Origins and Early Evolution of the tRNA Molecule

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Koji Tamura

2015-12-01

Full Text Available Modern transfer RNAs (tRNAs are composed of ~76 nucleotides and play an important role as “adaptor” molecules that mediate the translation of information from messenger RNAs (mRNAs. Many studies suggest that the contemporary full-length tRNA was formed by the ligation of half-sized hairpin-like RNAs. A minihelix (a coaxial stack of the acceptor stem on the T-stem of tRNA can function both in aminoacylation by aminoacyl tRNA synthetases and in peptide bond formation on the ribosome, indicating that it may be a vestige of the ancestral tRNA. The universal CCA-3′ terminus of tRNA is also a typical characteristic of the molecule. “Why CCA?” is the fundamental unanswered question, but several findings give a comprehensive picture of its origin. Here, the origins and early evolution of tRNA are discussed in terms of various perspectives, including nucleotide ligation, chiral selectivity of amino acids, genetic code evolution, and the organization of the ribosomal peptidyl transferase center (PTC. The proto-tRNA molecules may have evolved not only as adaptors but also as contributors to the composition of the ribosome.

From "Cellular" RNA to "Smart" RNA: Multiple Roles of RNA in Genome Stability and Beyond.

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Michelini, Flavia; Jalihal, Ameya P; Francia, Sofia; Meers, Chance; Neeb, Zachary T; Rossiello, Francesca; Gioia, Ubaldo; Aguado, Julio; Jones-Weinert, Corey; Luke, Brian; Biamonti, Giuseppe; Nowacki, Mariusz; Storici, Francesca; Carninci, Piero; Walter, Nils G; Fagagna, Fabrizio d'Adda di

2018-03-30

Coding for proteins has been considered the main function of RNA since the "central dogma" of biology was proposed. The discovery of noncoding transcripts shed light on additional roles of RNA, ranging from the support of polypeptide synthesis, to the assembly of subnuclear structures, to gene expression modulation. Cellular RNA has therefore been recognized as a central player in often unanticipated biological processes, including genomic stability. This ever-expanding list of functions inspired us to think of RNA as a "smart" phone, which has replaced the older obsolete "cellular" phone. In this review, we summarize the last two decades of advances in research on the interface between RNA biology and genome stability. We start with an account of the emergence of noncoding RNA, and then we discuss the involvement of RNA in DNA damage signaling and repair, telomere maintenance, and genomic rearrangements. We continue with the depiction of single-molecule RNA detection techniques, and we conclude by illustrating the possibilities of RNA modulation in hopes of creating or improving new therapies. The widespread biological functions of RNA have made this molecule a reoccurring theme in basic and translational research, warranting it the transcendence from classically studied "cellular" RNA to "smart" RNA.

Mapping the kinetic barriers of a Large RNA molecule's folding landscape.

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Jörg C Schlatterer

Full Text Available The folding of linear polymers into discrete three-dimensional structures is often required for biological function. The formation of long-lived intermediates is a hallmark of the folding of large RNA molecules due to the ruggedness of their energy landscapes. The precise thermodynamic nature of the barriers (whether enthalpic or entropic that leads to intermediate formation is still poorly characterized in large structured RNA molecules. A classic approach to analyzing kinetic barriers are temperature dependent studies analyzed with Eyring's transition state theory. We applied Eyring's theory to time-resolved hydroxyl radical (•OH footprinting kinetics progress curves collected at eight temperature from 21.5 °C to 51 °C to characterize the thermodynamic nature of folding intermediate formation for the Mg(2+-mediated folding of the Tetrahymena thermophila group I ribozyme. A common kinetic model configuration describes this RNA folding reaction over the entire temperature range studied consisting of primary (fast transitions to misfolded intermediates followed by much slower secondary transitions, consistent with previous studies. Eyring analysis reveals that the primary transitions are moderate in magnitude and primarily enthalpic in nature. In contrast, the secondary transitions are daunting in magnitude and entropic in nature. The entropic character of the secondary transitions is consistent with structural rearrangement of the intermediate species to the final folded form. This segregation of kinetic control reveals distinctly different molecular mechanisms during the two stages of RNA folding and documents the importance of entropic barriers to defining rugged RNA folding landscapes.

RNA damage in biological conflicts and the diversity of responding RNA repair systems

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Burroughs, A. Maxwell; Aravind, L.

2016-01-01

RNA is targeted in biological conflicts by enzymatic toxins or effectors. A vast diversity of systems which repair or ‘heal’ this damage has only recently become apparent. Here, we summarize the known effectors, their modes of action, and RNA targets before surveying the diverse systems which counter this damage from a comparative genomics viewpoint. RNA-repair systems show a modular organization with extensive shuffling and displacement of the constituent domains; however, a general ‘syntax’ is strongly maintained whereby systems typically contain: a RNA ligase (either ATP-grasp or RtcB superfamilies), nucleotidyltransferases, enzymes modifying RNA-termini for ligation (phosphatases and kinases) or protection (methylases), and scaffold or cofactor proteins. We highlight poorly-understood or previously-uncharacterized repair systems and components, e.g. potential scaffolding cofactors (Rot/TROVE and SPFH/Band-7 modules) with their respective cognate non-coding RNAs (YRNAs and a novel tRNA-like molecule) and a novel nucleotidyltransferase associating with diverse ligases. These systems have been extensively disseminated by lateral transfer between distant prokaryotic and microbial eukaryotic lineages consistent with intense inter-organismal conflict. Components have also often been ‘institutionalized’ for non-conflict roles, e.g. in RNA-splicing and in RNAi systems (e.g. in kinetoplastids) which combine a distinct family of RNA-acting prim-pol domains with DICER-like proteins. PMID:27536007

SINGLE MOLECULE APPROACHES TO BIOLOGY, 2010 GORDON RESEARCH CONFERENCE, JUNE 27-JULY 2, 2010, ITALY

Energy Technology Data Exchange (ETDEWEB)

Professor William Moerner

2010-07-09

The 2010 Gordon Conference on Single-Molecule Approaches to Biology focuses on cutting-edge research in single-molecule science. Tremendous technical developments have made it possible to detect, identify, track, and manipulate single biomolecules in an ambient environment or even in a live cell. Single-molecule approaches have changed the way many biological problems are addressed, and new knowledge derived from these approaches continues to emerge. The ability of single-molecule approaches to avoid ensemble averaging and to capture transient intermediates and heterogeneous behavior renders them particularly powerful in elucidating mechanisms of biomolecular machines: what they do, how they work individually, how they work together, and finally, how they work inside live cells. The burgeoning use of single-molecule methods to elucidate biological problems is a highly multidisciplinary pursuit, involving both force- and fluorescence-based methods, the most up-to-date advances in microscopy, innovative biological and chemical approaches, and nanotechnology tools. This conference seeks to bring together top experts in molecular and cell biology with innovators in the measurement and manipulation of single molecules, and will provide opportunities for junior scientists and graduate students to present their work in poster format and to exchange ideas with leaders in the field. A number of excellent poster presenters will be selected for short oral talks. Topics as diverse as single-molecule sequencing, DNA/RNA/protein interactions, folding machines, cellular biophysics, synthetic biology and bioengineering, force spectroscopy, new method developments, superresolution imaging in cells, and novel probes for single-molecule imaging will be on the program. Additionally, the collegial atmosphere of this Conference, with programmed discussion sessions as well as opportunities for informal gatherings in the afternoons and evenings in the beauty of the Il Ciocco site in

Intergenic mRNA molecules resulting from trans-splicing.

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Finta, Csaba; Zaphiropoulos, Peter G

2002-02-22

Accumulated recent evidence is indicating that alternative splicing represents a generalized process that increases the complexity of human gene expression. Here we show that mRNA production may not necessarily be limited to single genes, as human liver also has the potential to produce a variety of hybrid cytochrome P450 3A mRNA molecules. The four known cytochrome P450 3A genes in humans, CYP3A4, CYP3A5, CYP3A7, and CYP3A43, share a high degree of similarity, consist of 13 exons with conserved exon-intron boundaries, and form a cluster on chromosome 7. The chimeric CYP3A mRNA molecules described herein are characterized by CYP3A43 exon 1 joined at canonical splice sites to distinct sets of CYP3A4 or CYP3A5 exons. Because the CYP3A43 gene is in a head-to-head orientation with the CYP3A4 and CYP3A5 genes, bypassing transcriptional termination can not account for the formation of hybrid CYP3A mRNAs. Thus, the mechanism generating these molecules has to be an RNA processing event that joins exons of independent pre-mRNA molecules, i.e. trans-splicing. Using quantitative real-time polymerase chain reaction, the ratio of one CYP3A43/3A4 intergenic combination was estimated to be approximately 0.15% that of the CYP3A43 mRNAs. Moreover, trans-splicing has been found not to interfere with polyadenylation. Heterologous expression of the chimeric species composed of CYP3A43 exon 1 joined to exons 2-13 of CYP3A4 revealed catalytic activity toward testosterone.

Post-transcriptional bursting in genes regulated by small RNA molecules

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Rodrigo, Guillermo

2018-03-01

Gene expression programs in living cells are highly dynamic due to spatiotemporal molecular signaling and inherent biochemical stochasticity. Here we study a mechanism based on molecule-to-molecule variability at the RNA level for the generation of bursts of protein production, which can lead to heterogeneity in a cell population. We develop a mathematical framework to show numerically and analytically that genes regulated post transcriptionally by small RNA molecules can exhibit such bursts due to different states of translation activity (on or off), mostly revealed in a regime of few molecules. We exploit this framework to compare transcriptional and post-transcriptional bursting and also to illustrate how to tune the resulting protein distribution with additional post-transcriptional regulations. Moreover, because RNA-RNA interactions are predictable with an energy model, we define the kinetic constants of on-off switching as functions of the two characteristic free-energy differences of the system, activation and formation, with a nonequilibrium scheme. Overall, post-transcriptional bursting represents a distinctive principle linking gene regulation to gene expression noise, which highlights the importance of the RNA layer beyond the simple information transfer paradigm and significantly contributes to the understanding of the intracellular processes from a first-principles perspective.

Diverging affinity of tospovirus RNA silencing suppressor proteins, NSs, for various RNA duplex molecules.

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Schnettler, Esther; Hemmes, Hans; Huismann, Rik; Goldbach, Rob; Prins, Marcel; Kormelink, Richard

2010-11-01

The tospovirus NSs protein was previously shown to suppress the antiviral RNA silencing mechanism in plants. Here the biochemical analysis of NSs proteins from different tospoviruses, using purified NSs or NSs containing cell extracts, is described. The results showed that all tospoviral NSs proteins analyzed exhibited affinity to small double-stranded RNA molecules, i.e., small interfering RNAs (siRNAs) and micro-RNA (miRNA)/miRNA* duplexes. Interestingly, the NSs proteins from tomato spotted wilt virus (TSWV), impatiens necrotic spot virus (INSV), and groundnut ringspot virus (GRSV) also showed affinity to long double-stranded RNA (dsRNA), whereas tomato yellow ring virus (TYRV) NSs did not. The TSWV NSs protein was shown to be capable of inhibiting Dicer-mediated cleavage of long dsRNA in vitro. In addition, it suppressed the accumulation of green fluorescent protein (GFP)-specific siRNAs during coinfiltration with an inverted-repeat-GFP RNA construct in Nicotiana benthamiana. In vivo interference of TSWV NSs in the miRNA pathway was shown by suppression of an enhanced GFP (eGFP) miRNA sensor construct. The ability to stabilize miRNA/miRNA* by different tospovirus NSs proteins in vivo was demonstrated by increased accumulation and detection of both miRNA171c and miRNA171c* in tospovirus-infected N. benthamiana. All together, these data suggest that tospoviruses interfere in the RNA silencing pathway by sequestering siRNA and miRNA/miRNA* molecules before they are uploaded into their respective RNA-induced silencing complexes. The observed affinity to long dsRNA for only a subset of the tospoviruses studied is discussed in light of evolutional divergence and their ancestral relation to the animal-infecting members of the Bunyaviridae.

Secondary structure of 5S RNA: NMR experiments on RNA molecules partially labeled with Nitrogen-15

International Nuclear Information System (INIS)

Gewirth, D.T.; Abo, S.R.; Leontis, N.B.; Moore, P.B.

1987-01-01

A method has been found for reassembling fragment 1 of Escherichia coli 5S RNA from mixtures containing strand III (bases 69-87) and the complex consisting of strand II (bases 89-120) and strand IV (bases 1-11). The reassembled molecule is identical with unreconstituted fragment 1. With this technique, fragment 1 molecules have been constructed 15 N-labeled either in strand III or in the strand II-strand IV complex. Spectroscopic data obtained with these partially labeled molecules show that the terminal helix of 5S RNA includes the GU and GC base pairs at positions 9 and 10 which the standard model for 5S secondary structure predicts but that these base pairs are unstable both in the fragment and in native 5S RNA. The data also assign three resonances to the helix V region of the molecule (bases 70-77 and 99-106). None of these resonances has a normal chemical shift even though two of them correspond to AU or GU base pairs in the standard model. The implications of these findings for the authors understanding of the structure of 5S RNA and its complex with ribosomal protein L25 are discussed

The role of medicaments, exosomes and miRNA molecules in modulation of macrophage immune activity

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Katarzyna Nazimek

2015-01-01

Full Text Available Macrophages play an important role in innate immunity, in induction and orchestration of acquired immune response as well as in the maintenance of tissue homeostasis. Macrophages as antigen presenting cells induce or inhibit the development of immune response and as effector cells play an important role in innate immunity to infectious agents and in delayed--type hypersensitivity as well. Thus, either up- or down-regulation of their activity leads to the impairment of different biological processes. This often results in the development of immunological diseases or inflammatory response associated with metabolic, cardiovascular or neuroendocrine disorders. Therefore, the possibility of modulation of macrophage function should allow for elaboration of new effective therapeutic strategies. Noteworthy, interaction of medicaments with macrophages may directly mediate their therapeutic activity or is an additional beneficial effect increasing efficacy of treatment. Further, macrophage differentiation is regulated by miRNA-223, while expression of miRNA-146 and miRNA-155 may modulate and/or be a result of the current cell phenotype. Present review is focused on the current knowledge about the action of medicaments, microRNA molecules, exosomes and related vesicles on macrophages leading to modulation of their biological activity.

Single-Molecule Analysis of Pre-mRNA Splicing with Colocalization Single-Molecule Spectroscopy (CoSMoS).

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Braun, Joerg E; Serebrov, Victor

2017-01-01

Recent development of single-molecule techniques to study pre-mRNA splicing has provided insights into the dynamic nature of the spliceosome. Colocalization single-molecule spectroscopy (CoSMoS) allows following spliceosome assembly in real time at single-molecule resolution in the full complexity of cellular extracts. A detailed protocol of CoSMoS has been published previously (Anderson and Hoskins, Methods Mol Biol 1126:217-241, 2014). Here, we provide an update on the technical advances since the first CoSMoS studies including slide surface treatment, data processing, and representation. We describe various labeling strategies to generate RNA reporters with multiple dyes (or other moieties) at specific locations.

Electro-induced reactions of biologically important molecules

International Nuclear Information System (INIS)

Kocisek, J.

2010-01-01

The thesis presents the results of research activities in the field of electron interactions with biologically relevant molecules which was carried out during my PhD studies at the Department of Experimental Physics, Comenius University in Bratislava. Electron induced interactions with biologically relevant molecules were experimentally studied using crossed electron-molecule beams experiment. The obtained results, were presented in four publications in international scientific journals. First study of deals with electron impact ionisation of furanose alcohols [see 1. in list of author publications on page 22]. It has been motivated by most important works in the field of electron induced damages of DNA bases [4]. Studied 3-hydroxytetrahydrofuran and tetrahydrofurfuryl alcohol, are important model molecules for more complex biological systems (e.g. deoxyribose).The influence of hydroxyl group on stabilisation of the positive ions of the molecules, together with the stability of furan ring in ionized form are main themes of the study. The studies of small amides and aminoacids are connected to scientific studies in the field of formation of the aminoacids and other biologically relevant molecules in space and works trying to explain electron induced processes in more complex molecules[12, 13, 24]. The most important results were obtained for aminoacid Serine [see 2. in list of author publications on page 22]. We have showed that additional OH group of Serine considerably lower the reaction enthalpy limit of reactions resulting to formation of neutral water molecules, in comparison to other amino acids. Also the study of (M-H)- reaction channel using the electron beam with FWHM under 100 meV is of high importance in the field. The last part of the thesis is focused on the electron interactions with organosilane compounds. Materials prepared from organosilane molecules in plasmas have wide range of applications in both biology and medicine. We have studied electron

Finding Order in Randomness: Single-Molecule Studies Reveal Stochastic RNA Processing | Center for Cancer Research

Science.gov (United States)

Producing a functional eukaryotic messenger RNA (mRNA) requires the coordinated activity of several large protein complexes to initiate transcription, elongate nascent transcripts, splice together exons, and cleave and polyadenylate the 3’ end. Kinetic competition between these various processes has been proposed to regulate mRNA maturation, but this model could lead to multiple, randomly determined, or stochastic, pathways or outcomes. Regulatory checkpoints have been suggested as a means of ensuring quality control. However, current methods have been unable to tease apart the contributions of these processes at a single gene or on a time scale that could provide mechanistic insight. To begin to investigate the kinetic relationship between transcription and splicing, Daniel Larson, Ph.D., of CCR’s Laboratory of Receptor Biology and Gene Expression, and his colleagues employed a single-molecule RNA imaging approach to monitor production and processing of a human β-globin reporter gene in living cells.

Permuted tRNA genes of Cyanidioschyzon merolae, the origin of the tRNA molecule and the root of the Eukarya domain.

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Di Giulio, Massimo

2008-08-07

An evolutionary analysis is conducted on the permuted tRNA genes of Cyanidioschyzon merolae, in which the 5' half of the tRNA molecule is codified at the 3' end of the gene and its 3' half is codified at the 5' end. This analysis has shown that permuted genes cannot be considered as derived traits but seem to possess characteristics that suggest they are ancestral traits, i.e. they originated when tRNA molecule genes originated for the first time. In particular, if the hypothesis that permuted genes are a derived trait were true, then we should not have been able to observe that the most frequent class of permuted genes is that of the anticodon loop type, for the simple reason that this class would derive by random permutation from a class of non-permuted tRNA genes, which instead is the rarest. This would not explain the high frequency with which permuted tRNA genes with perfectly separate 5' and 3' halves were observed. Clearly the mechanism that produced this class of permuted genes would envisage the existence, in an advanced stage of evolution, of minigenes codifying for the 5' and 3' halves of tRNAs which were assembled in a permuted way at the origin of the tRNA molecule, thus producing a high frequency of permuted genes of the class here referred. Therefore, this evidence supports the hypothesis that the genes of the tRNA molecule were assembled by minigenes codifying for hairpin-like RNA molecules, as suggested by one model for the origin of tRNA [Di Giulio, M., 1992. On the origin of the transfer RNA molecule. J. Theor. Biol. 159, 199-214; Di Giulio, M., 1999. The non-monophyletic origin of tRNA molecule. J. Theor. Biol. 197, 403-414]. Moreover, the late assembly of the permuted genes of C. merolae, as well as their ancestrality, strengthens the hypothesis of the polyphyletic origins of these genes. Finally, on the basis of the uniqueness and the ancestrality of these permuted genes, I suggest that the root of the Eukarya domain is in the super

RNA targeting by small molecules: Binding of protoberberine ...

Indian Academy of Sciences (India)

2012-06-25

Jun 25, 2012 ... Studies on RNA targeting by small molecules to specifically control certain cellular functions is an .... form secondary structures such as stem-loop, hairpin, etc. ..... paired third strand of the triplex without affecting the stability.

