) aptamers.

    Science.gov (United States)

    McKeague, Maureen; Bradley, Charlotte R; De Girolamo, Annalisa; Visconti, Angelo; Miller, J David; Derosa, Maria C

    2010-01-01

    Fumonisins are mycotoxins produced by Fusarium verticillioides and F. proliferatum, fungi that are ubiquitous in corn (maize). Insect damage and some other environmental conditions result in the accumulation of fumonisins in corn-based products worldwide. Current methods of fumonisin detection rely on the use of immunoaffinity columns and high-performance liquid chromatography (HPLC). The use of aptamers offers a good alternative to the use of antibodies in fumonisin cleanup and detection due to lower costs and improved stability. Aptamers are single-stranded oligonucleotides that are selected using Systematic Evolution of Ligands by EXponential enrichment (SELEX) for their ability to bind to targets with high affinity and specificity. Sequences obtained after 18 rounds of SELEX were screened for their ability to bind to fumonisin B(1). Six unique sequences were obtained, each showing improved binding to fumonisin B(1) compared to controls. Sequence FB(1) 39 binds to fumonisin with a dissociation constant of 100 ± 30 nM and shows potential for use in fumonisin biosensors and solid phase extraction columns.

  7. Genomic SELEX: a discovery tool for genomic aptamers.

    Science.gov (United States)

    Zimmermann, Bob; Bilusic, Ivana; Lorenz, Christina; Schroeder, Renée

    2010-10-01

    Genomic SELEX is a discovery tool for genomic aptamers, which are genomically encoded functional domains in nucleic acid molecules that recognize and bind specific ligands. When combined with genomic libraries and using RNA-binding proteins as baits, Genomic SELEX used with high-throughput sequencing enables the discovery of genomic RNA aptamers and the identification of RNA-protein interaction networks. Here we describe how to construct and analyze genomic libraries, how to choose baits for selections, how to perform the selection procedure and finally how to analyze the enriched sequences derived from deep sequencing. As a control procedure, we recommend performing a "Neutral" SELEX experiment in parallel to the selection, omitting the selection step. This control experiment provides a background signal for comparison with the positively selected pool. We also recommend deep sequencing the initial library in order to facilitate the final in silico analysis of enrichment with respect to the initial levels. Counter selection procedures, using modified or inactive baits, allow strengthening the binding specificity of the winning selected sequences.

  8. Electrochemical Impedance Spectroscopic Sensing of Methamphetamine by a Specific Aptamer

    Science.gov (United States)

    Ebrahimi, Mohsen; Johari-Ahar, Mohammad; Hamzeiy, Hossein; Barar, Jaleh; Mashinchian, Omid; Omidi, Yadollah

    2012-01-01

    Introduction Electrochemical impedance spectroscopy (EIS) is a simple and highly sensitive technique that can be used for evaluation of the aptamer-target interaction even in a label-free approach. Methods To pursue the effectiveness of EIS, in the current study, the folding properties of specific aptamer for methamphetamine (METH) (i.e., aptaMETH) were evaluated in the presence of METH and amphetamine (Amph). Folded and unfolded aptaMETH was mounted on the gold electrode surface and the electron charge transfer was measured by EIS. Results The Ret of methamphetamine-aptaMETH was significantly increased in comparison with other folding conditions, indicating specific detection of METH by aptaMETH. Conclusion Based on these findings, methamphetamine-aptaMETH on the gold electrode surface displayed the most interfacial electrode resistance and thus the most folding situation. This clearly indicates that the aptaMETH can profoundly and specifically pinpoint METH; as a result we suggest utilization of this methodology for fast and cost-effective identification of METH. PMID:23678446

  9. Dissecting the effect of RNA aptamer binding on the dynamics of plasminogen activator inhibitor 1 using hydrogen/deuterium exchange mass spectrometry

    DEFF Research Database (Denmark)

    Trelle, Morten B; Dupont, Daniel Miotto; Madsen, Jeppe Buur

    2014-01-01

    , about their effects on protein conformation and dynamics. We have employed hydrogen/deuterium exchange (HDX) mass spectrometry to study the effect of RNA aptamers on the structural flexibility of the serpin plasminogen activator inhibitor-1 (PAI-1). The aptamers have characteristic effects...... of the aptamers to PAI-1 is associated with substantial and widespread protection against deuterium uptake in PAI-1. The aptamers induce protection against exchange with the solvent both in the protein-aptamer interface as well as in other specific areas. Interestingly, the aptamers induce substantial protection...