Comparison of methods for miRNA extraction from plasma and quantitative recovery of RNA from plasma and cerebrospinal fluid

Directory of Open Access Journals (Sweden)

Melissa A McAlexander

2013-05-01

Full Text Available Interest in extracellular RNA has intensified as evidence accumulates that these molecules may be useful as indicators of a wide variety of biological conditions. To establish specific extracellular RNA molecules as clinically relevant biomarkers, reproducible recovery from biological samples and reliable measurements of the isolated RNA are paramount. Towards these ends, careful and rigorous comparisons of technical procedures are needed at all steps from sample handling to RNA isolation to RNA measurement protocols. In the investigations described in this methods paper, RT-qPCR was used to examine the apparent recovery of specific endogenous miRNAs and a spiked-in synthetic RNA from blood plasma samples. RNA was isolated using several widely used RNA isolation kits, with or without the addition of glycogen as a carrier. Kits examined included total RNA isolation systems that have been commercially available for several years and commonly adapted for extraction of biofluid RNA, as well as more recently introduced biofluids-specific RNA methods. Our conclusions include the following: some RNA isolation methods appear to be superior to others for the recovery of RNA from biological fluids; addition of a carrier molecule seems to be beneficial for some but not all isolation methods; and partially or fully quantitative recovery of RNA is observed from increasing volumes of plasma and cerebrospinal fluid.

Analysis of extracellular RNA by digital PCR

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Kenji eTakahashi

2014-06-01

Full Text Available The transfer of extracellular RNA is emerging as an important mechanism for intracellular communication. The ability for the transfer of functionally active RNA molecules from one cell to another within vesicles such as exosomes enables a cell to modulate cellular signaling and biological processes within recipient cells. The study of extracellular RNA requires sensitive methods for the detection of these molecules. In this methods article, we will describe protocols for the detection of such extracellular RNA using sensitive detection technologies such as digital PCR. These protocols should be valuable to researchers interested in the role and contribution of extracellular RNA to tumor cell biology.

Circular RNA (circRNA) was an important bridge in the switch from the RNA world to the DNA world.

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Soslau, Gerald

2018-06-14

The concept that life on Earth began as an RNA world has been built upon extensive experimentation demonstrating that many of the building blocks required for living cells could be synthesized in the laboratory under conditions approximating our primordial world. Many of the building blocks for life have also been found in meteorites indicating that meteors may have been a source for these molecules, or more likely, that they represent the chemical library present in most/all bodies in the universe after the big bang. Perhaps the most important support for the concept comes from the fact that some RNA species possess catalytic activity, ribozymes, and that RNA could be reverse transcribe to DNA. The thrust of numerous papers on this topic has been to explore how the available molecules on Earth, at its birth, gave rise to life as we know it today. This paper focuses more on a reverse view of the topic. The "how" molecular building blocks were synthesized is not addressed nor how the "first" RNA molecules were synthesized. We can clearly speculate on the variable environmental conditions and chemistry available on Earth billions of years ago. However, we can never truly replicate the changing conditions or know the chemical composition of Earth at the beginning of time. We can, however, confirm that over millions, perhaps billions of years the basic building blocks for life accumulated sufficiently to initiate evolution to an RNA world followed by our RNA/DNA world. Here we are attempting to take the information from our current knowledge of biology and by inference and extrapolation work backward to hypothesize biological events in the march forward from RNA to DNA. It is proposed that the primordial replicating RNA cell, the ribocyte, evolved from liposomes encompassing required reactants and products for "life" and that ribonucleopeptide complexes formed membrane pores to support bidirectional ion and molecular transport to maintain biological functions and

Fast prediction of RNA-RNA interaction using heuristic algorithm.

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Montaseri, Soheila

2015-01-01

Interaction between two RNA molecules plays a crucial role in many medical and biological processes such as gene expression regulation. In this process, an RNA molecule prohibits the translation of another RNA molecule by establishing stable interactions with it. Some algorithms have been formed to predict the structure of the RNA-RNA interaction. High computational time is a common challenge in most of the presented algorithms. In this context, a heuristic method is introduced to accurately predict the interaction between two RNAs based on minimum free energy (MFE). This algorithm uses a few dot matrices for finding the secondary structure of each RNA and binding sites between two RNAs. Furthermore, a parallel version of this method is presented. We describe the algorithm's concurrency and parallelism for a multicore chip. The proposed algorithm has been performed on some datasets including CopA-CopT, R1inv-R2inv, Tar-Tar*, DIS-DIS, and IncRNA54-RepZ in Escherichia coli bacteria. The method has high validity and efficiency, and it is run in low computational time in comparison to other approaches.

Prediction of RNA secondary structures: from theory to models and real molecules

International Nuclear Information System (INIS)

Schuster, Peter

2006-01-01

RNA secondary structures are derived from RNA sequences, which are strings built form the natural four letter nucleotide alphabet, {AUGC}. These coarse-grained structures, in turn, are tantamount to constrained strings over a three letter alphabet. Hence, the secondary structures are discrete objects and the number of sequences always exceeds the number of structures. The sequences built from two letter alphabets form perfect structures when the nucleotides can form a base pair, as is the case with {GC} or {AU}, but the relation between the sequences and structures differs strongly from the four letter alphabet. A comprehensive theory of RNA structure is presented, which is based on the concepts of sequence space and shape space, being a space of structures. It sets the stage for modelling processes in ensembles of RNA molecules like evolutionary optimization or kinetic folding as dynamical phenomena guided by mappings between the two spaces. The number of minimum free energy (mfe) structures is always smaller than the number of sequences, even for two letter alphabets. Folding of RNA molecules into mfe energy structures constitutes a non-invertible mapping from sequence space onto shape space. The preimage of a structure in sequence space is defined as its neutral network. Similarly the set of suboptimal structures is the preimage of a sequence in shape space. This set represents the conformation space of a given sequence. The evolutionary optimization of structures in populations is a process taking place in sequence space, whereas kinetic folding occurs in molecular ensembles that optimize free energy in conformation space. Efficient folding algorithms based on dynamic programming are available for the prediction of secondary structures for given sequences. The inverse problem, the computation of sequences for predefined structures, is an important tool for the design of RNA molecules with tailored properties. Simultaneous folding or cofolding of two or more RNA

Tapping the RNA world for therapeutics.

Science.gov (United States)

Lieberman, Judy

2018-04-16

A recent revolution in RNA biology has led to the identification of new RNA classes with unanticipated functions, new types of RNA modifications, an unexpected multiplicity of alternative transcripts and widespread transcription of extragenic regions. This development in basic RNA biology has spawned a corresponding revolution in RNA-based strategies to generate new types of therapeutics. Here, I review RNA-based drug design and discuss barriers to broader applications and possible ways to overcome them. Because they target nucleic acids rather than proteins, RNA-based drugs promise to greatly extend the domain of 'druggable' targets beyond what can be achieved with small molecules and biologics.

antaRNA: ant colony-based RNA sequence design.

Science.gov (United States)

Kleinkauf, Robert; Mann, Martin; Backofen, Rolf

2015-10-01

RNA sequence design is studied at least as long as the classical folding problem. Although for the latter the functional fold of an RNA molecule is to be found ,: inverse folding tries to identify RNA sequences that fold into a function-specific target structure. In combination with RNA-based biotechnology and synthetic biology ,: reliable RNA sequence design becomes a crucial step to generate novel biochemical components. In this article ,: the computational tool antaRNA is presented. It is capable of compiling RNA sequences for a given structure that comply in addition with an adjustable full range objective GC-content distribution ,: specific sequence constraints and additional fuzzy structure constraints. antaRNA applies ant colony optimization meta-heuristics and its superior performance is shown on a biological datasets. http://www.bioinf.uni-freiburg.de/Software/antaRNA CONTACT: backofen@informatik.uni-freiburg.de Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press.

Three-dimensional model of a selective theophylline-binding RNA molecule

Energy Technology Data Exchange (ETDEWEB)

Tung, Chang-Shung; Oprea, T.I.; Hummer, G.; Garcia, A.E.

1995-07-01

We propose a three-dimensional (3D) model for an RNA molecule that selectively binds theophylline but not caffeine. This RNA, which was found using SELEX [Jenison, R.D., et al., Science (1994) 263:1425] is 10,000 times more specific for theophylline (Kd=320 nM) than for caffeine (Kd=3.5 mM), although the two ligands are identical except for a methyl group substituted at N7 (present only in caffeine). The binding affinity for ten xanthine-based ligands was used to derive a Comparative Molecular Field Analysis (CoMFA) model (R{sup 2} = 0.93 for 3 components, with cross-validated R{sup 2} of 0.73), using the SYBYL and GOLPE programs. A pharmacophoric map was generated to locate steric and electrostatic interactions between theophylline and the RNA binding site. This information was used to identify putative functional groups of the binding pocket and to generate distance constraints. Based on a model for the secondary structure (Jenison et al., idem), the 3D structure of this RNA was then generated using the following method: each helical region of the RNA molecule was treated as a rigid body; single-stranded loops with specific end-to-end distances were generated. The structures of RNA-xanthine complexes were studied using a modified Monte Carlo algorithm. The detailed structure of an RNA-ligand complex model, as well as possible explanations for the theophylline selectivity will be discussed.

Approaches to Validate and Manipulate RNA Targets with Small Molecules in Cells.

Science.gov (United States)

Childs-Disney, Jessica L; Disney, Matthew D

2016-01-01

RNA has become an increasingly important target for therapeutic interventions and for chemical probes that dissect and manipulate its cellular function. Emerging targets include human RNAs that have been shown to directly cause cancer, metabolic disorders, and genetic disease. In this review, we describe various routes to obtain bioactive compounds that target RNA, with a particular emphasis on the development of small molecules. We use these cases to describe approaches that are being developed for target validation, which include target-directed cleavage, classic pull-down experiments, and covalent cross-linking. Thus, tools are available to design small molecules to target RNA and to identify the cellular RNAs that are their targets.

Structural Analysis of ‘key’ Interactions in Functional RNA Molecules

KAUST Repository

Chawla, Mohit

2018-04-01

The main objective of the thesis is to carry out structural bioinformatics study along with usage of advanced quantum chemical methods to look at the structural stability and energetics of RNA building blocks. The main focus of the work described here lies on understanding the reasons behind the intrinsic stability of key interactions in nucleic acids. Crystal structures of RNA molecules exhibit fascinating variety of non-covalent interactions, which play an important role in maintaining the three dimensional structures. An accurate atomic level description of these interactions in the structural building blocks of RNA is a key to understand the structure-function relationship in these molecules. An effort has been made to link the conclusions drawn from quantum chemical computations on RNA base pairs in wide biochemical context of their occurrence in RNA structures. The initial attention was on the impact of natural and non-natural modifications of the nucleic acid bases on the structure and stability of base pairs that they are involved in. In the remaining sections we cover other molecular interactions shaping nucleic acids, as the interaction between ribose and the bases, and the fluoride sensing riboswitch system in order to investigate structure and dynamics of nucleic acids at the atomic level and to gain insight into the physical chemistry behind.

Structural Analysis of ‘key’ Interactions in Functional RNA Molecules

KAUST Repository

Chawla, Mohit

2018-01-01

The main objective of the thesis is to carry out structural bioinformatics study along with usage of advanced quantum chemical methods to look at the structural stability and energetics of RNA building blocks. The main focus of the work described here lies on understanding the reasons behind the intrinsic stability of key interactions in nucleic acids. Crystal structures of RNA molecules exhibit fascinating variety of non-covalent interactions, which play an important role in maintaining the three dimensional structures. An accurate atomic level description of these interactions in the structural building blocks of RNA is a key to understand the structure-function relationship in these molecules. An effort has been made to link the conclusions drawn from quantum chemical computations on RNA base pairs in wide biochemical context of their occurrence in RNA structures. The initial attention was on the impact of natural and non-natural modifications of the nucleic acid bases on the structure and stability of base pairs that they are involved in. In the remaining sections we cover other molecular interactions shaping nucleic acids, as the interaction between ribose and the bases, and the fluoride sensing riboswitch system in order to investigate structure and dynamics of nucleic acids at the atomic level and to gain insight into the physical chemistry behind.

Live Cell Genomics: RNA Exon-Specific RNA-Binding Protein Isolation.

Science.gov (United States)

Bell, Thomas J; Eberwine, James

2015-01-01

RNA-binding proteins (RBPs) are essential regulatory proteins that control all modes of RNA processing and regulation. New experimental approaches to isolate these indispensable proteins under in vivo conditions are needed to advance the field of RBP biology. Historically, in vitro biochemical approaches to isolate RBP complexes have been useful and productive, but biological relevance of the identified RBP complexes can be imprecise or erroneous. Here we review an inventive experimental to isolate RBPs under the in vivo conditions. The method is called peptide nucleic acid (PNA)-assisted identification of RBP (PAIR) technology and it uses cell-penetrating peptides (CPPs) to deliver photo-activatible RBP-capture molecule to the cytoplasm of the live cells. The PAIR methodology provides two significant advantages over the most commonly used approaches: (1) it overcomes the in vitro limitation of standard biochemical approaches and (2) the PAIR RBP-capture molecule is highly selective and adaptable which allows investigators to isolate exon-specific RBP complexes. Most importantly, the in vivo capture conditions and selectivity of the RBP-capture molecule yield biologically accurate and relevant RBP data.

NMR-study of dynamic structural transtions in RNA-molecules

OpenAIRE

Fürtig, Boris

2007-01-01

The following thesis is concerned with the elucidation of structural changes of RNA molecules during the time course of dynamic processes that are commonly denoted as folding reactions. In contrast to the field of protein folding, the concept of RNA folding comprises not only folding reactions itself but also refolding- or conformational switching- and assembly processes (see chapter III). The method in this thesis to monitor these diverse processes is high resolution liquid-state NMR spectro...

Alteration of RNA splicing by small molecule inhibitors of the interaction between NHP2L1 and U4

Science.gov (United States)

Diouf, Barthelemy; Lin, Wenwei; Goktug, Asli; Grace, Christy R. R.; Waddell, Michael Brett; Bao, Ju; Shao, Youming; Heath, Richard J.; Zheng, Jie J.; Shelat, Anang A.; Relling, Mary V.; Chen, Taosheng; Evans, William E.

2018-01-01

Splicing is an important eukaryotic mechanism for expanding the transcriptome and proteome, influencing a number of biological processes. Understanding its regulation and identifying small molecules that modulate this process remains a challenge. We developed an assay based on time-resolved FRET (TR-FRET) to detect the interaction between the protein NHP2L1 and U4 RNA, which are two key components of the spliceosome. We used this assay to identify small molecules that interfere with this interaction in a high-throughput screening (HTS) campaign. Topotecan and other camptothecin derivatives were among the top hits. We confirmed that topotecan disrupts the interaction between NHP2L1 and U4 by binding to U4 and inhibits RNA splicing. Our data reveal new functions of known drugs which could facilitate the development of therapeutic strategies to modify splicing and alter gene function. PMID:28985478

Single-molecule experiments in biological physics: methods and applications.

Science.gov (United States)

Ritort, F

2006-08-16

I review single-molecule experiments (SMEs) in biological physics. Recent technological developments have provided the tools to design and build scientific instruments of high enough sensitivity and precision to manipulate and visualize individual molecules and measure microscopic forces. Using SMEs it is possible to manipulate molecules one at a time and measure distributions describing molecular properties, characterize the kinetics of biomolecular reactions and detect molecular intermediates. SMEs provide additional information about thermodynamics and kinetics of biomolecular processes. This complements information obtained in traditional bulk assays. In SMEs it is also possible to measure small energies and detect large Brownian deviations in biomolecular reactions, thereby offering new methods and systems to scrutinize the basic foundations of statistical mechanics. This review is written at a very introductory level, emphasizing the importance of SMEs to scientists interested in knowing the common playground of ideas and the interdisciplinary topics accessible by these techniques. The review discusses SMEs from an experimental perspective, first exposing the most common experimental methodologies and later presenting various molecular systems where such techniques have been applied. I briefly discuss experimental techniques such as atomic-force microscopy (AFM), laser optical tweezers (LOTs), magnetic tweezers (MTs), biomembrane force probes (BFPs) and single-molecule fluorescence (SMF). I then present several applications of SME to the study of nucleic acids (DNA, RNA and DNA condensation) and proteins (protein-protein interactions, protein folding and molecular motors). Finally, I discuss applications of SMEs to the study of the nonequilibrium thermodynamics of small systems and the experimental verification of fluctuation theorems. I conclude with a discussion of open questions and future perspectives.

Single-molecule experiments in biological physics: methods and applications

International Nuclear Information System (INIS)

Ritort, F

2006-01-01

I review single-molecule experiments (SMEs) in biological physics. Recent technological developments have provided the tools to design and build scientific instruments of high enough sensitivity and precision to manipulate and visualize individual molecules and measure microscopic forces. Using SMEs it is possible to manipulate molecules one at a time and measure distributions describing molecular properties, characterize the kinetics of biomolecular reactions and detect molecular intermediates. SMEs provide additional information about thermodynamics and kinetics of biomolecular processes. This complements information obtained in traditional bulk assays. In SMEs it is also possible to measure small energies and detect large Brownian deviations in biomolecular reactions, thereby offering new methods and systems to scrutinize the basic foundations of statistical mechanics. This review is written at a very introductory level, emphasizing the importance of SMEs to scientists interested in knowing the common playground of ideas and the interdisciplinary topics accessible by these techniques. The review discusses SMEs from an experimental perspective, first exposing the most common experimental methodologies and later presenting various molecular systems where such techniques have been applied. I briefly discuss experimental techniques such as atomic-force microscopy (AFM), laser optical tweezers (LOTs), magnetic tweezers (MTs), biomembrane force probes (BFPs) and single-molecule fluorescence (SMF). I then present several applications of SME to the study of nucleic acids (DNA, RNA and DNA condensation) and proteins (protein-protein interactions, protein folding and molecular motors). Finally, I discuss applications of SMEs to the study of the nonequilibrium thermodynamics of small systems and the experimental verification of fluctuation theorems. I conclude with a discussion of open questions and future perspectives. (topical review)

Integrated structural biology to unravel molecular mechanisms of protein-RNA recognition.

Science.gov (United States)

Schlundt, Andreas; Tants, Jan-Niklas; Sattler, Michael

2017-04-15

Recent advances in RNA sequencing technologies have greatly expanded our knowledge of the RNA landscape in cells, often with spatiotemporal resolution. These techniques identified many new (often non-coding) RNA molecules. Large-scale studies have also discovered novel RNA binding proteins (RBPs), which exhibit single or multiple RNA binding domains (RBDs) for recognition of specific sequence or structured motifs in RNA. Starting from these large-scale approaches it is crucial to unravel the molecular principles of protein-RNA recognition in ribonucleoprotein complexes (RNPs) to understand the underlying mechanisms of gene regulation. Structural biology and biophysical studies at highest possible resolution are key to elucidate molecular mechanisms of RNA recognition by RBPs and how conformational dynamics, weak interactions and cooperative binding contribute to the formation of specific, context-dependent RNPs. While large compact RNPs can be well studied by X-ray crystallography and cryo-EM, analysis of dynamics and weak interaction necessitates the use of solution methods to capture these properties. Here, we illustrate methods to study the structure and conformational dynamics of protein-RNA complexes in solution starting from the identification of interaction partners in a given RNP. Biophysical and biochemical techniques support the characterization of a protein-RNA complex and identify regions relevant in structural analysis. Nuclear magnetic resonance (NMR) is a powerful tool to gain information on folding, stability and dynamics of RNAs and characterize RNPs in solution. It provides crucial information that is complementary to the static pictures derived from other techniques. NMR can be readily combined with other solution techniques, such as small angle X-ray and/or neutron scattering (SAXS/SANS), electron paramagnetic resonance (EPR), and Förster resonance energy transfer (FRET), which provide information about overall shapes, internal domain

NCI RNA Biology 2017 symposium recap | Center for Cancer Research

Science.gov (United States)

The recent discovery of new classes of RNAs and the demonstration that alterations in RNA metabolism underlie numerous human cancers have resulted in enormous interest among CCR investigators in RNA biology. In order to share the latest research in this exciting field, the CCR Initiative in RNA Biology held its second international symposium April 23-24, 2017, in Natcher

Efficient CRISPR/Cas9-Mediated Genome Editing Using a Chimeric Single-Guide RNA Molecule

KAUST Repository

Butt, Haroon; Eid, Ayman; Ali, Zahir; Atia, Mohamed A. M.; Mokhtar, Morad M.; Hassan, Norhan; Lee, Ciaran M.; Bao, Gang; Mahfouz, Magdy M.

2017-01-01

used CRISPR/Cas9 to generate targeted double-strand breaks and to deliver an RNA repair template for HDR in rice (Oryza sativa). We used chimeric single-guide RNA (cgRNA) molecules carrying both sequences for target site specificity (to generate

Enhanced NMR signal detection of imino protons in RNA molecules containing 3' dangling nucleotides

International Nuclear Information System (INIS)

Amborski, Andrew N.; Johnson, Philip E.

2008-01-01

We present a method for improving the quality of nuclear magnetic resonance (NMR) spectra involving exchangeable protons near the base of the stem of RNA hairpin molecules. NMR spectra of five different RNA hairpins were compared. These hairpins consisted of a native RNA structure and four molecules each having different unpaired, or dangling, nucleotides at the 3' end. NMR experiments were acquired in water for each construct and the quality of the imino proton spectral regions were examined. The imino resonances near the base of the stem of the wild type RNA structure were not observed due to breathing motions. However, a significant increase in spectral quality for molecules with dangling 3' adenosine or guanosine nucleotides was observed, with imino protons detected in these constructs that were not observed in the wild type construct. A modest improvement in spectral quality was seen for the construct with a 3' unpaired uridine, whereas no significant improvement was observed for a 3' unpaired cytidine. This improvement in NMR spectral quality mirrors the increased thermodynamic stability observed for 3' unpaired nucleotides which is dependant on the stacking interactions of these nucleotides against the base of the stem. The use of a dangling 3' adenosine nucleotide represents an easy method to significantly improve the quality of NMR spectra of RNA molecules

RNA secondary structure diagrams for very large molecules: RNAfdl

DEFF Research Database (Denmark)

Hecker, Nikolai; Wiegels, Tim; Torda, Andrew E.

2013-01-01

There are many programs that can read the secondary structure of an RNA molecule and draw a diagram, but hardly any that can cope with 10 3 bases. RNAfdl is slow but capable of producing intersection-free diagrams for ribosome-sized structures, has a graphical user interface for adjustments...

RNAspa: a shortest path approach for comparative prediction of the secondary structure of ncRNA molecules

Directory of Open Access Journals (Sweden)

Michaeli Shulamit

2007-10-01

Full Text Available Abstract Background In recent years, RNA molecules that are not translated into proteins (ncRNAs have drawn a great deal of attention, as they were shown to be involved in many cellular functions. One of the most important computational problems regarding ncRNA is to predict the secondary structure of a molecule from its sequence. In particular, we attempted to predict the secondary structure for a set of unaligned ncRNA molecules that are taken from the same family, and thus presumably have a similar structure. Results We developed the RNAspa program, which comparatively predicts the secondary structure for a set of ncRNA molecules in linear time in the number of molecules. We observed that in a list of several hundred suboptimal minimal free energy (MFE predictions, as provided by the RNAsubopt program of the Vienna package, it is likely that at least one suggested structure would be similar to the true, correct one. The suboptimal solutions of each molecule are represented as a layer of vertices in a graph. The shortest path in this graph is the basis for structural predictions for the molecule. We also show that RNA secondary structures can be compared very rapidly by a simple string Edit-Distance algorithm with a minimal loss of accuracy. We show that this approach allows us to more deeply explore the suboptimal structure space. Conclusion The algorithm was tested on three datasets which include several ncRNA families taken from the Rfam database. These datasets allowed for comparison of the algorithm with other methods. In these tests, RNAspa performed better than four other programs.

A large collapsed-state RNA can exhibit simple exponential single-molecule dynamics.

Science.gov (United States)

Smith, Glenna J; Lee, Kang Taek; Qu, Xiaohui; Xie, Zheng; Pesic, Jelena; Sosnick, Tobin R; Pan, Tao; Scherer, Norbert F

2008-05-09

The process of large RNA folding is believed to proceed from many collapsed structures to a unique functional structure requiring precise organization of nucleotides. The diversity of possible structures and stabilities of large RNAs could result in non-exponential folding kinetics (e.g. stretched exponential) under conditions where the molecules have not achieved their native state. We describe a single-molecule fluorescence resonance energy transfer (FRET) study of the collapsed-state region of the free energy landscape of the catalytic domain of RNase P RNA from Bacillus stearothermophilus (C(thermo)). Ensemble measurements have shown that this 260 residue RNA folds cooperatively to its native state at >or=1 mM Mg(2+), but little is known about the conformational dynamics at lower ionic strength. Our measurements of equilibrium conformational fluctuations reveal simple exponential kinetics that reflect a small number of discrete states instead of the expected inhomogeneous dynamics. The distribution of discrete dwell times, collected from an "ensemble" of 300 single molecules at each of a series of Mg(2+) concentrations, fit well to a double exponential, which indicates that the RNA conformational changes can be described as a four-state system. This finding is somewhat unexpected under [Mg(2+)] conditions in which this RNA does not achieve its native state. Observation of discrete well-defined conformations in this large RNA that are stable on the seconds timescale at low [Mg(2+)] (<0.1 mM) suggests that even at low ionic strength, with a tremendous number of possible (weak) interactions, a few critical interactions may produce deep energy wells that allow for rapid averaging of motions within each well, and yield kinetics that are relatively simple.

NCI RNA Biology 2017 symposium recap | Center for Cancer Research

Science.gov (United States)

The recent discovery of new classes of RNAs and the demonstration that alterations in RNA metabolism underlie numerous human cancers have resulted in enormous interest among CCR investigators in RNA biology. In order to share the latest research in this exciting field, the CCR Initiative in RNA Biology held its second international symposium April 23-24, 2017, in Natcher Auditorium. Learn more...

Double Stranded RNA in Human Seminal Plasma

Directory of Open Access Journals (Sweden)

Maxim V. Zagoskin

2017-10-01

Full Text Available Recently, human semen was shown to contain cell-free nucleic acids, such as DNA, long single stranded RNA, and small RNAs–miRNA and piRNA. The RNAs have been suggested to have potential biological roles as communication molecules between cells and in the temporal and spatial regulation of gene expression in the male reproductive system. Here we demonstrate that human seminal plasma contains a variety of cell-free dsRNAs, describe a robust method to isolate this type of nucleic acid in preparative amounts, and discuss the potential biological roles of these molecules in inheritance. dsRNA plays a role in a variety of biological processes, including gene regulation, is extremely stable and can gain access to cells from the extracellular medium. We suggest that one of the possible functions of dsRNA in human seminal plasma may be to influence human oocytes and therefore, influence the offspring. It also remains possible that these dsRNAs might have potential use as biomarkers for the study of human physiopathological conditions and genetic variation.

Cofactors in the RNA World

Science.gov (United States)

Ditzler, Mark A.

2014-01-01

RNA world theories figure prominently in many scenarios for the origin and early evolution of life. These theories posit that RNA molecules played a much larger role in ancient biology than they do now, acting both as the dominant biocatalysts and as the repository of genetic information. Many features of modern RNA biology are potential examples of molecular fossils from an RNA world, such as the pervasive involvement of nucleotides in coenzymes, the existence of natural aptamers that bind these coenzymes, the existence of natural ribozymes, a biosynthetic pathway in which deoxynucleotides are produced from ribonucleotides, and the central role of ribosomal RNA in protein synthesis in the peptidyl transferase center of the ribosome. Here, we uses both a top-down approach that evaluates RNA function in modern biology and a bottom-up approach that examines the capacities of RNA independent of modern biology. These complementary approaches exploit multiple in vitro evolution techniques coupled with high-throughput sequencing and bioinformatics analysis. Together these complementary approaches advance our understanding of the most primitive organisms, their early evolution, and their eventual transition to modern biochemistry.

Bioinformatics of cardiovascular miRNA biology.

Science.gov (United States)

Kunz, Meik; Xiao, Ke; Liang, Chunguang; Viereck, Janika; Pachel, Christina; Frantz, Stefan; Thum, Thomas; Dandekar, Thomas

2015-12-01

MicroRNAs (miRNAs) are small ~22 nucleotide non-coding RNAs and are highly conserved among species. Moreover, miRNAs regulate gene expression of a large number of genes associated with important biological functions and signaling pathways. Recently, several miRNAs have been found to be associated with cardiovascular diseases. Thus, investigating the complex regulatory effect of miRNAs may lead to a better understanding of their functional role in the heart. To achieve this, bioinformatics approaches have to be coupled with validation and screening experiments to understand the complex interactions of miRNAs with the genome. This will boost the subsequent development of diagnostic markers and our understanding of the physiological and therapeutic role of miRNAs in cardiac remodeling. In this review, we focus on and explain different bioinformatics strategies and algorithms for the identification and analysis of miRNAs and their regulatory elements to better understand cardiac miRNA biology. Starting with the biogenesis of miRNAs, we present approaches such as LocARNA and miRBase for combining sequence and structure analysis including phylogenetic comparisons as well as detailed analysis of RNA folding patterns, functional target prediction, signaling pathway as well as functional analysis. We also show how far bioinformatics helps to tackle the unprecedented level of complexity and systemic effects by miRNA, underlining the strong therapeutic potential of miRNA and miRNA target structures in cardiovascular disease. In addition, we discuss drawbacks and limitations of bioinformatics algorithms and the necessity of experimental approaches for miRNA target identification. This article is part of a Special Issue entitled 'Non-coding RNAs'. Copyright © 2014 Elsevier Ltd. All rights reserved.

Ethylene glycol monolayer protected nanoparticles: synthesis, characterization, and interactions with biological molecules.

Science.gov (United States)

Zheng, Ming; Li, Zhigang; Huang, Xueying

2004-05-11

The usefulness of the hybrid materials of nanoparticles and biological molecules on many occasions depends on how well one can achieve a rational design based on specific binding and programmable assembly. Nonspecific binding between nanoparticles and biomolecules is one of the major barriers for achieving their utilities in a biological system. In this paper, we demonstrate a new approach to eliminate nonspecific interactions between nanoparticles and biological molecules by shielding the nanoparticle with a monolayer of ethylene glycol. A direct synthesis of di-, tri-, and tetra(ethylene glycol)-protected gold nanoparticles (Au-S-EGn, n = 2, 3, and 4) was achieved under the condition that the water content was optimized in the range of 9-18% in the reaction mixture. With controlled ratio of [HAuCl4]/[EGn-SH] at 2, the synthesized particles have an average diameter of 3.5 nm and a surface plasma resonance band around 510 nm. Their surface structures were confirmed by 1H NMR spectra. These gold nanoparticles are bonded with a uniform monolayer with defined lengths of 0.8, 1.2, and 1.6 nm for Au-S-EG2, Au-S-EG3, and Au-S-EG4, respectively. They have great stabilities in aqueous solutions with a high concentration of electrolytes as well as in organic solvents. Thermogravimetric analysis revealed that the ethylene glycol monolayer coating is ca. 14% of the total nanoparticle weight. Biological binding tests by using ion-exchange chromatography and gel electrophoresis demonstrated that these Au-S-EGn (n = 2, 3, or 4) nanoparticles are free of any nonspecific bindings with various proteins, DNA, and RNA. These types of nanoparticles provide a fundamental starting material for designing hybrid materials composed of metallic nanoparticles and biomolecules.

An enhanced computational platform for investigating the roles of regulatory RNA and for identifying functional RNA motifs

OpenAIRE

Chang, Tzu-Hao; Huang, Hsi-Yuan; Hsu, Justin Bo-Kai; Weng, Shun-Long; Horng, Jorng-Tzong; Huang, Hsien-Da

2013-01-01

Background Functional RNA molecules participate in numerous biological processes, ranging from gene regulation to protein synthesis. Analysis of functional RNA motifs and elements in RNA sequences can obtain useful information for deciphering RNA regulatory mechanisms. Our previous work, RegRNA, is widely used in the identification of regulatory motifs, and this work extends it by incorporating more comprehensive and updated data sources and analytical approaches into a new platform. Methods ...

Detection of tmRNA molecules on microarrays at low temperatures using helper oligonucleotides

Directory of Open Access Journals (Sweden)

Palta Priit

2010-04-01

Full Text Available Abstract Background The hybridization of synthetic Streptococcus pneumoniae tmRNA on a detection microarray is slow at 34°C resulting in low signal intensities. Results We demonstrate that adding specific DNA helper oligonucleotides (chaperones to the hybridization buffer increases the signal strength at a given temperature and thus makes the specific detection of Streptococcus pneumoniae tmRNA more sensitive. No loss of specificity was observed at low temperatures compared to hybridization at 46°C. The effect of the chaperones can be explained by disruption of the strong secondary and tertiary structure of the target RNA by the selective hybridization of helper molecules. The amplification of the hybridization signal strength by chaperones is not necessarily local; we observed increased signal intensities in both local and distant regions of the target molecule. Conclusions The sensitivity of the detection of tmRNA at low temperature can be increased by chaperone oligonucleotides. Due to the complexity of RNA secondary and tertiary structures the effect of any individual chaperone is currently not predictable.

Small Molecule Targeting of a MicroRNA Associated with Hepatocellular Carcinoma.

Science.gov (United States)

Childs-Disney, Jessica L; Disney, Matthew D

2016-02-19

Development of precision therapeutics is of immense interest, particularly as applied to the treatment of cancer. By analyzing the preferred cellular RNA targets of small molecules, we discovered that 5"-azido neomycin B binds the Drosha processing site in the microRNA (miR)-525 precursor. MiR-525 confers invasive properties to hepatocellular carcinoma (HCC) cells. Although HCC is one of the most common cancers, treatment options are limited, making the disease often fatal. Herein, we find that addition of 5"-azido neomycin B and its FDA-approved precursor, neomycin B, to an HCC cell line selectively inhibits production of the mature miRNA, boosts a downstream protein, and inhibits invasion. Interestingly, neomycin B is a second-line agent for hepatic encephalopathy (HE) and bacterial infections due to cirrhosis. Our results provocatively suggest that neomycin B, or second-generation derivatives, may be dual functioning molecules to treat both HE and HCC. Collectively, these studies show that rational design approaches can be tailored to disease-associated RNAs to afford potential lead therapeutics.

A Capture-SELEX Strategy for Multiplexed Selection of RNA Aptamers Against Small Molecules

DEFF Research Database (Denmark)

Lauridsen, Lasse Holm; Doessing, Holger B.; Long, Katherine S.

2018-01-01

-SELEX, a selection strategy that uses an RNA library to yield ligand-responsive RNA aptamers targeting small organic molecules in solution. To demonstrate the power of this method we selected several aptamers with specificity towards either the natural sweetener rebaudioside A or the food-coloring agent carminic...

Identification of RNA molecules by specific enzyme digestion and mass spectrometry: software for and implementation of RNA mass mapping

DEFF Research Database (Denmark)

Matthiesen, Rune; Kirpekar, Finn

2009-01-01

The idea of identifying or characterizing an RNA molecule based on a mass spectrum of specifically generated RNA fragments has been used in various forms for well over a decade. We have developed software-named RRM for 'RNA mass mapping'-which can search whole prokaryotic genomes or RNA FASTA...... sequence databases to identify the origin of a given RNA based on a mass spectrum of RNA fragments. As input, the program uses the masses of specific RNase cleavage of the RNA under investigation. RNase T1 digestion is used here as a demonstration of the usability of the method for RNA identification....... The concept for identification is that the masses of the digestion products constitute a specific fingerprint, which characterize the given RNA. The search algorithm is based on the same principles as those used in peptide mass fingerprinting, but has here been extended to work for both RNA sequence databases...

Associations of mRNA:microRNA for the shared downstream molecules of EGFR and alternative tyrosine kinase receptors in Non-Small Cell Lung Cancer

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Fengfeng Wang

2016-10-01

Full Text Available Lung cancer is the top cancer killer worldwide with high mortality rate. Majority belong to non-small cell lung cancers (NSCLCs. The epidermal growth factor receptor (EGFR has been broadly explored as a drug target for therapy. However, the drug responses are not durable due to the acquired resistance. MicroRNAs (miRNAs are small noncoding and endogenous molecules that can inhibit mRNA translation initiation and degrade mRNAs. We wonder if some downstream molecules shared by EGFR and the other tyrosine kinase receptors (TKRs further transduce the signals alternatively, and some miRNAs play the key roles in affecting the expression of these downstream molecules. In this study, we investigated the mRNA:miRNA associations for the direct EGFR downstream molecules in the EGFR signaling pathway shared with the other TKRs, including c-MET (hepatocyte growth factor receptor, Ron (a protein tyrosine kinase related to c-MET, PDGFR (platelet-derived growth factor receptor, and IGF-1R (insulin-like growth factor receptor-1. The multiple linear regression and support vector regression (SVR models were used to discover the statistically significant and the best weighted miRNAs regulating the mRNAs of these downstream molecules. These two models revealed the similar mRNA:miRNA associations. It was found that the miRNAs significantly affecting the mRNA expressions in the multiple regression model were also those with the largest weights in the SVR model. To conclude, we effectively identified a list of meaningful mRNA:miRNA associations: phospholipase C, gamma 1 (PLCG1 with miR-34a, phosphoinositide-3-kinase, regulatory subunit 2 (PIK3R2 with miR-30a-5p, growth factor receptor-bound protein 2 (GRB2 with miR-27a, and Janus kinase 1 (JAK1 with miR-302b and miR-520e. These associations could make great contributions to explore new mechanism in NSCLCs. These candidate miRNAs may be regarded as the potential drug targets for treating NSCLCs with acquired drug

Design of a Bioactive Small Molecule that Targets the Myotonic Dystrophy Type 1 RNA Via an RNA Motif-Ligand Database & Chemical Similarity Searching

Science.gov (United States)

Parkesh, Raman; Childs-Disney, Jessica L.; Nakamori, Masayuki; Kumar, Amit; Wang, Eric; Wang, Thomas; Hoskins, Jason; Tran, Tuan; Housman, David; Thornton, Charles A.; Disney, Matthew D.

2012-01-01

Myotonic dystrophy type 1 (DM1) is a triplet repeating disorder caused by expanded CTG repeats in the 3′ untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. The transcribed repeats fold into an RNA hairpin with multiple copies of a 5′CUG/3′GUC motif that binds the RNA splicing regulator muscleblind-like 1 protein (MBNL1). Sequestration of MBNL1 by expanded r(CUG) repeats causes splicing defects in a subset of pre-mRNAs including the insulin receptor, the muscle-specific chloride ion channel, Sarco(endo)plasmic reticulum Ca2+ ATPase 1 (Serca1/Atp2a1), and cardiac troponin T (cTNT). Based on these observations, the development of small molecule ligands that target specifically expanded DM1 repeats could serve as therapeutics. In the present study, computational screening was employed to improve the efficacy of pentamidine and Hoechst 33258 ligands that have been shown previously to target the DM1 triplet repeat. A series of inhibitors of the RNA-protein complex with low micromolar IC50’s, which are >20-fold more potent than the query compounds, were identified. Importantly, a bis-benzimidazole identified from the Hoechst query improves DM1-associated pre-mRNA splicing defects in cell and mouse models of DM1 (when dosed with 1 mM and 100 mg/kg, respectively). Since Hoechst 33258 was identified as a DM1 binder through analysis of an RNA motif-ligand database, these studies suggest that lead ligands targeting RNA with improved biological activity can be identified by using a synergistic approach that combines analysis of known RNA-ligand interactions with virtual screening. PMID:22300544

Functional characterization of endogenous siRNA target genes in Caenorhabditis elegans

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Heikkinen Liisa

2008-06-01

Full Text Available Abstract Background Small interfering RNA (siRNA molecules mediate sequence specific silencing in RNA interference (RNAi, a gene regulatory phenomenon observed in almost all organisms. Large scale sequencing of small RNA libraries obtained from C. elegans has revealed that a broad spectrum of siRNAs is endogenously transcribed from genomic sequences. The biological role and molecular diversity of C. elegans endogenous siRNA (endo-siRNA molecules, nonetheless, remain poorly understood. In order to gain insight into their biological function, we annotated two large libraries of endo-siRNA sequences, identified their cognate targets, and performed gene ontology analysis to identify enriched functional categories. Results Systematic trends in categorization of target genes according to the specific length of siRNA sequences were observed: 18- to 22-mer siRNAs were associated with genes required for embryonic development; 23-mers were associated uniquely with post-embryonic development; 24–26-mers were associated with phosphorus metabolism or protein modification. Moreover, we observe that some argonaute related genes associate with siRNAs with multiple reads. Sequence frequency graphs suggest that different lengths of siRNAs share similarities in overall sequence structure: the 5' end begins with G, while the body predominates with U and C. Conclusion These results suggest that the lengths of endogenous siRNA molecules are consequential to their biological functions since the gene ontology categories for their cognate mRNA targets vary depending upon their lengths.