  10. Modifications of the chromophore of Spinach aptamer based on QM:MM calculations.

    Science.gov (United States)

    Skúpa, Katarína; Urban, Ján

    2017-02-01

    Spinach aptamer was developed as an RNA analog of the green fluorescent protein. The aptamer interacts with its ligand and modifies its electronic spectrum so that it fluoresces brightly at the wavelength of 501 nm. Song et al. investigated modifications of the ligand in their experimental study and found a molecule emitting at 523 nm upon creating a complex with the Spinach aptamer. The crystal structure of the aptamer in complex with its original ligand has been published, which enabled us to study the system computationally. In this article, we suggest several new modifications of the ligand that shift the emission maximum of the complex to even longer wavelengths. Our results are based on combined quantum mechanical/molecular mechanical calculations with DFT method used for geometry optimization and TD-DFT for calculations of absorption and emission energies.

  11. Efficient isolation and elution of cellular proteins using aptamer-mediated protein precipitation assay.

    Science.gov (United States)

    Kim, Kiseok; Lee, SeungJin; Ryu, Sungho; Han, Dongil

    2014-05-23

    Protein precipitation is one of the most widely used methods for antigen detection and purification in biological research. We developed a reproducible aptamer-mediated magnetic protein precipitation method that is able to efficiently capture, purify and isolate the target proteins. We discovered DNA aptamers having individually high affinity and specificity against human epidermal growth factor receptor (EGFR) and human insulin receptor (INSR). Using aptamers and magnetic beads, we showed it is highly efficient technique to enrich endogenous proteins complex and is applicable to identify physiologically relevant protein-protein interactions with minimized nonspecific binding of proteins. The results presented here indicate that aptamers would be applicable as a useful and cost-effective tool to identify the presence of the particular target protein with their specific protein partners.

  12. Biomedical Applications of Quantum Dots, Nucleic Acid-Based Aptamers, and Nanostructures in Biosensors.

    Science.gov (United States)

    Meshik, Xenia; Farid, Sidra; Choi, Min; Lan, Yi; Mukherjee, Souvik; Datta, Debopam; Dutta, Mitra; Stroscio, Michael A

    2015-01-01

    This review is a survey of the biomedical applications of semiconductor quantum dots, nucleic acid-based aptamers, and nanosensors as molecular biosensors. It focuses on the detection of analytes in biomedical applications using (1) advances in molecular beacons incorporating semiconductor quantum dots and nanoscale quenching elements; (2) aptamer-based nanosensors on a variety of platforms, including graphene; (3) Raman scattering and surface-enhanced Raman scattering (SERS) using nanostructures for enhanced SERS spectra of biomolecules, including aptamers; and (4) the electrical and optical properties of nanostructures incorporated into molecular beacons and aptamer-based nanosensors. Research done at the University of Illinois at Chicago (UIC) is highlighted throughout since it emphasizes the specific approaches taken by the bioengineering department at UIC.

  13. Rational design of a structure-switching DNA aptamer for potassium ions

    Science.gov (United States)

    Catherine, Andrew T.; Shishido, Stephanie N.; Robbins-Welty, Gregg A.; Diegelman-Parente, Amy

    2014-01-01

    Structure-switching molecules provide a unique means for analyte detection, generating a response to analyte concentration through a binding-specific conformational change between non-binding and binding-competent states. While most ligand-binding molecules are not structure switching by default, many can be engineered to be so through the introduction of an alternative non-binding (and thus non-signalling) conformation. This population-shift mechanism is particularly effective with oligonucleotides and has led to the creation of structure-switching aptamers for many target ligands. Here, we report the rational design of structure-switching DNA aptamers, based on the thrombin binding aptamer (TBA), that bind potassium with affinities that bridge the gap between previously reported weak-binding and strong-binding aptamers. We also demonstrate a correlation between the free energy of the experimentally determined binding affinity for potassium and the computationally estimated free energy of the alternative (non-binding) structure. PMID:25352996