Fluorescent labeling of NASBA amplified tmRNA molecules for microarray applications

Directory of Open Access Journals (Sweden)

Kaplinski Lauris

2009-05-01

Full Text Available Abstract Background Here we present a novel promising microbial diagnostic method that combines the sensitivity of Nucleic Acid Sequence Based Amplification (NASBA with the high information content of microarray technology for the detection of bacterial tmRNA molecules. The NASBA protocol was modified to include aminoallyl-UTP (aaUTP molecules that were incorporated into nascent RNA during the NASBA reaction. Post-amplification labeling with fluorescent dye was carried out subsequently and tmRNA hybridization signal intensities were measured using microarray technology. Significant optimization of the labeled NASBA protocol was required to maintain the required sensitivity of the reactions. Results Two different aaUTP salts were evaluated and optimum final concentrations were identified for both. The final 2 mM concentration of aaUTP Li-salt in NASBA reaction resulted in highest microarray signals overall, being twice as high as the strongest signals with 1 mM aaUTP Na-salt. Conclusion We have successfully demonstrated efficient combination of NASBA amplification technology with microarray based hybridization detection. The method is applicative for many different areas of microbial diagnostics including environmental monitoring, bio threat detection, industrial process monitoring and clinical microbiology.

Recoding aminoacyl-tRNA synthetases for synthetic biology by rational protein-RNA engineering.

Science.gov (United States)

Hadd, Andrew; Perona, John J

2014-12-19

We have taken a rational approach to redesigning the amino acid binding and aminoacyl-tRNA pairing specificities of bacterial glutaminyl-tRNA synthetase. The four-stage engineering incorporates generalizable design principles and improves the pairing efficiency of noncognate glutamate with tRNA(Gln) by over 10(5)-fold compared to the wild-type enzyme. Better optimized designs of the protein-RNA complex include substantial reengineering of the globular core region of the tRNA, demonstrating a role for specific tRNA nucleotides in specifying the identity of the genetically encoded amino acid. Principles emerging from this engineering effort open new prospects for combining rational and genetic selection approaches to design novel aminoacyl-tRNA synthetases that ligate noncanonical amino acids onto tRNAs. This will facilitate reconstruction of the cellular translation apparatus for applications in synthetic biology.

Using a Specific RNA-Protein Interaction To Quench the Fluorescent RNA Spinach.

Science.gov (United States)

Roszyk, Laura; Kollenda, Sebastian; Hennig, Sven

2017-12-15

RNAs are involved in interaction networks with other biomolecules and are crucial for proper cell function. Yet their biochemical analysis remains challenging. For Förster Resonance Energy Transfer (FRET), a common tool to study such interaction networks, two interacting molecules have to be fluorescently labeled. "Spinach" is a genetically encodable RNA aptamer that starts to fluoresce upon binding of an organic molecule. Therefore, it is a biological fluorophore tag for RNAs. However, spinach has never been used in a FRET assembly before. Here, we describe how spinach is quenched when close to acceptors. We used RNA-DNA hybridization to bring quenchers or red organic dyes in close proximity to spinach. Furthermore, we investigate RNA-protein interactions quantitatively on the example of Pseudomonas aeruginosa phage coat protein 7 (PP7) and its interacting pp7-RNA. We utilize spinach quenching as a fully genetically encodable system even under lysate conditions. Therefore, this work represents a direct method to analyze RNA-protein interactions by quenching the spinach aptamer.

TOPICAL REVIEW: Single-molecule experiments in biological physics: methods and applications

Science.gov (United States)

Ritort, F.

2006-08-01

I review single-molecule experiments (SMEs) in biological physics. Recent technological developments have provided the tools to design and build scientific instruments of high enough sensitivity and precision to manipulate and visualize individual molecules and measure microscopic forces. Using SMEs it is possible to manipulate molecules one at a time and measure distributions describing molecular properties, characterize the kinetics of biomolecular reactions and detect molecular intermediates. SMEs provide additional information about thermodynamics and kinetics of biomolecular processes. This complements information obtained in traditional bulk assays. In SMEs it is also possible to measure small energies and detect large Brownian deviations in biomolecular reactions, thereby offering new methods and systems to scrutinize the basic foundations of statistical mechanics. This review is written at a very introductory level, emphasizing the importance of SMEs to scientists interested in knowing the common playground of ideas and the interdisciplinary topics accessible by these techniques. The review discusses SMEs from an experimental perspective, first exposing the most common experimental methodologies and later presenting various molecular systems where such techniques have been applied. I briefly discuss experimental techniques such as atomic-force microscopy (AFM), laser optical tweezers (LOTs), magnetic tweezers (MTs), biomembrane force probes (BFPs) and single-molecule fluorescence (SMF). I then present several applications of SME to the study of nucleic acids (DNA, RNA and DNA condensation) and proteins (protein-protein interactions, protein folding and molecular motors). Finally, I discuss applications of SMEs to the study of the nonequilibrium thermodynamics of small systems and the experimental verification of fluctuation theorems. I conclude with a discussion of open questions and future perspectives.

Psmir: a database of potential associations between small molecules and miRNAs.

Science.gov (United States)

Meng, Fanlin; Wang, Jing; Dai, Enyu; Yang, Feng; Chen, Xiaowen; Wang, Shuyuan; Yu, Xuexin; Liu, Dianming; Jiang, Wei

2016-01-13

miRNAs are key post-transcriptional regulators of many essential biological processes, and their dysregulation has been validated in almost all human cancers. Restoring aberrantly expressed miRNAs might be a novel therapeutics. Recently, many studies have demonstrated that small molecular compounds can affect miRNA expression. Thus, prediction of associations between small molecules and miRNAs is important for investigation of miRNA-targeted drugs. Here, we analyzed 39 miRNA-perturbed gene expression profiles, and then calculated the similarity of transcription responses between miRNA perturbation and drug treatment to predict drug-miRNA associations. At the significance level of 0.05, we obtained 6501 candidate associations between 1295 small molecules and 25 miRNAs, which included 624 FDA approved drugs. Finally, we constructed the Psmir database to store all potential associations and the related materials. In a word, Psmir served as a valuable resource for dissecting the biological significance in small molecules' effects on miRNA expression, which will facilitate developing novel potential therapeutic targets or treatments for human cancers. Psmir is supported by all major browsers, and is freely available at http://www.bio-bigdata.com/Psmir/.

A versatile method to design stem-loop primer-based quantitative PCR assays for detecting small regulatory RNA molecules.

Directory of Open Access Journals (Sweden)

Zsolt Czimmerer

Full Text Available Short regulatory RNA-s have been identified as key regulators of gene expression in eukaryotes. They have been involved in the regulation of both physiological and pathological processes such as embryonal development, immunoregulation and cancer. One of their relevant characteristics is their high stability, which makes them excellent candidates for use as biomarkers. Their number is constantly increasing as next generation sequencing methods reveal more and more details of their synthesis. These novel findings aim for new detection methods for the individual short regulatory RNA-s in order to be able to confirm the primary data and characterize newly identified subtypes in different biological conditions. We have developed a flexible method to design RT-qPCR assays that are very sensitive and robust. The newly designed assays were tested extensively in samples from plant, mouse and even human formalin fixed paraffin embedded tissues. Moreover, we have shown that these assays are able to quantify endogenously generated shRNA molecules. The assay design method is freely available for anyone who wishes to use a robust and flexible system for the quantitative analysis of matured regulatory RNA-s.

Nucleic Acids as Information Molecules.

Science.gov (United States)

McInerney, Joseph D.

1996-01-01

Presents an activity that aims at enabling students to recognize that DNA and RNA are information molecules whose function is to store, copy, and make available the information in biological systems, without feeling overwhelmed by the specialized vocabulary and the minutia of the central dogma. (JRH)

Single molecule force spectroscopy: methods and applications in biology

International Nuclear Information System (INIS)

Shen Yi; Hu Jun

2012-01-01

Single molecule measurements have transformed our view of biomolecules. Owing to the ability of monitoring the activity of individual molecules, we now see them as uniquely structured, fluctuating molecules that stochastically transition between frequently many substrates, as two molecules do not follow precisely the same trajectory. Indeed, it is this discovery of critical yet short-lived substrates that were often missed in ensemble measurements that has perhaps contributed most to the better understanding of biomolecular functioning resulting from single molecule experiments. In this paper, we give a review on the three major techniques of single molecule force spectroscopy, and their applications especially in biology. The single molecular study of biotin-streptavidin interactions is introduced as a successful example. The problems and prospects of the single molecule force spectroscopy are discussed, too. (authors)

The Poitiers School of Mathematical and Theoretical Biology: Besson-Gavaudan-Schützenberger's Conjectures on Genetic Code and RNA Structures.

Science.gov (United States)

Demongeot, J; Hazgui, H

2016-12-01

The French school of theoretical biology has been mainly initiated in Poitiers during the sixties by scientists like J. Besson, G. Bouligand, P. Gavaudan, M. P. Schützenberger and R. Thom, launching many new research domains on the fractal dimension, the combinatorial properties of the genetic code and related amino-acids as well as on the genetic regulation of the biological processes. Presently, the biological science knows that RNA molecules are often involved in the regulation of complex genetic networks as effectors, e.g., activators (small RNAs as transcription factors), inhibitors (micro-RNAs) or hybrids (circular RNAs). Examples of such networks will be given showing that (1) there exist RNA "relics" that have played an important role during evolution and have survived in many genomes, whose probability distribution of their sub-sequences is quantified by the Shannon entropy, and (2) the robustness of the dynamics of the networks they regulate can be characterized by the Kolmogorov-Sinaï dynamic entropy and attractor entropy.

Nano- and micro-fabrication for single-molecule biological studies

NARCIS (Netherlands)

Huang, Z.

2012-01-01

Heterogeneity is a general feature in biological system. In order to avoid possible misleading effects of ensemble averaging, and to ensure a correct understanding of the biological system, it is very important to look into individuals, such as a single bio-molecule or a single cell, for details.

Electrochemically etched nanoporous silicon membrane for separation of biological molecules in mixture

Science.gov (United States)

Burham, Norhafizah; Azlan Hamzah, Azrul; Yunas, Jumril; Yeop Majlis, Burhanuddin

2017-07-01

This paper presents a technique for separating biological molecules in mixture using nanoporous silicon membrane. Nanopores were formed using electrochemical etching process (ECE) by etching a prefabricated silicon membrane in hydrofluoric acid (HF) and ethanol, and then directly bonding it with PDMS to form a complete filtration system for separating biological molecules. Tygon S3™ tubings were used as fluid interconnection between PDMS molds and silicon membrane during testing. Electrochemical etching parameters were manipulated to control pore structure and size. In this work, nanopores with sizes of less than 50 nm, embedded on top of columnar structures have been fabricated using high current densities and variable HF concentrations. Zinc oxide was diluted with deionized (DI) water and mixed with biological molecules and non-biological particles, namely protein standard, serum albumin and sodium chloride. Zinc oxide particles were trapped on the nanoporous silicon surface, while biological molecules of sizes up to 12 nm penetrated the nanoporous silicon membrane. The filtered particles were inspected using a Zetasizer Nano SP for particle size measurement and count. The Zetasizer Nano SP results revealed that more than 95% of the biological molecules in the mixture were filtered out by the nanoporous silicon membrane. The nanoporous silicon membrane fabricated in this work is integratable into bio-MEMS and Lab-on-Chip components to separate two or more types of biomolecules at once. The membrane is especially useful for the development of artificial kidney.

Single Fluorescent Molecules as Nano-Illuminators for Biological Structure and Function

Science.gov (United States)

Moerner, W. E.

2011-03-01

Since the first optical detection and spectroscopy of a single molecule in a solid (Phys. Rev. Lett. {62}, 2535 (1989)), much has been learned about the ability of single molecules to probe local nanoenvironments and individual behavior in biological and nonbiological materials in the absence of ensemble averaging that can obscure heterogeneity. Because each single fluorophore acts a light source roughly 1 nm in size, microscopic imaging of individual fluorophores leads naturally to superlocalization, or determination of the position of the molecule with precision beyond the optical diffraction limit, simply by digitization of the point-spread function from the single emitter. For example, the shape of single filaments in a living cell can be extracted simply by allowing a single molecule to move through the filament (PNAS {103}, 10929 (2006)). The addition of photoinduced control of single-molecule emission allows imaging beyond the diffraction limit (super-resolution) and a new array of acronyms (PALM, STORM, F-PALM etc.) and advances have appeared. We have used the native blinking and switching of a common yellow-emitting variant of green fluorescent protein (EYFP) reported more than a decade ago (Nature {388}, 355 (1997)) to achieve sub-40 nm super-resolution imaging of several protein structures in the bacterium Caulobacter crescentus: the quasi-helix of the actin-like protein MreB (Nat. Meth. {5}, 947 (2008)), the cellular distribution of the DNA binding protein HU (submitted), and the recently discovered division spindle composed of ParA filaments (Nat. Cell Biol. {12}, 791 (2010)). Even with these advances, better emitters would provide more photons and improved resolution, and a new photoactivatable small-molecule emitter has recently been synthesized and targeted to specific structures in living cells to provide super-resolution images (JACS {132}, 15099 (2010)). Finally, a new optical method for extracting three-dimensional position information based on

A semantic web ontology for small molecules and their biological targets.

Science.gov (United States)

Choi, Jooyoung; Davis, Melissa J; Newman, Andrew F; Ragan, Mark A

2010-05-24

A wide range of data on sequences, structures, pathways, and networks of genes and gene products is available for hypothesis testing and discovery in biological and biomedical research. However, data describing the physical, chemical, and biological properties of small molecules have not been well-integrated with these resources. Semantically rich representations of chemical data, combined with Semantic Web technologies, have the potential to enable the integration of small molecule and biomolecular data resources, expanding the scope and power of biomedical and pharmacological research. We employed the Semantic Web technologies Resource Description Framework (RDF) and Web Ontology Language (OWL) to generate a Small Molecule Ontology (SMO) that represents concepts and provides unique identifiers for biologically relevant properties of small molecules and their interactions with biomolecules, such as proteins. We instanced SMO using data from three public data sources, i.e., DrugBank, PubChem and UniProt, and converted to RDF triples. Evaluation of SMO by use of predetermined competency questions implemented as SPARQL queries demonstrated that data from chemical and biomolecular data sources were effectively represented and that useful knowledge can be extracted. These results illustrate the potential of Semantic Web technologies in chemical, biological, and pharmacological research and in drug discovery.

Illuminating Messengers: An Update and Outlook on RNA Visualization in Bacteria

Directory of Open Access Journals (Sweden)

Lieke A. van Gijtenbeek

2017-06-01

Full Text Available To be able to visualize the abundance and spatiotemporal features of RNAs in bacterial cells would permit obtaining a pivotal understanding of many mechanisms underlying bacterial cell biology. The first methods that allowed observing single mRNA molecules in individual cells were introduced by Bertrand et al. (1998 and Femino et al. (1998. Since then, a plethora of techniques to image RNA molecules with the aid of fluorescence microscopy has emerged. Many of these approaches are useful for the large eukaryotic cells but their adaptation to study RNA, specifically mRNA molecules, in bacterial cells progressed relatively slow. Here, an overview will be given of fluorescent techniques that can be used to reveal specific RNA molecules inside fixed and living single bacterial cells. It includes a critical evaluation of their caveats as well as potential solutions.

SERS-based inverse molecular sentinel (iMS) nanoprobes for multiplexed detection of microRNA cancer biomarkers in biological samples

Science.gov (United States)

Crawford, Bridget M.; Wang, Hsin-Neng; Fales, Andrew M.; Bowie, Michelle L.; Seewaldt, Victoria L.; Vo-Dinh, Tuan

2017-02-01

The development of sensitive and selective biosensing techniques is of great interest for clinical diagnostics. Here, we describe the development and application of a surface enhanced Raman scattering (SERS) sensing technology, referred to as "inverse Molecular Sentinel (iMS)" nanoprobes, for the detection of nucleic acid biomarkers in biological samples. This iMS nanoprobe involves the use of plasmonic-active nanostars as the sensing platform for a homogenous assay for multiplexed detection of nucleic acid biomarkers, including DNA, RNA and microRNA (miRNA). The "OFF-to-ON" signal switch is based on a non-enzymatic strand-displacement process and the conformational change of stem-loop (hairpin) oligonucleotide probes upon target binding. Here, we demonstrate the development of iMS nanoprobes for the detection of DNA sequences as well as a modified design of the nanoprobe for the detection of short (22-nt) microRNA sequences. The application of iMS nanoprobes to detect miRNAs in real biological samples was performed with total small RNA extracted from breast cancer cell lines. The multiplex capability of the iMS technique was demonstrated using a mixture of the two differently labeled nanoprobes to detect miR-21 and miR-34a miRNA biomarkers for breast cancer. The results of this study demonstrate the feasibility of applying the iMS technique for multiplexed detection of nucleic acid biomarkers, including short miRNAs molecules.

A combinatorial enumeration problem of RNA secondary structures

African Journals Online (AJOL)

use

2011-12-21

Dec 21, 2011 ... interesting combinatorial questions (Chen et al., 2005;. Liu, 2006; Schmitt and Waterman 1994; Stein and. Waterman 1978). The research on the enumeration of. RNA secondary structures becomes one of the hot topics in Computational Molecular Biology. An RNA molecule is described by its sequences of.

A combinatorial enumeration problem of RNA secondary structures

African Journals Online (AJOL)

use

2011-12-21

Dec 21, 2011 ... connection between Discrete Mathematics and Compu- tational Molecular Biology (Chen et al, 2005; Hofacker et ... in Computational Molecular Biology. An RNA molecule is described by its sequences of bases ... Here, a mathematical definition of secondary structure is given (Stein and Waterman 1978).

Computational Modeling of Biological Systems From Molecules to Pathways

CERN Document Server

2012-01-01

Computational modeling is emerging as a powerful new approach for studying and manipulating biological systems. Many diverse methods have been developed to model, visualize, and rationally alter these systems at various length scales, from atomic resolution to the level of cellular pathways. Processes taking place at larger time and length scales, such as molecular evolution, have also greatly benefited from new breeds of computational approaches. Computational Modeling of Biological Systems: From Molecules to Pathways provides an overview of established computational methods for the modeling of biologically and medically relevant systems. It is suitable for researchers and professionals working in the fields of biophysics, computational biology, systems biology, and molecular medicine.

Suppression and enhancement of non-native molecules within biological systems

Energy Technology Data Exchange (ETDEWEB)

Jones, E.A. [Surface Analysis Research Centre, CEAS, School of Chemical Engineering and Analytical Science, University of Manchester, Manchester M60 1QD (United Kingdom)]. E-mail: e.jones@postgrad.manchester.ac.uk; Lockyer, N.P. [Surface Analysis Research Centre, CEAS, School of Chemical Engineering and Analytical Science, University of Manchester, Manchester M60 1QD (United Kingdom); Vickerman, J.C. [Surface Analysis Research Centre, CEAS, School of Chemical Engineering and Analytical Science, University of Manchester, Manchester M60 1QD (United Kingdom)

2006-07-30

With the aim of evaluating the potential of SIMS to provide molecular information from small molecules within biological systems, here we investigate the effect of different biological compounds as they act as matrices. The results highlight the fact that the chemical environment of a molecule can have a significant effect on its limit of detection. This has implications for the imaging of drugs and xenobiotics in tissue sections and other biological matrices. A 1:1 mixture of the organic acid 2,4,6-trihydroxyacetophenone and the dipeptide valine-valine demonstrates that almost complete suppression of the [M + H]{sup +} ion of one compound can be caused by the presence of a compound of higher proton affinity. The significance of this is highlighted when two similar drug molecules, atropine (a neutral molecule) and ipratropium bromide (a quaternary nitrogen containing salt) are mixed with brain homogenate. The atropine [M + H]{sup +} ion shows significant suppression whilst the [M - Br]{sup +} of ipratopium bromide is detected at an intensity that can be rationalised by its decreased surface concentration. By investigating the effect of two abundant tissue lipids, cholesterol and dipalmitoylphosphatidyl choline (DPPC), on the atropine [M + H]{sup +} signal detected in mixtures with these lipids we see that the DPPC has a strong suppressing effect, which may be attributed to gas phase proton transfer.

BIOLOGICAL FUNCTION OF TOMBUSVIRUS-ENCODED SUPPRESSOR OF RNA SILENCING IN PLANTS

Directory of Open Access Journals (Sweden)

Omarov R.T.

2012-08-01

Full Text Available RNA interference (RNAi plays multiple biological roles in eukaryotic organisms to regulate gene expression. RNAi also operates as a conserved adaptive molecular immune mechanism against invading viruses. The antiviral RNAi pathway is initiated with the generation of virus-derived short-interfering RNAs (siRNAs that are used for subsequent sequence-specific recognition and degradation of the cognate viral RNA molecules. As an efficient counter-defensive strategy, most plant viruses evolved the ability to encode specific proteins capable of interfering with RNAi, and this process is commonly known as RNA silencing suppression. Virus-encoded suppressors of RNAi (VSRs operate at different steps in the RNAi pathway and display distinct biochemical properties that enable these proteins to efficiently interfere with the host-defense system. Tombusvirus-encoded P19 is an important pathogenicity factor, required for symptom development and elicitation of a hypersensitive response in a host-dependent manner. Protein plays a crucial role of TBSV P19 in protecting viral RNA during systemic infection on Nicotiana benthamiana. The X-ray crystallographic studies conducted by two independent groups revealed the existence of a P19-siRNA complex; a conformation whereby caliper tryptophan residues on two subunits of P19 dimers measure and bind 21-nt siRNA duplexes. These structural studies provided the first details on the possible molecular mechanism of any viral suppressor to block RNAi. The association between P19 and siRNAs was also shown to occur in infected plants These and related studies revealed that in general the ability of P19 to efficiently sequester siRNAs influences symptom severity, however this is not a strict correlation in all hosts.The current working model is that during TBSV infection of plants, P19 appropriates abundantly circulating Tombusvirus-derived siRNAs thereby rendering these unavailable to program RISC, to prevent degradation of

Coinhibitory molecules in cancer biology and therapy.

Science.gov (United States)

Mocellin, Simone; Benna, Clara; Pilati, Pierluigi

2013-04-01

The adaptive immune response is controlled by checkpoints represented by coinhibitory molecules, which are crucial for maintaining self-tolerance and minimizing collateral tissue damage under physiological conditions. A growing body of preclinical evidence supports the hypothesis that unleashing this immunological break might be therapeutically beneficial in the fight against cancer, as it would elicit an effective antitumor immune response. Remarkably, recent clinical trials have demonstrated that this novel strategy can be highly effective in the treatment of patients with cancer, as shown by the paradigmatic case of ipilimumab (a monoclonal antibody blocking the coinhibitory molecule cytotoxic T lymphocyte associated antigen-4 [CTLA4]) that is opening a new era in the therapeutic approach to a chemoresistant tumor such as cutaneous melanoma. In this review we summarize the biology of coinhibitory molecules, overview the experimental and clinical attempts to interfere with these immune checkpoints to treat cancer and critically discuss the challenges posed by such a promising antitumor modality. Copyright © 2013 Elsevier Ltd. All rights reserved.

Human microRNA oncogenes and tumor suppressors show significantly different biological patterns: from functions to targets.

Directory of Open Access Journals (Sweden)

Dong Wang

Full Text Available MicroRNAs (miRNAs are small noncoding RNAs which play essential roles in many important biological processes. Therefore, their dysfunction is associated with a variety of human diseases, including cancer. Increasing evidence shows that miRNAs can act as oncogenes or tumor suppressors, and although there is great interest in research into these cancer-associated miRNAs, little is known about them. In this study, we performed a comprehensive analysis of putative human miRNA oncogenes and tumor suppressors. We found that miRNA oncogenes and tumor suppressors clearly show different patterns in function, evolutionary rate, expression, chromosome distribution, molecule size, free energy, transcription factors, and targets. For example, miRNA oncogenes are located mainly in the amplified regions in human cancers, whereas miRNA tumor suppressors are located mainly in the deleted regions. miRNA oncogenes tend to cleave target mRNAs more frequently than miRNA tumor suppressors. These results indicate that these two types of cancer-associated miRNAs play different roles in cancer formation and development. Moreover, the patterns identified here can discriminate novel miRNA oncogenes and tumor suppressors with a high degree of accuracy. This study represents the first large-scale bioinformatic analysis of human miRNA oncogenes and tumor suppressors. Our findings provide help for not only understanding of miRNAs in cancer but also for the specific identification of novel miRNAs as miRNA oncogenes and tumor suppressors. In addition, the data presented in this study will be valuable for the study of both miRNAs and cancer.

Selective small-molecule inhibition of an RNA structural element

Energy Technology Data Exchange (ETDEWEB)

Howe, John A.; Wang, Hao; Fischmann, Thierry O.; Balibar, Carl J.; Xiao, Li; Galgoci, Andrew M.; Malinverni, Juliana C.; Mayhood, Todd; Villafania, Artjohn; Nahvi, Ali; Murgolo, Nicholas; Barbieri, Christopher M.; Mann, Paul A.; Carr, Donna; Xia, Ellen; Zuck, Paul; Riley, Dan; Painter, Ronald E.; Walker, Scott S.; Sherborne, Brad; de Jesus, Reynalda; Pan, Weidong; Plotkin, Michael A.; Wu, Jin; Rindgen, Diane; Cummings, John; Garlisi, Charles G.; Zhang, Rumin; Sheth, Payal R.; Gill, Charles J.; Tang, Haifeng; Roemer , Terry (Merck)

2015-09-30

Riboswitches are non-coding RNA structures located in messenger RNAs that bind endogenous ligands, such as a specific metabolite or ion, to regulate gene expression. As such, riboswitches serve as a novel, yet largely unexploited, class of emerging drug targets. Demonstrating this potential, however, has proven difficult and is restricted to structurally similar antimetabolites and semi-synthetic analogues of their cognate ligand, thus greatly restricting the chemical space and selectivity sought for such inhibitors. Here we report the discovery and characterization of ribocil, a highly selective chemical modulator of bacterial riboflavin riboswitches, which was identified in a phenotypic screen and acts as a structurally distinct synthetic mimic of the natural ligand, flavin mononucleotide, to repress riboswitch-mediated ribB gene expression and inhibit bacterial cell growth. Our findings indicate that non-coding RNA structural elements may be more broadly targeted by synthetic small molecules than previously expected.

The origin of the 5S ribosomal RNA molecule could have been caused by a single inverse duplication: strong evidence from its sequences.

Science.gov (United States)

Branciamore, Sergio; Di Giulio, Massimo

2012-04-01

The secondary structure of the 5S ribosomal RNA (5S rRNA) molecule shows a high degree of symmetry. In order to explain the origin of this symmetry, it has been conjectured that one half of the 5S rRNA molecule was its precursor and that an indirect duplication of this precursor created the other half and thus the current symmetry of the molecule. Here, we have subjected to an empirical test both the indirect duplication model, analysing a total of 684 5S rRNA sequences for complementarity between the two halves of the 5S rRNA, and the direct duplication model analysing in this case the similarity between the two halves of this molecule. In intra- and inter-molecule and intra- and inter-domain comparisons, we find a high statistical support to the hypothesis of a complementarity relationship between the two halves of the 5S rRNA molecule, denying vice versa the hypothesis of similarity between these halves. Therefore, these observations corroborate the indirect duplication model at the expense of the direct duplication model, as reason of the origin of the 5S rRNA molecule. More generally, we discuss and favour the hypothesis that all RNAs and proteins, which present symmetry, did so through gene duplication and not by gradualistic accumulation of few monomers or segments of molecule into a gradualistic growth process. This would be the consequence of the very high propensity that nucleic acids have to be subjected to duplications.

2012 Gordon Research Conference, Single molecule approaches to biology, July 15-20 2012

Energy Technology Data Exchange (ETDEWEB)

Fernandez, Julio M. [Columbia Univ., New York, NY (United States)

2012-04-20

Single molecule techniques are rapidly occupying a central role in biological research at all levels. This transition was made possible by the availability and dissemination of robust techniques that use fluorescence and force probes to track the conformation of molecules one at a time, in vitro as well as in live cells. Single-molecule approaches have changed the way many biological problems are studied. These novel techniques provide previously unobtainable data on fundamental biochemical processes that are essential for all forms of life. The ability of single-molecule approaches to avoid ensemble averaging and to capture transient intermediates and heterogeneous behavior renders them particularly powerful in elucidating mechanisms of the molecular systems that underpin the functioning of living cells. Hence, our conference seeks to disseminate the implementation and use of single molecule techniques in the pursuit of new biological knowledge. Topics covered include: Molecular Motors on the Move; Origin And Fate Of Proteins; Physical Principles Of Life; Molecules and Super-resolution Microscopy; Nanoswitches In Action; Active Motion Or Random Diffusion?; Building Blocks Of Living Cells; From Molecular Mechanics To Physiology; Tug-of-war: Force Spectroscopy Of Single Proteins.

SimRNA: a coarse-grained method for RNA folding simulations and 3D structure prediction.

Science.gov (United States)

Boniecki, Michal J; Lach, Grzegorz; Dawson, Wayne K; Tomala, Konrad; Lukasz, Pawel; Soltysinski, Tomasz; Rother, Kristian M; Bujnicki, Janusz M

2016-04-20

RNA molecules play fundamental roles in cellular processes. Their function and interactions with other biomolecules are dependent on the ability to form complex three-dimensional (3D) structures. However, experimental determination of RNA 3D structures is laborious and challenging, and therefore, the majority of known RNAs remain structurally uncharacterized. Here, we present SimRNA: a new method for computational RNA 3D structure prediction, which uses a coarse-grained representation, relies on the Monte Carlo method for sampling the conformational space, and employs a statistical potential to approximate the energy and identify conformations that correspond to biologically relevant structures. SimRNA can fold RNA molecules using only sequence information, and, on established test sequences, it recapitulates secondary structure with high accuracy, including correct prediction of pseudoknots. For modeling of complex 3D structures, it can use additional restraints, derived from experimental or computational analyses, including information about secondary structure and/or long-range contacts. SimRNA also can be used to analyze conformational landscapes and identify potential alternative structures. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Synthesis of molecules of biological interest labelled with high specific activity tritium

International Nuclear Information System (INIS)

Petillot, Yves

1975-01-01

Labelled molecules are artificial organic compounds possessing one or several radioactive or steady isotopic atoms. Using tritium to label molecules presents several benefits: a raw material easy to obtain with a high purity and at reasonable cost; synthesised labelled molecules displaying high specific activities very interesting in molecular biology; high resolution of radiographies; relatively simple and quick introduction of tritium atoms in complex molecules. Thus, this report for graduation in organic chemistry addresses the synthesis and study of new labelled molecules which belong to families of organic compounds which have fundamental activities in biology: uridine 3 H-5,6 and thymidine 3 H-methyl which are nucleotides which intervene under the form of phosphates in the synthesis of nucleic acids, oestradiol 3 H-2,4,6,7 which is a powerful estrogenic hormone which naturally secreted by the ovary; and noradrenaline 3 H-1,1' and dopamine 3 H-1,2 which are usually secreted by adrenal medulla and have multiple actions on the nervous system

Single molecule tools for enzymology, structural biology, systems biology and nanotechnology: an update

Science.gov (United States)

Widom, Julia R.; Dhakal, Soma; Heinicke, Laurie A.; Walter, Nils G.

2015-01-01

Toxicology is the highly interdisciplinary field studying the adverse effects of chemicals on living organisms. It requires sensitive tools to detect such effects. After their initial implementation during the 1990s, single-molecule fluorescence detection tools were quickly recognized for their potential to contribute greatly to many different areas of scientific inquiry. In the intervening time, technical advances in the field have generated ever-improving spatial and temporal resolution, and have enabled the application of single-molecule fluorescence to increasingly complex systems, such as live cells. In this review, we give an overview of the optical components necessary to implement the most common versions of single-molecule fluorescence detection. We then discuss current applications to enzymology and structural studies, systems biology, and nanotechnology, presenting the technical considerations that are unique to each area of study, along with noteworthy recent results. We also highlight future directions that have the potential to revolutionize these areas of study by further exploiting the capabilities of single-molecule fluorescence microscopy. PMID:25212907

HIV-1 in the RNA world: Transcription regulation, miRNAs and antiviral RNAs

NARCIS (Netherlands)

Harwig, A.

2015-01-01

All organisms, from bacteria to human, use three biological molecules that each serve critical functions in the expression of genes in the cell. These are deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and proteins. RNA is synthesized from DNA in a process called transcription. RNA differs from

Oncomirs: from tumor biology to molecularly targeted anticancer strategies.

Science.gov (United States)

Mocellin, Simone; Pasquali, Sandro; Pilati, Pierluigi

2009-01-01

Deregulation of microRNA (miRNA) promotes carcinogenesis, as these molecules can act as oncogenes or tumor suppressor genes. Here we provide an overview of miRNA biology, discuss the most recent findings on miRNA and cancer development/progression, and report on how tumor-related miRNAs (oncomirs) are being used to develop novel cancer specific therapeutic approaches.

Experimental and Computational Characterization of Biological Liquid Crystals: A Review of Single-Molecule Bioassays

Directory of Open Access Journals (Sweden)

Sungsoo Na

2009-09-01

Full Text Available Quantitative understanding of the mechanical behavior of biological liquid crystals such as proteins is essential for gaining insight into their biological functions, since some proteins perform notable mechanical functions. Recently, single-molecule experiments have allowed not only the quantitative characterization of the mechanical behavior of proteins such as protein unfolding mechanics, but also the exploration of the free energy landscape for protein folding. In this work, we have reviewed the current state-of-art in single-molecule bioassays that enable quantitative studies on protein unfolding mechanics and/or various molecular interactions. Specifically, single-molecule pulling experiments based on atomic force microscopy (AFM have been overviewed. In addition, the computational simulations on single-molecule pulling experiments have been reviewed. We have also reviewed the AFM cantilever-based bioassay that provides insight into various molecular interactions. Our review highlights the AFM-based single-molecule bioassay for quantitative characterization of biological liquid crystals such as proteins.

Small Molecule, Big Prospects: MicroRNA in Pregnancy and Its Complications

Directory of Open Access Journals (Sweden)

Meng Cai

2017-01-01

Full Text Available MicroRNAs are small, noncoding RNA molecules that regulate target gene expression in the posttranscriptional level. Unlike siRNA, microRNAs are “fine-tuners” rather than “switches” in the regulation of gene expression; thus they play key roles in maintaining tissue homeostasis. The aberrant microRNA expression is implicated in the disease process. To date, numerous studies have demonstrated the regulatory roles of microRNAs in various pathophysiological conditions. In contrast, the study of microRNA in pregnancy and its associated complications, such as preeclampsia (PE, fetal growth restriction (FGR, and preterm labor, is a young field. Over the last decade, the knowledge of pregnancy-related microRNAs has increased and the molecular mechanisms by which microRNAs regulate pregnancy or its associated complications are emerging. In this review, we focus on the recent advances in the research of pregnancy-related microRNAs, especially their function in pregnancy-associated complications and the potential clinical applications. Here microRNAs that associate with pregnancy are classified as placenta-specific, placenta-associated, placenta-derived circulating, and uterine microRNA according to their localization and origin. MicroRNAs offer a great potential for developing diagnostic and therapeutic targets in pregnancy-related disorders.

Along the Central Dogma-Controlling Gene Expression with Small Molecules.

Science.gov (United States)

Schneider-Poetsch, Tilman; Yoshida, Minoru

2018-05-04

The central dogma of molecular biology, that DNA is transcribed into RNA and RNA translated into protein, was coined in the early days of modern biology. Back in the 1950s and 1960s, bacterial genetics first opened the way toward understanding life as the genetically encoded interaction of macromolecules. As molecular biology progressed and our knowledge of gene control deepened, it became increasingly clear that expression relied on many more levels of regulation. In the process of dissecting mechanisms of gene expression, specific small-molecule inhibitors played an important role and became valuable tools of investigation. Small molecules offer significant advantages over genetic tools, as they allow inhibiting a process at any desired time point, whereas mutating or altering the gene of an important regulator would likely result in a dead organism. With the advent of modern sequencing technology, it has become possible to monitor global cellular effects of small-molecule treatment and thereby overcome the limitations of classical biochemistry, which usually looks at a biological system in isolation. This review focuses on several molecules, especially natural products, that have played an important role in dissecting gene expression and have opened up new fields of investigation as well as clinical venues for disease treatment. Expected final online publication date for the Annual Review of Biochemistry Volume 87 is June 20, 2018. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

RNA structure alignment by a unit-vector approach.

Science.gov (United States)

Capriotti, Emidio; Marti-Renom, Marc A

2008-08-15

The recent discovery of tiny RNA molecules such as microRNAs and small interfering RNA are transforming the view of RNA as a simple information transfer molecule. Similar to proteins, the native three-dimensional structure of RNA determines its biological activity. Therefore, classifying the current structural space is paramount for functionally annotating RNA molecules. The increasing numbers of RNA structures deposited in the PDB requires more accurate, automatic and benchmarked methods for RNA structure comparison. In this article, we introduce a new algorithm for RNA structure alignment based on a unit-vector approach. The algorithm has been implemented in the SARA program, which results in RNA structure pairwise alignments and their statistical significance. The SARA program has been implemented to be of general applicability even when no secondary structure can be calculated from the RNA structures. A benchmark against the ARTS program using a set of 1275 non-redundant pairwise structure alignments results in inverted approximately 6% extra alignments with at least 50% structurally superposed nucleotides and base pairs. A first attempt to perform RNA automatic functional annotation based on structure alignments indicates that SARA can correctly assign the deepest SCOR classification to >60% of the query structures. The SARA program is freely available through a World Wide Web server http://sgu.bioinfo.cipf.es/services/SARA/. Supplementary data are available at Bioinformatics online.

Co-LncRNA: investigating the lncRNA combinatorial effects in GO annotations and KEGG pathways based on human RNA-Seq data.

Science.gov (United States)

Zhao, Zheng; Bai, Jing; Wu, Aiwei; Wang, Yuan; Zhang, Jinwen; Wang, Zishan; Li, Yongsheng; Xu, Juan; Li, Xia

2015-01-01

Long non-coding RNAs (lncRNAs) are emerging as key regulators of diverse biological processes and diseases. However, the combinatorial effects of these molecules in a specific biological function are poorly understood. Identifying co-expressed protein-coding genes of lncRNAs would provide ample insight into lncRNA functions. To facilitate such an effort, we have developed Co-LncRNA, which is a web-based computational tool that allows users to identify GO annotations and KEGG pathways that may be affected by co-expressed protein-coding genes of a single or multiple lncRNAs. LncRNA co-expressed protein-coding genes were first identified in publicly available human RNA-Seq datasets, including 241 datasets across 6560 total individuals representing 28 tissue types/cell lines. Then, the lncRNA combinatorial effects in a given GO annotations or KEGG pathways are taken into account by the simultaneous analysis of multiple lncRNAs in user-selected individual or multiple datasets, which is realized by enrichment analysis. In addition, this software provides a graphical overview of pathways that are modulated by lncRNAs, as well as a specific tool to display the relevant networks between lncRNAs and their co-expressed protein-coding genes. Co-LncRNA also supports users in uploading their own lncRNA and protein-coding gene expression profiles to investigate the lncRNA combinatorial effects. It will be continuously updated with more human RNA-Seq datasets on an annual basis. Taken together, Co-LncRNA provides a web-based application for investigating lncRNA combinatorial effects, which could shed light on their biological roles and could be a valuable resource for this community. Database URL: http://www.bio-bigdata.com/Co-LncRNA/. © The Author(s) 2015. Published by Oxford University Press.