  14. Selection of DMA aptamer that specific binding human carcinoembryonic antigen in vitro

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective:To select the specific aptamer of carcinoembryonic antigen (CEA), one of the most attractive molecule for cancer target therapy and imaging. Methods: Seven rounds in vitro selection were performed against the purified CEA protein. Ligand-mediated target purification and Co-immunoprecipitation were adopted to verify the specific binding of the aptamer to the purified and native protein separately. Results:The CEA-specific aptamer which can bind both the purified and native protein with the high specificity was obtained. Conclusion:This is the first time the CEA specific apatmer was produced. The results in this study provides the preliminary evidence for further investigation and application of CEA-aptamer in the future.

  15. Generating aptamers by cell-SELEX for applications in molecular medicine.

    Science.gov (United States)

    Ye, Mao; Hu, Jun; Peng, Minyuan; Liu, Jing; Liu, Jun; Liu, Huixia; Zhao, Xielan; Tan, Weihong

    2012-01-01

    Aptamers are single-stranded oligonucleotides of DNA or RNA that bind to target molecules with high affinity and specificity. Typically, aptamers are generated by an iterative selection process, called systematic evolution of ligands by exponential enrichment (SELEX). Recent advancements in SELEX technology have extended aptamer selection from comparatively simple mixtures of purified proteins to whole living cells, and now cell-based SELEX (or cell-SELEX) can isolate aptamers that bind to specific target cells. Combined with nanotechnology, microchips, microfluidic devices, RNAi and other advanced technologies, cell-SELEX represents an integrated platform providing ultrasensitive and highly specific tools for clinical medicine. In this review, we describe the recent progress made in the application of cell-SELEX for diagnosis, therapy and biomarker discovery.

  16. Generating Aptamers by Cell-SELEX for Applications in Molecular Medicine

    Directory of Open Access Journals (Sweden)

    Huixia Liu

    2012-03-01

    Full Text Available Aptamers are single-stranded oligonucleotides of DNA or RNA that bind to target molecules with high affinity and specificity. Typically, aptamers are generated by an iterative selection process, called systematic evolution of ligands by exponential enrichment (SELEX. Recent advancements in SELEX technology have extended aptamer selection from comparatively simple mixtures of purified proteins to whole living cells, and now cell-based SELEX (or cell-SELEX can isolate aptamers that bind to specific target cells. Combined with nanotechnology, microchips, microfluidic devices, RNAi and other advanced technologies, cell-SELEX represents an integrated platform providing ultrasensitive and highly specific tools for clinical medicine. In this review, we describe the recent progress made in the application of cell-SELEX for diagnosis, therapy and biomarker discovery.

  17. Identification of DNA aptamers toward epithelial cell adhesion molecule via cell-SELEX.

    Science.gov (United States)

    Kim, Ji Won; Kim, Eun Young; Kim, Sun Young; Byun, Sang Kyung; Lee, Dasom; Oh, Kyoung-Jin; Kim, Won Kon; Han, Baek Soo; Chi, Seung-Wook; Lee, Sang Chul; Bae, Kwang-Hee

    2014-10-31

    The epithelial cell adhesion molecule (EpCAM, also known as CD326) is a transmembrane glycoprotein that is specifically detected in most adenocarcinomas and cancer stem cells. In this study, we performed a Cell systematic evolution of ligands by exponential enrichment (SELEX) experiment to isolate the aptamers against EpCAM. After seven round of Cell SELEX, we identified several aptamer candidates. Among the selected aptamers, EP166 specifically binds to cells expressing EpCAM with an equilibrium dissociation constant (Kd) in a micromolar range. On the other hand, it did not bind to negative control cells. Moreover, EP166 binds to J1ES cells, a mouse embryonic stem cell line. Therefore, the isolated aptamers against EpCAM could be used as a stem cell marker or in other applications in both stem cell and cancer studies.