An insight into the biological activities of heterocyclic-fatty acid hybrid molecules.

Science.gov (United States)

Venepally, Vijayendar; Reddy Jala, Ram Chandra

2017-12-01

Heterocyclic compounds are the interesting core structures for the development of new bioactive compounds. Fatty acids are derived from renewable raw materials and exhibit various biological activities. Several researchers are amalgamating these two bioactive components to yield bioactive hybrid molecules with some desirable features. Heterocyclic-fatty acid hybrid derivatives are a new class of heterocyclic compounds with a broad range of biological activities and significance in the field of medicinal chemistry. Over the last few years, many research articles emphasized the significance of heterocyclic-fatty acid hybrid derivatives. The present review article focuses the developments in designing and biological evaluation of heterocyclic-fatty acid hybrid molecules. Copyright © 2017. Published by Elsevier Masson SAS.

Cytochrome c oxidase subunit 1-based human RNA quantification to enhance mRNA profiling in forensic biology

Directory of Open Access Journals (Sweden)

Dong Zhao

2017-01-01

Full Text Available RNA analysis offers many potential applications in forensic science, and molecular identification of body fluids by analysis of cell-specific RNA markers represents a new technique for use in forensic cases. However, due to the nature of forensic materials that often admixed with nonhuman cellular components, human-specific RNA quantification is required for the forensic RNA assays. Quantification assay for human RNA has been developed in the present study with respect to body fluid samples in forensic biology. The quantitative assay is based on real-time reverse transcription-polymerase chain reaction of mitochondrial RNA cytochrome c oxidase subunit I and capable of RNA quantification with high reproducibility and a wide dynamic range. The human RNA quantification improves the quality of mRNA profiling in the identification of body fluids of saliva and semen because the quantification assay can exclude the influence of nonhuman components and reduce the adverse affection from degraded RNA fragments.

Nucleic Acid Templated Reactions for Chemical Biology.

Science.gov (United States)

Di Pisa, Margherita; Seitz, Oliver

2017-06-21

Nucleic acid directed bioorthogonal reactions offer the fascinating opportunity to unveil and redirect a plethora of intracellular mechanisms. Nano- to picomolar amounts of specific RNA molecules serve as templates and catalyze the selective formation of molecules that 1) exert biological effects, or 2) provide measurable signals for RNA detection. Turnover of reactants on the template is a valuable asset when concentrations of RNA templates are low. The idea is to use RNA-templated reactions to fully control the biodistribution of drugs and to push the detection limits of DNA or RNA analytes to extraordinary sensitivities. Herein we review recent and instructive examples of conditional synthesis or release of compounds for in cellulo protein interference and intracellular nucleic acid imaging. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Detection of NASBA amplified bacterial tmRNA molecules on SLICSel designed microarray probes

Directory of Open Access Journals (Sweden)

Toome Kadri

2011-02-01

Full Text Available Abstract Background We present a comprehensive technological solution for bacterial diagnostics using tmRNA as a marker molecule. A robust probe design algorithm for microbial detection microarray is implemented. The probes were evaluated for specificity and, combined with NASBA (Nucleic Acid Sequence Based Amplification amplification, for sensitivity. Results We developed a new web-based program SLICSel for the design of hybridization probes, based on nearest-neighbor thermodynamic modeling. A SLICSel minimum binding energy difference criterion of 4 kcal/mol was sufficient to design of Streptococcus pneumoniae tmRNA specific microarray probes. With lower binding energy difference criteria, additional hybridization specificity tests on the microarray were needed to eliminate non-specific probes. Using SLICSel designed microarray probes and NASBA we were able to detect S. pneumoniae tmRNA from a series of total RNA dilutions equivalent to the RNA content of 0.1-10 CFU. Conclusions The described technological solution and both its separate components SLICSel and NASBA-microarray technology independently are applicative for many different areas of microbial diagnostics.

Detection of NASBA amplified bacterial tmRNA molecules on SLICSel designed microarray probes

LENUS (Irish Health Repository)

Scheler, Ott

2011-02-28

Abstract Background We present a comprehensive technological solution for bacterial diagnostics using tmRNA as a marker molecule. A robust probe design algorithm for microbial detection microarray is implemented. The probes were evaluated for specificity and, combined with NASBA (Nucleic Acid Sequence Based Amplification) amplification, for sensitivity. Results We developed a new web-based program SLICSel for the design of hybridization probes, based on nearest-neighbor thermodynamic modeling. A SLICSel minimum binding energy difference criterion of 4 kcal\\/mol was sufficient to design of Streptococcus pneumoniae tmRNA specific microarray probes. With lower binding energy difference criteria, additional hybridization specificity tests on the microarray were needed to eliminate non-specific probes. Using SLICSel designed microarray probes and NASBA we were able to detect S. pneumoniae tmRNA from a series of total RNA dilutions equivalent to the RNA content of 0.1-10 CFU. Conclusions The described technological solution and both its separate components SLICSel and NASBA-microarray technology independently are applicative for many different areas of microbial diagnostics.

RNA biology in a test tube--an overview of in vitro systems/assays.

Science.gov (United States)

Roca, Xavier; Karginov, Fedor V

2012-01-01

In vitro systems have provided a wealth of information in the field of RNA biology, as they constitute a superior and sometimes the unique approach to address many important questions. Such cell-free methods can be sorted by the degree of complexity of the preparation of enzymatic and/or regulatory activity. Progress in the study of pre-mRNA processing has largely relied on traditional in vitro methods, as these reactions have been recapitulated in cell-free systems. The pre-mRNA capping, editing, and cleavage/polyadenylation reactions have even been reconstituted using purified components, and the enzymes responsible for catalysis have been characterized by such techniques. In vitro splicing using nuclear or cytoplasmic extracts has yielded clues on spliceosome assembly, kinetics, and mechanisms of splicing and has been essential to elucidate the function of splicing factors. Coupled systems have been important to functionally connect distinct processes, like transcription and splicing. Extract preparation has also been adapted to cells from a variety of tissues and species, revealing general versus species-specific mechanisms. Cell-free assays have also been applied to newly discovered pathways such as those involving small RNAs, including microRNAs (miRNAs), small interfering RNAs (siRNAs), and Piwi-interacting RNAs (piRNAs). The first two pathways have been well characterized largely by in vitro methods, which need to be developed for piRNAs. Finally, new techniques, such as single-molecule studies, are continuously being established, providing new and important insights into the field. Thus, in vitro approaches have been, are, and will continue being at the forefront of RNA research. Copyright © 2012 John Wiley & Sons, Ltd.

RNA-PAIRS: RNA probabilistic assignment of imino resonance shifts

International Nuclear Information System (INIS)

Bahrami, Arash; Clos, Lawrence J.; Markley, John L.; Butcher, Samuel E.; Eghbalnia, Hamid R.

2012-01-01

The significant biological role of RNA has further highlighted the need for improving the accuracy, efficiency and the reach of methods for investigating RNA structure and function. Nuclear magnetic resonance (NMR) spectroscopy is vital to furthering the goals of RNA structural biology because of its distinctive capabilities. However, the dispersion pattern in the NMR spectra of RNA makes automated resonance assignment, a key step in NMR investigation of biomolecules, remarkably challenging. Herein we present RNA Probabilistic Assignment of Imino Resonance Shifts (RNA-PAIRS), a method for the automated assignment of RNA imino resonances with synchronized verification and correction of predicted secondary structure. RNA-PAIRS represents an advance in modeling the assignment paradigm because it seeds the probabilistic network for assignment with experimental NMR data, and predicted RNA secondary structure, simultaneously and from the start. Subsequently, RNA-PAIRS sets in motion a dynamic network that reverberates between predictions and experimental evidence in order to reconcile and rectify resonance assignments and secondary structure information. The procedure is halted when assignments and base-parings are deemed to be most consistent with observed crosspeaks. The current implementation of RNA-PAIRS uses an initial peak list derived from proton-nitrogen heteronuclear multiple quantum correlation ( 1 H– 15 N 2D HMQC) and proton–proton nuclear Overhauser enhancement spectroscopy ( 1 H– 1 H 2D NOESY) experiments. We have evaluated the performance of RNA-PAIRS by using it to analyze NMR datasets from 26 previously studied RNAs, including a 111-nucleotide complex. For moderately sized RNA molecules, and over a range of comparatively complex structural motifs, the average assignment accuracy exceeds 90%, while the average base pair prediction accuracy exceeded 93%. RNA-PAIRS yielded accurate assignments and base pairings consistent with imino resonances for a

RNA-PAIRS: RNA probabilistic assignment of imino resonance shifts

Energy Technology Data Exchange (ETDEWEB)

Bahrami, Arash; Clos, Lawrence J.; Markley, John L.; Butcher, Samuel E. [National Magnetic Resonance Facility at Madison (United States); Eghbalnia, Hamid R., E-mail: eghbalhd@uc.edu [University of Cincinnati, Department of Molecular and Cellular Physiology (United States)

2012-04-15

The significant biological role of RNA has further highlighted the need for improving the accuracy, efficiency and the reach of methods for investigating RNA structure and function. Nuclear magnetic resonance (NMR) spectroscopy is vital to furthering the goals of RNA structural biology because of its distinctive capabilities. However, the dispersion pattern in the NMR spectra of RNA makes automated resonance assignment, a key step in NMR investigation of biomolecules, remarkably challenging. Herein we present RNA Probabilistic Assignment of Imino Resonance Shifts (RNA-PAIRS), a method for the automated assignment of RNA imino resonances with synchronized verification and correction of predicted secondary structure. RNA-PAIRS represents an advance in modeling the assignment paradigm because it seeds the probabilistic network for assignment with experimental NMR data, and predicted RNA secondary structure, simultaneously and from the start. Subsequently, RNA-PAIRS sets in motion a dynamic network that reverberates between predictions and experimental evidence in order to reconcile and rectify resonance assignments and secondary structure information. The procedure is halted when assignments and base-parings are deemed to be most consistent with observed crosspeaks. The current implementation of RNA-PAIRS uses an initial peak list derived from proton-nitrogen heteronuclear multiple quantum correlation ({sup 1}H-{sup 15}N 2D HMQC) and proton-proton nuclear Overhauser enhancement spectroscopy ({sup 1}H-{sup 1}H 2D NOESY) experiments. We have evaluated the performance of RNA-PAIRS by using it to analyze NMR datasets from 26 previously studied RNAs, including a 111-nucleotide complex. For moderately sized RNA molecules, and over a range of comparatively complex structural motifs, the average assignment accuracy exceeds 90%, while the average base pair prediction accuracy exceeded 93%. RNA-PAIRS yielded accurate assignments and base pairings consistent with imino

Genetic control of mammalian T-cell proliferation with synthetic RNA regulatory systems

OpenAIRE

Chen, Yvonne Y.; Jensen, Michael C.; Smolke, Christina D.

2010-01-01

RNA molecules perform diverse regulatory functions in natural biological systems, and numerous synthetic RNA-based control devices that integrate sensing and gene-regulatory functions have been demonstrated, predominantly in bacteria and yeast. Despite potential advantages of RNA-based genetic control strategies in clinical applications, there has been limited success in extending engineered RNA devices to mammalian gene-expression control and no example of their application to functional res...

Efficient CRISPR/Cas9-Mediated Genome Editing Using a Chimeric Single-Guide RNA Molecule

KAUST Repository

Butt, Haroon

2017-08-24

The CRISPR/Cas9 system has been applied in diverse eukaryotic organisms for targeted mutagenesis. However, targeted gene editing is inefficient and requires the simultaneous delivery of a DNA template for homology-directed repair (HDR). Here, we used CRISPR/Cas9 to generate targeted double-strand breaks and to deliver an RNA repair template for HDR in rice (Oryza sativa). We used chimeric single-guide RNA (cgRNA) molecules carrying both sequences for target site specificity (to generate the double-strand breaks) and repair template sequences (to direct HDR), flanked by regions of homology to the target. Gene editing was more efficient in rice protoplasts using repair templates complementary to the non-target DNA strand, rather than the target strand. We applied this cgRNA repair method to generate herbicide resistance in rice, which showed that this cgRNA repair method can be used for targeted gene editing in plants. Our findings will facilitate applications in functional genomics and targeted improvement of crop traits.

From early lessons to new frontiers: the worm as a treasure trove of small RNA biology.

Science.gov (United States)

Youngman, Elaine M; Claycomb, Julie M

2014-01-01

In the past 20 years, the tiny soil nematode Caenorhabditis elegans has provided critical insights into our understanding of the breadth of small RNA-mediated gene regulatory activities. The first microRNA was identified in C. elegans in 1993, and the understanding that dsRNA was the driving force behind RNA-mediated gene silencing came from experiments performed in C. elegans in 1998. Likewise, early genetic screens in C. elegans for factors involved in RNA interference pointed to conserved mechanisms for small RNA-mediated gene silencing pathways, placing the worm squarely among the founding fathers of a now extensive field of molecular biology. Today, the worm continues to be at the forefront of ground-breaking insight into small RNA-mediated biology. Recent studies have revealed with increasing mechanistic clarity that C. elegans possesses an extensive nuclear small RNA regulatory network that encompasses not only gene silencing but also gene activating roles. Further, a portrait is emerging whereby small RNA pathways play key roles in integrating responses to environmental stimuli and transmitting epigenetic information about such responses from one generation to the next. Here we discuss endogenous small RNA pathways in C. elegans and the insight worm biology has provided into the mechanisms employed by these pathways. We touch on the increasingly spectacular diversity of small RNA biogenesis and function, and discuss the relevance of lessons learned in the worm for human biology.

From cell biology to immunology: Controlling metastatic progression of cancer via microRNA regulatory networks.

Science.gov (United States)

Park, Jae Hyon; Theodoratou, Evropi; Calin, George A; Shin, Jae Il

2016-01-01

Recently, the study of microRNAs has expanded our knowledge of the fundamental processes of cancer biology and the underlying mechanisms behind tumor metastasis. Extensive research in the fields of microRNA and its novel mechanisms of actions against various cancers has more recently led to the trial of a first cancer-targeted microRNA drug, MRX34. Yet, these microRNAs are mostly being studied and clinically trialed solely based on the understanding of their cell biologic effects, thus, neglecting the important immunologic effects that are sometimes opposite of the cell biologic effects. Here, we summarize both the cell biologic and immunologic effects of various microRNAs and discuss the importance of considering both effects before using them in clinical settings. We stress the importance of understanding the miRNA's effect on cancer metastasis from a "systems" perspective before developing a miRNA-targeted therapeutic in treating cancer metastasis.

Novel nuclear magnetic resonance techniques for studying biological molecules

International Nuclear Information System (INIS)

Laws, David D.

2000-01-01

Over the fifty-five year history of Nuclear Magnetic Resonance (NMR), considerable progress has been made in the development of techniques for studying the structure, function, and dynamics of biological molecules. The majority of this research has involved the development of multi-dimensional NMR experiments for studying molecules in solution, although in recent years a number of groups have begun to explore NMR methods for studying biological systems in the solid-state. Despite this new effort, a need still exists for the development of techniques that improve sensitivity, maximize information, and take advantage of all the NMR interactions available in biological molecules. In this dissertation, a variety of novel NMR techniques for studying biomolecules are discussed. A method for determining backbone (φ/ψ) dihedral angles by comparing experimentally determined 13 C a , chemical-shift anisotropies with theoretical calculations is presented, along with a brief description of the theory behind chemical-shift computation in proteins and peptides. The utility of the Spin-Polarization Induced Nuclear Overhauser Effect (SPINOE) to selectively enhance NMR signals in solution is examined in a variety of systems, as are methods for extracting structural information from cross-relaxation rates that can be measured in SPINOE experiments. Techniques for the production of supercritical and liquid laser-polarized xenon are discussed, as well as the prospects for using optically pumped xenon as a polarizing solvent. In addition, a detailed study of the structure of PrP 89-143 is presented. PrP 89-143 is a 54 residue fragment of the prion proteins which, upon mutation and aggregation, can induce prion diseases in transgenic mice. Whereas the structure of the wild-type PrP 89-143 is a generally unstructured mixture of α-helical and β-sheet conformers in the solid state, the aggregates formed from the PrP 89-143 mutants appear to be mostly β-sheet.

3-Econsystems: MicroRNAs, Receptors, and Latent Viruses; Some Insights Biology Can Gain from Economic Theory.

Science.gov (United States)

Polansky, Hanan; Javaherian, Adrian

2016-01-01

This mini-review describes three biological systems. All three include competing molecules and a limiting molecule that binds the competing molecules. Such systems are extensively researched by economists. In fact, the issue of limited resources is the defining feature of economic systems. Therefore, we call these systems "econsystems." In an econsystem, the allocation of the limiting molecule between the competing molecules determines the behavior of the system. A cell is an example of an econsystem. Therefore, a change in the allocation of a limiting molecule as a result of, for instance, an abnormal change in the concentration of one of the competing molecules, may result in abnormal cellular behavior, and disease. The first econsystem described in this mini-review includes a long non-coding RNA and a messenger RNA (lncRNA and mRNA). The limiting molecule is a microRNA (miRNA). The lncRNA and mRNA are known as competing endogenous RNAs (ceRNAs). The second econsystem includes two receptors, and the limiting molecule is a ligand. The third econsystem includes a cis-regulatory element of a latent virus and that of a human gene. The limiting molecule is a transcription complex that binds both cis-elements.

Aberrant microRNA expression in multiple myeloma

DEFF Research Database (Denmark)

Dimopoulos, Konstantinos; Gimsing, Peter; Grønbæk, Kirsten

2013-01-01

Multiple myeloma (MM) is a devastating disease with a complex biology, and in spite of improved survivability by novel treatment strategies over the last decade, MM is still incurable by current therapy. MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate gene expression at a post...

From early lessons to new frontiers: The worm as a treasure trove of small RNA biology

Directory of Open Access Journals (Sweden)

Elaine M. Youngman

2014-11-01

Full Text Available In the past twenty years, the tiny soil nematode C. elegans has provided critical insights into our understanding of the breadth of small RNA-mediated gene regulatory activities. The first microRNA was identified in C. elegans in 1993, and the understanding that dsRNA was the driving force behind RNA-mediated gene silencing came from experiments performed in C. elegans in 1998. Likewise, early genetic screens in C. elegans for factors involved in RNAi pointed to conserved mechanisms for small RNA-mediated gene silencing pathways, placing the worm squarely among the founding fathers of a now extensive field of molecular biology. Today, the worm continues to be at the forefront of ground-breaking insight into small RNA-mediated biology. Recent studies have revealed with increasing mechanistic clarity that C. elegans possesses an extensive nuclear small RNA regulatory network that encompasses not only gene silencing but also gene activating roles. Further, a portrait is emerging whereby small RNA pathways play key roles in integrating responses to environmental stimuli and transmitting epigenetic information about such responses from one generation to the next. Here we discuss endogenous small RNA pathways in C. elegans and the insight worm biology has provided into the mechanisms employed by these pathways. We touch on the increasingly spectacular diversity of small RNA biogenesis and function, and discuss the relevance of lessons learned in the worm for human biology.

A Comparative Taxonomy of Parallel Algorithms for RNA Secondary Structure Prediction

Science.gov (United States)

Al-Khatib, Ra’ed M.; Abdullah, Rosni; Rashid, Nur’Aini Abdul

2010-01-01

RNA molecules have been discovered playing crucial roles in numerous biological and medical procedures and processes. RNA structures determination have become a major problem in the biology context. Recently, computer scientists have empowered the biologists with RNA secondary structures that ease an understanding of the RNA functions and roles. Detecting RNA secondary structure is an NP-hard problem, especially in pseudoknotted RNA structures. The detection process is also time-consuming; as a result, an alternative approach such as using parallel architectures is a desirable option. The main goal in this paper is to do an intensive investigation of parallel methods used in the literature to solve the demanding issues, related to the RNA secondary structure prediction methods. Then, we introduce a new taxonomy for the parallel RNA folding methods. Based on this proposed taxonomy, a systematic and scientific comparison is performed among these existing methods. PMID:20458364

Unzippers, Resolvers and Sensors: A Structural and Functional Biochemistry Tale of RNA Helicases

Directory of Open Access Journals (Sweden)

Ana Lúcia Leitão

2015-01-01

Full Text Available The centrality of RNA within the biological world is an irrefutable fact that currently attracts increasing attention from the scientific community. The panoply of functional RNAs requires the existence of specific biological caretakers, RNA helicases, devoted to maintain the proper folding of those molecules, resolving unstable structures. However, evolution has taken advantage of the specific position and characteristics of RNA helicases to develop new functions for these proteins, which are at the interface of the basic processes for transference of information from DNA to proteins. RNA helicases are involved in many biologically relevant processes, not only as RNA chaperones, but also as signal transducers, scaffolds of molecular complexes, and regulatory elements. Structural biology studies during the last decade, founded in X-ray crystallography, have characterized in detail several RNA-helicases. This comprehensive review summarizes the structural knowledge accumulated in the last two decades within this family of proteins, with special emphasis on the structure-function relationships of the most widely-studied families of RNA helicases: the DEAD-box, RIG-I-like and viral NS3 classes.

A systems biology approach for miRNA-mRNA expression patterns analysis in non-small cell lung cancer.

Science.gov (United States)

Najafi, Ali; Tavallaei, Mahmood; Hosseini, Sayed Mostafa

2016-01-01

Non-small cell lung cancers (NSCLCs) is a prevalent and heterogeneous subtype of lung cancer accounting for 85 percent of patients. MicroRNAs (miRNAs), a class of small endogenous non-coding RNAs, incorporate into regulation of gene expression post-transcriptionally. Therefore, deregulation of miRNAs' expression has provided further layers of complexity to the molecular etiology and pathogenesis of different diseases and malignancies. Although, until now considerable number of studies has been carried out to illuminate this complexity in NSCLC, they have remained less effective in their goal due to lack of a holistic and integrative systems biology approach which considers all natural elaborations of miRNAs' function. It is able to reliably nominate most affected signaling pathways and therapeutic target genes by deregulated miRNAs during a particular pathological condition. Herein, we utilized a holistic systems biology approach, based on appropriate re-analyses of microarray datasets followed by reliable data filtering, to analyze integrative and combinatorial deregulated miRNA-mRNA interaction network in NSCLC, aiming to ascertain miRNA-dysregulated signaling pathway and potential therapeutic miRNAs and mRNAs which represent a lion' share during various aspects of NSCLC's pathogenesis. Our systems biology approach introduced and nominated 1) important deregulated miRNAs in NSCLCs compared with normal tissue 2) significant and confident deregulated mRNAs which were anti-correlatively targeted by deregulated miRNA in NSCLCs and 3) dysregulated signaling pathways in association with deregulated miRNA-mRNAs interactions in NSCLCs. These results introduce possible mechanism of function of deregulated miRNAs and mRNAs in NSCLC that could be used as potential therapeutic targets.

Small Molecule Binding, Docking, and Characterization of the Interaction between Pth1 and Peptidyl-tRNA

Directory of Open Access Journals (Sweden)

Mary C. Hames

2013-11-01

Full Text Available Bacterial Pth1 is essential for viability. Pth1 cleaves the ester bond between the peptide and nucleotide of peptidyl-tRNA generated from aborted translation, expression of mini-genes, and short ORFs. We have determined the shape of the Pth1:peptidyl-tRNA complex using small angle neutron scattering. Binding of piperonylpiperazine, a small molecule constituent of a combinatorial synthetic library common to most compounds with inhibitory activity, was mapped to Pth1 via NMR spectroscopy. We also report computational docking results, modeling piperonylpiperazine binding based on chemical shift perturbation mapping. Overall these studies promote Pth1 as a novel antibiotic target, contribute to understanding how Pth1 interacts with its substrate, advance the current model for cleavage, and demonstrate feasibility of small molecule inhibition.

MicroRNA functional network in pancreatic cancer: From biology to ...

Indian Academy of Sciences (India)

[Wang J and Sen S 2011 MicroRNA functional network in pancreatic cancer: From biology to biomarkers of disease. ... growth factor type I receptor; INSR, insulin receptor; IPA, Ingenuity Pathway Analysis; IPMN, ..... Prostate cancer signalling.

Mapping posttranscriptional regulation of the human glycome uncovers microRNA defining the glycocode

OpenAIRE

Agrawal, Praveen; Kurcon, Tomasz; Pilobello, Kanoelani T.; Rakus, John F.; Koppolu, Sujeethraj; Liu, Zhongyin; Batista, Bianca S.; Eng, William S.; Hsu, Ku-Lung; Liang, Yaxuan; Mahal, Lara K.

2014-01-01

Carbohydrates (glycans) are complex cell surface molecules that control multiple aspects of cell biology, including cell–cell communication, cancer metastasis, and inflammation. Glycan biosynthesis requires the coordination of many enzymes, but how this is regulated is not well understood. Herein we show that microRNA (miRNA), small noncoding RNA, are a major regulator of cell surface glycosylation. We map miRNA expression onto carbohydrate signatures obtained by using lectin microarrays, a g...

Studying RNA-protein interactions in vivo by RNA immunoprecipitation

DEFF Research Database (Denmark)

Selth, Luke A; Close, Pierre; Svejstrup, Jesper Q

2011-01-01

and have significant effects on gene expression. RNA immunoprecipitation (RIP) is a powerful technique used to detect direct and indirect interactions between individual proteins and specific RNA molecules in vivo. Here, we describe RIP methods for both yeast and mammalian cells.......The crucial roles played by RNA-binding proteins in all aspects of RNA metabolism, particularly in the regulation of transcription, have become increasingly evident. Moreover, other factors that do not directly interact with RNA molecules can nevertheless function proximally to RNA polymerases...

Quantitative PCR--new diagnostic tool for quantifying specific mRNA and DNA molecules

DEFF Research Database (Denmark)

Schlemmer, B O; Sorensen, B S; Overgaard, J

2004-01-01

of a subset of ligands from the EGF system is increased in bladder cancer. Furthermore, measurement of the mRNA concentration gives important information such as the expression of these ligands correlated to the survival of the patients. In addition to the alterations at the mRNA level, changes also can occur...... at the DNA level in the EGF system. Thus, it has been demonstrated that the number of genes coding for the human epidermal growth factor receptor 2 (HER2) is increased in a number of breast tumors. It is now possible to treat breast cancer patients with a humanized antibody reacting with HER2...... of mRNA or DNA in biological samples. In this study quantitative PCR was used to investigate the role of the EGF (epidermal growth factor) system in cancer both for measurements of mRNA concentrations and for measurements of the number of copies of specific genes. It is shown that the mRNA expression...

Central dogma at the single-molecule level in living cells.

Science.gov (United States)

Li, Gene-Wei; Xie, X Sunney

2011-07-20

Gene expression originates from individual DNA molecules within living cells. Like many single-molecule processes, gene expression and regulation are stochastic, that is, sporadic in time. This leads to heterogeneity in the messenger-RNA and protein copy numbers in a population of cells with identical genomes. With advanced single-cell fluorescence microscopy, it is now possible to quantify transcriptomes and proteomes with single-molecule sensitivity. Dynamic processes such as transcription-factor binding, transcription and translation can be monitored in real time, providing quantitative descriptions of the central dogma of molecular biology and the demonstration that a stochastic single-molecule event can determine the phenotype of a cell.

Structural insight into RNA recognition motifs: versatile molecular Lego building blocks for biological systems.

Science.gov (United States)

Muto, Yutaka; Yokoyama, Shigeyuki

2012-01-01

'RNA recognition motifs (RRMs)' are common domain-folds composed of 80-90 amino-acid residues in eukaryotes, and have been identified in many cellular proteins. At first they were known as RNA binding domains. Through discoveries over the past 20 years, however, the RRMs have been shown to exhibit versatile molecular recognition activities and to behave as molecular Lego building blocks to construct biological systems. Novel RNA/protein recognition modes by RRMs are being identified, and more information about the molecular recognition by RRMs is becoming available. These RNA/protein recognition modes are strongly correlated with their biological significance. In this review, we would like to survey the recent progress on these versatile molecular recognition modules. Copyright © 2012 John Wiley & Sons, Ltd.

RNA detection in situ with FISH-STICs.

Science.gov (United States)

Sinnamon, John R; Czaplinski, Kevin

2014-02-01

The ability to detect RNA molecules in situ has long had important applications for molecular biological studies. Enzyme or dye-labeled antisense in vitro runoff transcripts and synthetic oligodeoxynucleotides (ODN) both have a proven track record of success, but each of these also has scientific and practical drawbacks and limitations to its use. We devised a means to use commercially synthesized oligonucleotides as RNA-FISH probes without further modification and show that such probes work well for detection of RNA in cultured cells. This approach can bind a high concentration of fluorescent ODN to a short stretch of an RNA using commercial DNA synthesis outlets available to any laboratory. We call this approach for creating in situ hybridization probes Fluorescence In Situ Hybridization with Sequential Tethered and Intertwined ODN Complexes (FISH-STICs). We demonstrate that one FISH-STIC probe can detect mRNA molecules in culture, and that probe detection can be improved by the addition of multiple probes that can be easily adapted for robust mRNA quantification. Using FISH-STICs, we demonstrate a nonoverlapping distribution for β-actin and γ-actin mRNA in cultured fibroblasts, and the detection of neuron-specific transcripts within cultured primary hippocampal neurons.

Novel nuclear magnetic resonance techniques for studying biological molecules

Energy Technology Data Exchange (ETDEWEB)

Laws, David Douglas [Univ. of California, Berkeley, CA (United States)

2000-06-01

Over the fifty-five year history of Nuclear Magnetic Resonance (NMR), considerable progress has been made in the development of techniques for studying the structure, function, and dynamics of biological molecules. The majority of this research has involved the development of multi-dimensional NMR experiments for studying molecules in solution, although in recent years a number of groups have begun to explore NMR methods for studying biological systems in the solid-state. Despite this new effort, a need still exists for the development of techniques that improve sensitivity, maximize information, and take advantage of all the NMR interactions available in biological molecules. In this dissertation, a variety of novel NMR techniques for studying biomolecules are discussed. A method for determining backbone (Φ/Ψ) dihedral angles by comparing experimentally determined ¹³C_a, chemical-shift anisotropies with theoretical calculations is presented, along with a brief description of the theory behind chemical-shift computation in proteins and peptides. The utility of the Spin-Polarization Induced Nuclear Overhauser Effect (SPINOE) to selectively enhance NMR signals in solution is examined in a variety of systems, as are methods for extracting structural information from cross-relaxation rates that can be measured in SPINOE experiments. Techniques for the production of supercritical and liquid laser-polarized xenon are discussed, as well as the prospects for using optically pumped xenon as a polarizing solvent. In addition, a detailed study of the structure of PrP 89-143 is presented. PrP 89-143 is a 54 residue fragment of the prion proteins which, upon mutation and aggregation, can induce prion diseases in transgenic mice. Whereas the structure of the wild-type PrP 89-143 is a generally unstructured mixture of α-helical and β-sheet conformers in the solid state, the aggregates formed from the PrP 89-143 mutants appear to be mostly β-sheet.

Entropy-based model for miRNA isoform analysis.

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Shengqin Wang

Full Text Available MiRNAs have been widely studied due to their important post-transcriptional regulatory roles in gene expression. Many reports have demonstrated the evidence of miRNA isoform products (isomiRs in high-throughput small RNA sequencing data. However, the biological function involved in these molecules is still not well investigated. Here, we developed a Shannon entropy-based model to estimate isomiR expression profiles of high-throughput small RNA sequencing data extracted from miRBase webserver. By using the Kolmogorov-Smirnov statistical test (KS test, we demonstrated that the 5p and 3p miRNAs present more variants than the single arm miRNAs. We also found that the isomiR variant, except the 3' isomiR variant, is strongly correlated with Minimum Free Energy (MFE of pre-miRNA, suggesting the intrinsic feature of pre-miRNA should be one of the important factors for the miRNA regulation. The functional enrichment analysis showed that the miRNAs with high variation, particularly the 5' end variation, are enriched in a set of critical functions, supporting these molecules should not be randomly produced. Our results provide a probabilistic framework for miRNA isoforms analysis, and give functional insights into pre-miRNA processing.

The Secret Life of RNA: Lessons from Emerging Methodologies.

Science.gov (United States)

Medioni, Caroline; Besse, Florence

2018-01-01

The last past decade has witnessed a revolution in our appreciation of transcriptome complexity and regulation. This remarkable expansion in our knowledge largely originates from the advent of high-throughput methodologies, and the consecutive discovery that up to 90% of eukaryotic genomes are transcribed, thus generating an unanticipated large range of noncoding RNAs (Hangauer et al., 15(4):112, 2014). Besides leading to the identification of new noncoding RNA species, transcriptome-wide studies have uncovered novel layers of posttranscriptional regulatory mechanisms controlling RNA processing, maturation or translation, and each contributing to the precise and dynamic regulation of gene expression. Remarkably, the development of systems-level studies has been accompanied by tremendous progress in the visualization of individual RNA molecules in single cells, such that it is now possible to image RNA species with a single-molecule resolution from birth to translation or decay. Monitoring quantitatively, with unprecedented spatiotemporal resolution, the fate of individual molecules has been key to understanding the molecular mechanisms underlying the different steps of RNA regulation. This has also revealed biologically relevant, intracellular and intercellular heterogeneities in RNA distribution or regulation. More recently, the convergence of imaging and high-throughput technologies has led to the emergence of spatially resolved transcriptomic techniques that provide a means to perform large-scale analyses while preserving spatial information. By generating transcriptome-wide data on single-cell RNA content, or even subcellular RNA distribution, these methodologies are opening avenues to a wide range of network-level studies at the cell and organ-level, and promise to strongly improve disease diagnostic and treatment.In this introductory chapter, we highlight how recently developed technologies aiming at detecting and visualizing RNA molecules have contributed to

Counting molecules in cell-free DNA and single cells RNA

OpenAIRE

Karlsson, Kasper

2016-01-01

The field of Molecular Biology got started in earnest with the discovery of the molecular structure of DNA. This lead to a surge of interest into the relationships between DNA, RNA and proteins, and to the development of fundamental tools for manipulating those substances, such as cutting, ligating, amplifying, visualizing and size-selecting DNA. With these tools at hand it was possible to begin sequencing DNA, a process that took a leap forward in 2005 with the advent of Next Generation Sequ...

Single Molecule Fluorescence: from Physical Fascination to Biological Relevance

OpenAIRE

Segers-Nolten, Gezina M.J.

2003-01-01

Confocal fluorescence microscopy is particularly well-known from the beautiful images that have been obtained with this technique from cells. Several cellular components could be nicely visualized simultaneously by staining them with different fluorophores. Not only for ensemble applications but also in single molecule research confocal fluorescence microscopy became a popular technique. In this thesis the possibilities are shown to study a complicated biological process, which is Nucleotide ...

Structural basis of RNA folding and recognition in an AMP-RNA aptamer complex.

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Jiang, F; Kumar, R A; Jones, R A; Patel, D J

1996-07-11

The catalytic properties of RNA and its well known role in gene expression and regulation are the consequence of its unique solution structures. Identification of the structural determinants of ligand recognition by RNA molecules is of fundamental importance for understanding the biological functions of RNA, as well as for the rational design of RNA Sequences with specific catalytic activities. Towards this latter end, Szostak et al. used in vitro selection techniques to isolate RNA sequences ('aptamers') containing a high-affinity binding site for ATP, the universal currency of cellular energy, and then used this motif to engineer ribozymes with polynucleotide kinase activity. Here we present the solution structure, as determined by multidimensional NMR spectroscopy and molecular dynamics calculations, of both uniformly and specifically 13C-, 15N-labelled 40-mer RNA containing the ATP-binding motif complexed with AMP. The aptamer adopts an L-shaped structure with two nearly orthogonal stems, each capped proximally by a G x G mismatch pair, binding the AMP ligand at their junction in a GNRA-like motif.

MicroRNA Expression Varies according to Glucose Tolerance, Measurement Platform, and Biological Source

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S. Dias

2017-01-01

Full Text Available Dysregulated microRNA (miRNA expression is observed during type 2 diabetes (T2D, although the consistency of miRNA expression across measurement platform and biological source is uncertain. Here we report miRNA profiling in the whole blood and serum of South African women with different levels of glucose tolerance, using next generation sequencing (NGS and quantitative real time PCR (qRT-PCR. Whole blood-derived miRNAs from women with newly diagnosed T2D (n=4, impaired glucose tolerance (IGT (n=4, and normal glucose tolerance (NGT (n=4 were subjected to NGS, whereafter transcript levels of selected miRNAs were quantified in the whole blood and serum of these women using qRT-PCR. Of the five significantly differentially expressed miRNAs identified by NGS, only the directional increase of miR-27b in women with IGT compared to NGT was confirmed in whole blood and serum, using qRT-PCR. Functional enrichment of miR-27b gene targets identified biological pathways associated with glucose transport and insulin regulation. In conclusion, this study showed poor correlation in miRNA expression profiled using NGS and qRT-PCR and in whole blood and serum. The consistent increased expression of miR-27b in women with IGT compared to NGT across measurement platform and biological source holds potential as a biomarker for risk stratification in our population.

Surface functionalization of bioactive glasses with natural molecules of biological significance, Part I: Gallic acid as model molecule

Science.gov (United States)

Zhang, Xin; Ferraris, Sara; Prenesti, Enrico; Verné, Enrica

2013-12-01

Gallic acid (3,4,5-trihydroxybenzoic acid, GA) and its derivatives are a group of biomolecules (polyphenols) obtained from plants. They have effects which are potentially beneficial to heath, for example they are antioxidant, anticarcinogenic and antibacterial, as recently investigated in many fields such as medicine, food and plant sciences. The main drawbacks of these molecules are both low stability and bioavailability. In this research work the opportunity to graft GA to bioactive glasses is investigated, in order to deliver the undamaged biological molecule into the body, using the biomaterial surfaces as a localized carrier. GA was considered for functionalization since it is a good model molecule for polyphenols and presents several interesting biological activities, like antibacterial, antioxidant and anticarcinogenic properties. Two different silica based bioactive glasses (SCNA and CEL2), with different reactivity, were employed as substrates. UV photometry combined with the Folin&Ciocalteu reagent was adopted to test the concentration of GA in uptake solution after functionalization. This test verified how much GA consumption occurred with surface modification and it was also used on solid samples to test the presence of GA on functionalized glasses. XPS and SEM-EDS techniques were employed to characterize the modification of material surface properties and functional group composition before and after functionalization.

A focus on polarity: Investigating the role of orientation cues in mediating student performance on mRNA synthesis tasks in an introductory cell and molecular biology course.

Science.gov (United States)

Olimpo, Jeffrey T; Quijas, Daniel A; Quintana, Anita M

2017-11-01

The central dogma has served as a foundational model for information flow, exchange, and storage in the biological sciences for several decades. Despite its continued importance, however, recent research suggests that novices in the domain possess several misconceptions regarding the aforementioned processes, including those pertaining specifically to the formation of messenger ribonucleic acid (mRNA) transcripts. In the present study, we sought to expand upon these observations through exploration of the influence of orientation cues on students' aptitude at synthesizing mRNAs from provided deoxyribonucleic acid (DNA) template strands. Data indicated that participants (n = 45) were proficient at solving tasks of this nature when the DNA template strand and the mRNA molecule were represented in an antiparallel orientation. In contrast, participants' performance decreased significantly on items in which the mRNA was depicted in a parallel orientation relative to the DNA template strand. Furthermore, participants' Grade Point Average, self-reported confidence in understanding the transcriptional process, and spatial ability were found to mediate their performance on the mRNA synthesis tasks. Collectively, these data reaffirm the need for future research and pedagogical interventions designed to enhance students' comprehension of the central dogma in a manner that makes transparent its relevance to real-world scientific phenomena. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(6):501-508, 2017. © 2017 The International Union of Biochemistry and Molecular Biology.