  18. Asymmetric PCR for good quality ssDNA generation towards DNA aptamer production

    Directory of Open Access Journals (Sweden)

    Junji Tominaga4

    2012-04-01

    Full Text Available Aptamers are ssDNA or RNA that binds to wide variety of target molecules with high affinity and specificity producedby systematic evolution of ligands by exponential enrichment (SELEX. Compared to RNA aptamer, DNA aptamer is muchmore stable, favourable to be used in many applications. The most critical step in DNA SELEX experiment is the conversion ofdsDNA to ssDNA. The purpose of this study was to develop an economic and efficient approach of generating ssDNA byusing asymmetric PCR. Our results showed that primer ratio (sense primer:antisense primer of 20:1 and sense primer amountof 10 to 100 pmol, up to 20 PCR cycles using 20 ng of initial template, in combination with polyacrylamide gel electrophoresis,were the optimal conditions for generating good quality and quantity of ssDNA. The generation of ssDNA via this approachcan greatly enhance the success rate of DNA aptamer generation.

  19. Preparation of a Specific ssDNA Aptamer for Brevetoxin-2 Using SELEX

    National Research Council Canada - National Science Library

    Tian, Rui-Yun; Lin, Chao; Yu, Shi-Yu; Gong, Sheng; Hu, Pan; Li, Yan-Song; Wu, Zong-Cheng; Gao, Yang; Zhou, Yu; Liu, Zeng-Shan; Ren, Hong-Lin; Lu, Shi-Ying

    2016-01-01

    .... The development of an alternative detection probe is another promising research direction. This paper reports the use of aptamers binding to BTX-2 in an analytical assay using the systematic evolution of ligands by exponential enrichment (SELEX...

  20. Efficient reverse transcription using locked nucleic acid nucleotides towards the evolution of nuclease resistant RNA aptamers

    DEFF Research Database (Denmark)

    Crouzier, Lucile; Dubois, Camille; Edwards, Stacey L

    2012-01-01

    We found that SuperScript® III Reverse Transcriptase is an efficient enzyme for the recognition of LNA nucleotides, making it a prime candidate to be used in de novo selection of LNA containing RNA aptamers....

  1. Through-bond effects in the ternary complexes of thrombin sandwiched by two DNA aptamers

    Science.gov (United States)

    Pica, Andrea; Russo Krauss, Irene; Parente, Valeria; Tateishi-Karimata, Hisae; Nagatoishi, Satoru; Tsumoto, Kouhei; Sugimoto, Naoki; Sica, Filomena

    2017-01-01

    Aptamers directed against human thrombin can selectively bind to two different exosites on the protein surface. The simultaneous use of two DNA aptamers, HD1 and HD22, directed to exosite I and exosite II respectively, is a very powerful approach to exploit their combined affinity. Indeed, strategies to link HD1 and HD22 together have been proposed in order to create a single bivalent molecule with an enhanced ability to control thrombin activity. In this work, the crystal structures of two ternary complexes, in which thrombin is sandwiched between two DNA aptamers, are presented and discussed. The structures shed light on the cross talk between the two exosites. The through-bond effects are particularly evident at exosite II, with net consequences on the HD22 structure. Moreover, thermodynamic data on the binding of the two aptamers are also reported and analyzed. PMID:27899589

  2. Selection of LNA-containing DNA aptamers against recombinant human CD73

    DEFF Research Database (Denmark)

    Elle, Ida C; Karlsen, Kasper K; Terp, Mikkel G

    2015-01-01

    tested by surface plasmon resonance. Truncated variants of these aptamers and variants where the LNA nucleotides were substituted for the DNA equivalent also exhibited affinity for the recombinant CD73 in the low nanomolar range. In enzyme inhibition assays with recombinant CD73 the aptamer sequences......LNA-containing DNA aptamers against CD73 (human ecto-5'-nucleotidase), a protein frequently overexpressed in solid tumours, were isolated by SELEX. A pre-defined stem-loop library, containing LNA in the forward primer region, was enriched with CD73 binding sequences through six rounds of SELEX...... with recombinant his-tagged CD73 immobilised on anti-his plates. Enriched pools isolated from rounds one, three and six were subjected to next-generation sequencing and analysed for enrichment using custom bioinformatics software. The software identified aptamer sequences via the primers and then performed several...

  3. Through-bond effects in the ternary complexes of thrombin sandwiched by two DNA aptamers.

    Science.gov (United States)

    Pica, Andrea; Russo Krauss, Irene; Parente, Valeria; Tateishi-Karimata, Hisae; Nagatoishi, Satoru; Tsumoto, Kouhei; Sugimoto, Naoki; Sica, Filomena

    2017-01-09

    Aptamers directed against human thrombin can selectively bind to two different exosites on the protein surface. The simultaneous use of two DNA aptamers, HD1 and HD22, directed to exosite I and exosite II respectively, is a very powerful approach to exploit their combined affinity. Indeed, strategies to link HD1 and HD22 together have been proposed in order to create a single bivalent molecule with an enhanced ability to control thrombin activity. In this work, the crystal structures of two ternary complexes, in which thrombin is sandwiched between two DNA aptamers, are presented and discussed. The structures shed light on the cross talk between the two exosites. The through-bond effects are particularly evident at exosite II, with net consequences on the HD22 structure. Moreover, thermodynamic data on the binding of the two aptamers are also reported and analyzed. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. High Efficiency Binding Aptamers for a Wide Range of Bacterial Sepsis Agents.

    Science.gov (United States)

    Graziani, Ana Cláudia; Stets, Maria Isabel; Lopes, Ana Luisa Kalb; Schluga, Pedro Henrique Caires; Marton, Soledad; Mendes, Ieda Ferreira; Andrade, Antero Silva Ribeiro de; Krieger, Marco Aurélio; Cardoso, Josiane

    2017-04-28

    Sepsis is a major health problem worldwide, with an extremely high rate of morbidity and mortality, partly due to delayed diagnosis during early disease. Currently, sepsis diagnosis requires bacterial culturing of blood samples over several days, whereas PCR-based molecular diagnosis methods are faster but lack sensitivity. The use of biosensors containing nucleic acid aptamers that bind targets with high affinity and specificity could accelerate sepsis diagnosis. Previously, we used the systematic evolution of ligands by exponential enrichment technique to develop the aptamers Antibac1 and Antibac2, targeting the ubiquitous bacterial peptidoglycan. Here, we show that these aptamers bind to four gram-positive and seven gram-negative bacterial sepsis agents with high binding efficiency. Thus, these aptamers could be used in combination as biological recognition elements in the development of biosensors that are an alternative to rapid bacteria detection, since they could provide culture and amplification-free tests for rapid clinical sepsis diagnosis.

  5. [Atomic force microscopy fishing of gp120 on immobilized aptamer and its mass spectrometry identification].

    Science.gov (United States)

    Bukharina, N S; Ivanov, Yu D; Pleshakova, T O; Frantsuzov, P A; Andreeva, E Yu; Kaysheva, A L; Izotov, A A; Pavlova, T I; Ziborov, V S; Radko, S P; Archakov, A I

    2015-01-01

    A method of atomic force microscopy-based fishing (AFM fishing) has been developed for protein detection in the analyte solution using a chip with an immobilized aptamer. This method is based on the biospecific fishing of a target protein from a bulk solution onto the small AFM chip area with the immobilized aptamer to this protein used as the molecular probe. Such aptamer-based approach allows to increase an AFM image contrast compared to the antibody-based approach. Mass spectrometry analysis used after the biospecific fishing to identify the target protein on the AFM chip has proved complex formation. Use of the AFM chip with the immobilized aptamer avoids interference of the antibody and target protein peaks in a mass spectrum.

  6. In vitro evolution of chemically-modified nucleic acid aptamers: Pros and cons, and comprehensive selection strategies.

    Science.gov (United States)

    Lipi, Farhana; Chen, Suxiang; Chakravarthy, Madhuri; Rakesh, Shilpa; Veedu, Rakesh N

    2016-12-01

    Nucleic acid aptamers are single-stranded DNA or RNA oligonucleotide sequences that bind to a specific target molecule with high affinity and specificity through their ability to adopt 3-dimensional structure in solution. Aptamers have huge potential as targeted therapeutics, diagnostics, delivery agents and as biosensors. However, aptamers composed of natural nucleotide monomers are quickly degraded in vivo and show poor pharmacodynamic properties. To overcome this, chemically-modified nucleic acid aptamers are developed by incorporating modified nucleotides after or during the selection process by Systematic Evolution of Ligands by EXponential enrichment (SELEX). This review will discuss the development of chemically-modified aptamers and provide the pros and cons, and new insights on in vitro aptamer selection strategies by using chemically-modified nucleic acid libraries.

  7. Development of RNA aptamers as molecular probes for HER2+ breast cancer study using cell-SELEX

    Directory of Open Access Journals (Sweden)

    Seyedeh Alia Moosavian

    2015-06-01

    Full Text Available Objective(s: Development of molecules that specifically recognize cancer cells is one of the major areas in cancer research. Human epidermal growth factor receptor 2 (HER2 is specifically expressed on the surface of breast cancer cells. HER2 is associated with an aggressive phenotype and poor prognosis. In this study we aimed to isolate RNA aptamers that specifically bind to HER2 overexpressing TUBO cell line. Materials and Methods: Panel of aptamers was selected using cell-based systematic evolution of ligands by exponential enrichment (cell-SELEX. Results: Binding studies showed that selected aptamers can identify TUBO cell line with high affinity and selectivity. Our preliminary investigation of the target of aptamers suggested that aptamers bind with HER2 proteins on the surface of TUBO cells. Conclusion: We believe the selected aptamers could be useful ligands for targeted breast cancer therapy.

  8. Solution NMR determination of hydrogen bonding and base pairing between the glyQS T box riboswitch Specifier domain and the anticodon loop of tRNA(Gly).

    Science.gov (United States)

    Chang, Andrew T; Nikonowicz, Edward P

    2013-11-01

    In Gram-positive bacteria the tRNA-dependent T box riboswitch regulates the expression of many amino acid biosynthetic and aminoacyl-tRNA synthetase genes through a transcription attenuation mechanism. The Specifier domain of the T box riboswitch contains the Specifier sequence that is complementary to the tRNA anticodon and is flanked by a highly conserved purine nucleotide that could result in a fourth base pair involving the invariant U33 of tRNA. We show that the interaction between the T box Specifier domain and tRNA consists of three Watson-Crick base pairs and that U33 confers stability to the complex through intramolecular hydrogen bonding. Enhanced packing within the Specifier domain loop E motif may stabilize the complex and contribute to cognate tRNA selection.

  9. Mechanistic Insights into Cofactor-Dependent Coupling of RNA Folding and mRNA Transcription/Translation by a Cobalamin Riboswitch

    Directory of Open Access Journals (Sweden)

    Jacob T. Polaski

    2016-05-01

    Full Text Available Riboswitches are mRNA elements regulating gene expression in response to direct binding of a metabolite. While these RNAs are increasingly well understood with respect to interactions between receptor domains and their cognate effector molecules, little is known about the specific mechanistic relationship between metabolite binding and gene regulation by the downstream regulatory domain. Using a combination of cell-based, biochemical, and biophysical techniques, we reveal the specific RNA architectural features enabling a cobalamin-dependent hairpin loop docking interaction between receptor and regulatory domains. Furthermore, these data demonstrate that docking kinetics dictate a regulatory response involving the coupling of translation initiation to general mechanisms that control mRNA abundance. These results yield a comprehensive picture of how RNA structure in the riboswitch regulatory domain enables kinetically constrained ligand-dependent regulation of gene expression.

  10. Aptamer-based label-free impedimetric biosensor for detection of progesterone.

    Science.gov (United States)

    Contreras Jiménez, Gastón; Eissa, Shimaa; Ng, Andy; Alhadrami, Hani; Zourob, Mohammed; Siaj, Mohamed

    2015-01-20

    Rising progesterone (P4) levels in humans due to its overconsumption through hormonal therapy, food products, or drinking water can lead to many negative health effects. Thus, the simple and accurate assessment of P4 in both environmental and clinical samples is highly important to protect public health. In this work, we present the selection, identification, and characterization of ssDNA aptamers with high binding affinity to P4. The aptamers were selected in vitro from a single-stranded DNA library of 1.8 × 10(15) oligonucleotides showing dissociation constants (KD) in the low nanomolar range. The dissociation constant of the best aptamer, designated as P4G13, was estimated to be 17 nM by electrochemical impedance spectroscopy (EIS) as well as fluorometric assay. Moreover, the aptamer P4G13 did not show cross-reactivity to analogues similar to progesterone such as 17β-estradiol (E2) and norethisterone (NET). An impedimetric aptasensor for progesterone was then fabricated based on the conformational change of P4G13 aptamer, immobilized on the gold electrode by self-assembly, upon binding to P4, which results in an increase in electron transfer resistance. Aptamer-complementary DNA (cDNA) oligonucleotides were tested to maximize the signal gain of the aptasensor after binding with progesterone. Significant signal enhancement was observed when the aptamer hybridized with a short complementary sequence at specific site was used instead of pure aptamer. This signal gain is likely due to the more significant conformational change of the aptamer-cDNA than the pure aptamer upon binding with P4, as confirmed by circular dichroism (CD) spectroscopy. The developed aptasensor exhibited a linear range for concentrations of P4 from 10 to 60 ng/mL with a detection limit of 0.90 ng/mL. Moreover, the aptasensor was applied in spiked tap water samples and showed good recovery percentages. The new selected progesterone aptamers can be exploited in further biosensing applications

  11. FRET-Aptamer Assays for Bone Marker Assessment, C-Telopeptide, Creatinine, and Vitamin D

    Science.gov (United States)

    Bruno, John G.

    2013-01-01

    Astronauts lose 1.0 to 1.5% of their bone mass per month on long-duration spaceflights. NASA wishes to monitor the bone loss onboard spacecraft to develop nutritional and exercise countermeasures, and make adjustments during long space missions. On Earth, the same technology could be used to monitor osteoporosis and its therapy. Aptamers bind to targets against which they are developed, much like antibodies. However, aptamers do not require animal hosts or cell culture and are therefore easier, faster, and less expensive to produce. In addition, aptamers sometimes exhibit greater affinity and specificity vs. comparable antibodies. In this work, fluorescent dyes and quenchers were added to the aptamers to enable pushbutton, one-step, bind-and-detect fluorescence resonance energy transfer (FRET) assays or tests that can be freeze-dried, rehydrated with body fluids, and used to quantitate bone loss of vitamin D levels with a handheld fluorometer in the spacecraft environment. This work generated specific, rapid, one-step FRET assays for the bone loss marker C-telopeptide (CTx) when extracted from urine, creatinine from urine, and vitamin D congeners in diluted serum. The assays were quantified in nanograms/mL using a handheld fluorometer connected to a laptop computer to convert the raw fluorescence values into concentrations of each analyte according to linear standard curves. DNA aptamers were selected and amplified for several rounds against a 26- amino acid form of CTx, creatinine, and vitamin D. The commonalities between loop structures were studied, and several common loop structures were converted into aptamer beacons with a fluorophore and quencher on each end. In theory, when the aptamer beacon binds its cognate target (CTx bone peptide, creatinine, or vitamin D), it is forced open and no longer quenched, so it gives off fluorescent light (when excited) in proportion to the amount of target present in a sample. This proportional increase in fluorescence is

  12. Aptamers generated from cell-SELEX for molecular medicine: a chemical biology approach.

    Science.gov (United States)

    Fang, Xiaohong; Tan, Weihong

    2010-01-19

    Molecular medicine is an emerging field focused on understanding the molecular basis of diseases and translating this information into strategies for diagnosis and therapy. This approach could lead to personalized medical treatments. Currently, our ability to understand human diseases at the molecular level is limited by the lack of molecular tools to identify and characterize the distinct molecular features of the disease state, especially for diseases such as cancer. Among the new tools being developed by researchers including chemists, engineers, and other scientists is a new class of nucleic acid probes called aptamers, which are ssDNA/RNA molecules selected to target a wide range of molecules and even cells. In this Account, we will focus on the use of aptamers, generated from cell-based selections, as a novel molecular tool for cancer research. Cancers originate from mutations of human genes. These genetic alterations result in molecular changes to diseased cells, which, in turn, lead to changes in cell morphology and physiology. For decades, clinicians have diagnosed cancers primarily based on the morphology of tumor cells or tissues. However,