Inhibition of Xenograft tumor growth by gold nanoparticle-DNA oligonucleotide conjugates-assisted delivery of BAX mRNA.

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Ji-Hyun Yeom

Full Text Available Use of non-biological agents for mRNA delivery into living systems in order to induce heterologous expression of functional proteins may provide more advantages than the use of DNA and/or biological vectors for delivery. However, the low efficiency of mRNA delivery into live animals, using non-biological systems, has hampered the use of mRNA as a therapeutic molecule. Here, we show that gold nanoparticle-DNA oligonucleotide (AuNP-DNA conjugates can serve as universal vehicles for more efficient delivery of mRNA into human cells, as well as into xenograft tumors generated in mice. Injections of BAX mRNA loaded on AuNP-DNA conjugates into xenograft tumors resulted in highly efficient mRNA delivery. The delivered mRNA directed the efficient production of biologically functional BAX protein, a pro-apoptotic factor, consequently inhibiting tumor growth. These results demonstrate that mRNA delivery by AuNP-DNA conjugates can serve as a new platform for the development of safe and efficient gene therapy.

Use of various ionization modes for the study of molecules of biological interest

International Nuclear Information System (INIS)

Forest, E.

1987-01-01

For the last ten years a revolutionary advance in mass spectrometry applied to molecules of biological interest occurred, chiefly concerning ionization with the emergence of many new modes allowing non volatile, polar or thermally labile sample analysis. Some examples of spectra obtained on high mass molecules such as vitamins, protein fragments, porphyrins (chlorophyll or hemoglobin active site), polysaccharides, are presented using some of the new modes [fr

Rclick: a web server for comparison of RNA 3D structures.

Science.gov (United States)

Nguyen, Minh N; Verma, Chandra

2015-03-15

RNA molecules play important roles in key biological processes in the cell and are becoming attractive for developing therapeutic applications. Since the function of RNA depends on its structure and dynamics, comparing and classifying the RNA 3D structures is of crucial importance to molecular biology. In this study, we have developed Rclick, a web server that is capable of superimposing RNA 3D structures by using clique matching and 3D least-squares fitting. Our server Rclick has been benchmarked and compared with other popular servers and methods for RNA structural alignments. In most cases, Rclick alignments were better in terms of structure overlap. Our server also recognizes conformational changes between structures. For this purpose, the server produces complementary alignments to maximize the extent of detectable similarity. Various examples showcase the utility of our web server for comparison of RNA, RNA-protein complexes and RNA-ligand structures. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Regulatory effects of cotranscriptional RNA structure formation and transitions.

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Liu, Sheng-Rui; Hu, Chun-Gen; Zhang, Jin-Zhi

2016-09-01

RNAs, which play significant roles in many fundamental biological processes of life, fold into sophisticated and precise structures. RNA folding is a dynamic and intricate process, which conformation transition of coding and noncoding RNAs form the primary elements of genetic regulation. The cellular environment contains various intrinsic and extrinsic factors that potentially affect RNA folding in vivo, and experimental and theoretical evidence increasingly indicates that the highly flexible features of the RNA structure are affected by these factors, which include the flanking sequence context, physiochemical conditions, cis RNA-RNA interactions, and RNA interactions with other molecules. Furthermore, distinct RNA structures have been identified that govern almost all steps of biological processes in cells, including transcriptional activation and termination, transcriptional mutagenesis, 5'-capping, splicing, 3'-polyadenylation, mRNA export and localization, and translation. Here, we briefly summarize the dynamic and complex features of RNA folding along with a wide variety of intrinsic and extrinsic factors that affect RNA folding. We then provide several examples to elaborate RNA structure-mediated regulation at the transcriptional and posttranscriptional levels. Finally, we illustrate the regulatory roles of RNA structure and discuss advances pertaining to RNA structure in plants. WIREs RNA 2016, 7:562-574. doi: 10.1002/wrna.1350 For further resources related to this article, please visit the WIREs website. © 2016 Wiley Periodicals, Inc.

Biochemical and single-molecule analyses of the RNA silencing suppressing activity of CrPV-1A.

Science.gov (United States)

Watanabe, Mariko; Iwakawa, Hiro-Oki; Tadakuma, Hisashi; Tomari, Yukihide

2017-10-13

Viruses often encode viral silencing suppressors (VSSs) to counteract the hosts' RNA silencing activity. The cricket paralysis virus 1A protein (CrPV-1A) is a unique VSS that binds to a specific Argonaute protein (Ago)-the core of the RNA-induced silencing complex (RISC)-in insects to suppress its target cleavage reaction. However, the precise molecular mechanism of CrPV-1A action remains unclear. Here we utilized biochemical and single-molecule imaging approaches to analyze the effect of CrPV-1A during target recognition and cleavage by Drosophila Ago2-RISC. Our results suggest that CrPV-1A obstructs the initial target searching by Ago2-RISC via base pairing in the seed region. The combination of biochemistry and single-molecule imaging may help to pave the way for mechanistic understanding of VSSs with diverse functions. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Conceptual modeling in systems biology fosters empirical findings: the mRNA lifecycle.

Directory of Open Access Journals (Sweden)

Dov Dori

Full Text Available One of the main obstacles to understanding complex biological systems is the extent and rapid evolution of information, way beyond the capacity individuals to manage and comprehend. Current modeling approaches and tools lack adequate capacity to model concurrently structure and behavior of biological systems. Here we propose Object-Process Methodology (OPM, a holistic conceptual modeling paradigm, as a means to model both diagrammatically and textually biological systems formally and intuitively at any desired number of levels of detail. OPM combines objects, e.g., proteins, and processes, e.g., transcription, in a way that is simple and easily comprehensible to researchers and scholars. As a case in point, we modeled the yeast mRNA lifecycle. The mRNA lifecycle involves mRNA synthesis in the nucleus, mRNA transport to the cytoplasm, and its subsequent translation and degradation therein. Recent studies have identified specific cytoplasmic foci, termed processing bodies that contain large complexes of mRNAs and decay factors. Our OPM model of this cellular subsystem, presented here, led to the discovery of a new constituent of these complexes, the translation termination factor eRF3. Association of eRF3 with processing bodies is observed after a long-term starvation period. We suggest that OPM can eventually serve as a comprehensive evolvable model of the entire living cell system. The model would serve as a research and communication platform, highlighting unknown and uncertain aspects that can be addressed empirically and updated consequently while maintaining consistency.

Single-Molecule Sensing with Nanopore Confinement: from Chemical Reactions to Biological Interactions.

Science.gov (United States)

Lin, Yao; Ying, Yi-Lun; Gao, Rui; Long, Yi-Tao

2018-03-25

The nanopore can generate an electrochemical confinement for single-molecule sensing which help understand the fundamental chemical principle in nanoscale dimensions. By observing the generated ionic current, individual bond-making and bond-breaking steps, single biomolecule dynamic conformational changes and electron transfer processes that occur within pore can be monitored with high temporal and current resolution. These single-molecule studies in nanopore confinement are revealing information about the fundamental chemical and biological processes that cannot be extracted from ensemble measurements. In this concept, we introduce and discuss the electrochemical confinement effects on single-molecule covalent reactions, conformational dynamics of individual molecules and host-guest interactions in protein nanopores. Then, we extend the concept of nanopore confinement effects to confine electrochemical redox reactions in solid-state nanopores for developing new sensing mechanisms. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Finding optimal interaction interface alignments between biological complexes

KAUST Repository

Cui, Xuefeng

2015-06-13

Motivation: Biological molecules perform their functions through interactions with other molecules. Structure alignment of interaction interfaces between biological complexes is an indispensable step in detecting their structural similarities, which are keys to understanding their evolutionary histories and functions. Although various structure alignment methods have been developed to successfully access the similarities of protein structures or certain types of interaction interfaces, existing alignment tools cannot directly align arbitrary types of interfaces formed by protein, DNA or RNA molecules. Specifically, they require a \\'blackbox preprocessing\\' to standardize interface types and chain identifiers. Yet their performance is limited and sometimes unsatisfactory. Results: Here we introduce a novel method, PROSTA-inter, that automatically determines and aligns interaction interfaces between two arbitrary types of complex structures. Our method uses sequentially remote fragments to search for the optimal superimposition. The optimal residue matching problem is then formulated as a maximum weighted bipartite matching problem to detect the optimal sequence order-independent alignment. Benchmark evaluation on all non-redundant protein-DNA complexes in PDB shows significant performance improvement of our method over TM-align and iAlign (with the \\'blackbox preprocessing\\'). Two case studies where our method discovers, for the first time, structural similarities between two pairs of functionally related protein-DNA complexes are presented. We further demonstrate the power of our method on detecting structural similarities between a protein-protein complex and a protein-RNA complex, which is biologically known as a protein-RNA mimicry case. © The Author 2015. Published by Oxford University Press.

Assigned and unassigned distance geometry: applications to biological molecules and nanostructures

Energy Technology Data Exchange (ETDEWEB)

Billinge, Simon J. L. [Columbia Univ., New York, NY (United States). Applied Physics and Applied Mathematics; Brookhaven National Lab. (BNL), Upton, NY (United States). X-ray Scattering Group; Duxbury, Phillip M. [Michigan State Univ., East Lansing, MI (United States). Dept. of Physics and Astronomy; Gonçalves, Douglas S. [Univ. Federal de Santa Catarina,; Lavor, Carlile [Univ. of Campinas (UNICAMP), Sao Paulo (Brazil). Dept. of Applied Mathematics (IMECC-UNICAMP); Mucherino, Antonio [Univ. de Rennes, Rennes (France). Institut de Recherche en Informatique et Systemes Aleatoires

2016-04-04

Here, considering geometry based on the concept of distance, the results found by Menger and Blumenthal originated a body of knowledge called distance geometry. This survey covers some recent developments for assigned and unassigned distance geometry and focuses on two main applications: determination of three-dimensional conformations of biological molecules and nanostructures.

Nanoparticle-based delivery of small interfering RNA: challenges for cancer therapy

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Miele E

2012-07-01

Full Text Available Evelina Miele,1,* Gian Paolo Spinelli,2,* Ermanno Miele,3 Enzo Di Fabrizio,3,6 Elisabetta Ferretti,4 Silverio Tomao,2 Alberto Gulino,1,5 1Department of Molecular Medicine, 2Department of Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome, Rome, 3Nanostructures, Istituto Italiano di Tecnologia, via Morego, 30, 16163 Genova, 4Department of Experimental Medicine, Sapienza University of Rome, Rome, 5Center for Life Nanoscience, Istituto Italiano di Tecnologia, Rome, Italy, 6BIONEM lab, University of Magna Graecia, Campus S. Venuta, Viale Europa 88100 Catanzaro, Italy *These authors contributed equally to this workAbstract: During recent decades there have been remarkable advances and profound changes in cancer therapy. Many therapeutic strategies learned at the bench, including monoclonal antibodies and small molecule inhibitors, have been used at the bedside, leading to important successes. One of the most important advances in biology has been the discovery that small interfering RNA (siRNA is able to regulate the expression of genes, by a phenomenon known as RNA interference (RNAi. RNAi is one of the most rapidly growing fields of research in biology and therapeutics. Much research effort has gone into the application of this new discovery in the treatment of various diseases, including cancer. However, even though these molecules may have potential and strong utility, some limitations make their clinical application difficult, including delivery problems, side effects due to off-target actions, disturbance of physiological functions of the cellular machinery involved in gene silencing, and induction of the innate immune response. Many researchers have attempted to overcome these limitations and to improve the safety of potential RNAi-based therapeutics. Nanoparticles, which are nanostructured entities with tunable size, shape, and surface, as well as biological behavior, provide an ideal opportunity to modify current

Assessing Specific Oligonucleotides and Small Molecule Antibiotics for the Ability to Inhibit the CRD-BP-CD44 RNA Interaction

Science.gov (United States)

Thomsen, Dana; Lee, Chow H.

2014-01-01

Studies on Coding Region Determinant-Binding Protein (CRD-BP) and its orthologs have confirmed their functional role in mRNA stability and localization. CRD-BP is present in extremely low levels in normal adult tissues, but it is over-expressed in many types of aggressive human cancers and in neonatal tissues. Although the exact role of CRD-BP in tumour progression is unclear, cumulative evidence suggests that its ability to physically associate with target mRNAs is an important criterion for its oncogenic role. CRD-BP has high affinity for the 3′UTR of the oncogenic CD44 mRNA and depletion of CRD-BP in cells led to destabilization of CD44 mRNA, decreased CD44 expression, reduced adhesion and disruption of invadopodia formation. Here, we further characterize the CRD-BP-CD44 RNA interaction and assess specific antisense oligonucleotides and small molecule antibiotics for their ability to inhibit the CRD-BP-CD44 RNA interaction. CRD-BP has a high affinity for binding to CD44 RNA nts 2862–3055 with a Kd of 645 nM. Out of ten antisense oligonucleotides spanning nts 2862–3055, only three antisense oligonucleotides (DD4, DD7 and DD10) were effective in competing with CRD-BP for binding to 32P-labeled CD44 RNA. The potency of DD4, DD7 and DD10 in inhibiting the CRD-BP-CD44 RNA interaction in vitro correlated with their ability to specifically reduce the steady-state level of CD44 mRNA in cells. The aminoglycoside antibiotics neomycin, paramomycin, kanamycin and streptomycin effectively inhibited the CRD-BP-CD44 RNA interaction in vitro. Assessing the potential inhibitory effect of aminoglycoside antibiotics including neomycin on the CRD-BP-CD44 mRNA interaction in cells proved difficult, likely due to their propensity to non-specifically bind nucleic acids. Our results have important implications for future studies in finding small molecules and nucleic acid-based inhibitors that interfere with protein-RNA interactions. PMID:24622399

High-throughput platform assay technology for the discovery of pre-microrna-selective small molecule probes.

Science.gov (United States)

Lorenz, Daniel A; Song, James M; Garner, Amanda L

2015-01-21

MicroRNAs (miRNA) play critical roles in human development and disease. As such, the targeting of miRNAs is considered attractive as a novel therapeutic strategy. A major bottleneck toward this goal, however, has been the identification of small molecule probes that are specific for select RNAs and methods that will facilitate such discovery efforts. Using pre-microRNAs as proof-of-concept, herein we report a conceptually new and innovative approach for assaying RNA-small molecule interactions. Through this platform assay technology, which we term catalytic enzyme-linked click chemistry assay or cat-ELCCA, we have designed a method that can be implemented in high throughput, is virtually free of false readouts, and is general for all nucleic acids. Through cat-ELCCA, we envision the discovery of selective small molecule ligands for disease-relevant miRNAs to promote the field of RNA-targeted drug discovery and further our understanding of the role of miRNAs in cellular biology.

Study of radionuclides speciation with biological molecules of interest by spectrometric techniques

International Nuclear Information System (INIS)

Lourenco, V.

2007-07-01

Mechanisms of complexation and accumulation of the radionuclides at the cellular and molecular level are complex and poorly known because the studies on these subjects are scarce. Within the framework of this thesis, we studied the interactions of these cations with biological molecules of interest. We chose to focus on an actinide: uranium (VI) as well as europium as an analogue of trivalent actinides. The selected biological molecules are the phyto-chelatins: their role is to protect cells against intrusions from nonessential heavy metals (thus toxic). These proteins are likely to be implied in the mechanisms of sequestration of radionuclides in living organisms. However, their structure is complex, this is why, in order to better include/understand their reactivity, we extended our studies to lower entities which constitute them (amino acid: glycine, glutamic acid and cysteine; polypeptides: glutathione reduced and oxidized forms). In particular, we determined solution speciation (stoichiometry, structure) as well as the complexing constants associated with the formation with these species. These studies were undertaken by Time Resolved Laser induced Fluorescence (TRLIF), Electro-Spray-Mass Spectrometry (ES-MS), Nuclear Magnetic Resonance (NMR), Fourier Transform Infra-Rouge spectroscopy (FTIR) and Extended X-ray Absorption Fine Structure Spectroscopy (EXAFS).The determination of the complexation constants enabled us to conclude that the complexing capacity of these molecules with respect to radionuclides was moderate (log 10 K 1 ≤ 3, pH 3 or 6), the formed species are mononuclear with only one ligand molecule (1:1). The interaction is performed via oxygenated (hard) groups. The direct complexation of europium with phyto-chelatins at acidic pH was studied jointly by TRLIF and ES-MS. The complexing capacity of these molecules is much higher than that of GSH from which they result. The interaction of europium with metallothioneins is, on the contrary, lower than

Porcine blood mononuclear cell cytokine responses to PAMP molecules: comparison of mRNA and protein production

DEFF Research Database (Denmark)

Sørensen, Nanna Skall; Skovgaard, Kerstin; Heegaard, Peter M. H.

2011-01-01

Pathogen-associated molecular patterns (PAMPs) are conserved molecules of microorganisms inducing innate immune cells to secrete distinct patterns of cytokines. In veterinary species, due to a lack of specific antibodies, cytokines are often monitored as expressed mRNA only. This study investigated...... the induction of IFN-α, IL-12 p40, IL-1β, TNF-α, IL-6 and IL-10 by PAMP-molecules [CpG oligonucleotide D19 (CpG), peptidoglycan (PGN), lipopolysaccharide (LPS), Pam3Cys and poly-U] in porcine blood mononuclear cells (BMC) within a 24h period. As expected, cytokine responses were PAMP-specific, CpG inducing IFN...

RNA Crystallization

Science.gov (United States)

Golden, Barbara L.; Kundrot, Craig E.

2003-01-01

RNA molecules may be crystallized using variations of the methods developed for protein crystallography. As the technology has become available to syntheisize and purify RNA molecules in the quantities and with the quality that is required for crystallography, the field of RNA structure has exploded. The first consideration when crystallizing an RNA is the sequence, which may be varied in a rational way to enhance crystallizability or prevent formation of alternate structures. Once a sequence has been designed, the RNA may be synthesized chemically by solid-state synthesis, or it may be produced enzymatically using RNA polymerase and an appropriate DNA template. Purification of milligram quantities of RNA can be accomplished by HPLC or gel electrophoresis. As with proteins, crystallization of RNA is usually accomplished by vapor diffusion techniques. There are several considerations that are either unique to RNA crystallization or more important for RNA crystallization. Techniques for design, synthesis, purification, and crystallization of RNAs will be reviewed here.

Pragmatic turn in biology: From biological molecules to genetic content operators.

Science.gov (United States)

Witzany, Guenther

2014-08-26

Erwin Schrödinger's question "What is life?" received the answer for decades of "physics + chemistry". The concepts of Alain Turing and John von Neumann introduced a third term: "information". This led to the understanding of nucleic acid sequences as a natural code. Manfred Eigen adapted the concept of Hammings "sequence space". Similar to Hilbert space, in which every ontological entity could be defined by an unequivocal point in a mathematical axiomatic system, in the abstract "sequence space" concept each point represents a unique syntactic structure and the value of their separation represents their dissimilarity. In this concept molecular features of the genetic code evolve by means of self-organisation of matter. Biological selection determines the fittest types among varieties of replication errors of quasi-species. The quasi-species concept dominated evolution theory for many decades. In contrast to this, recent empirical data on the evolution of DNA and its forerunners, the RNA-world and viruses indicate cooperative agent-based interactions. Group behaviour of quasi-species consortia constitute de novo and arrange available genetic content for adaptational purposes within real-life contexts that determine epigenetic markings. This review focuses on some fundamental changes in biology, discarding its traditional status as a subdiscipline of physics and chemistry.

Promoter binding, initiation, and elongation by bacteriophage T7 RNA polymerase. A single-molecule view of the transcription cycle.

Science.gov (United States)

Skinner, Gary M; Baumann, Christoph G; Quinn, Diana M; Molloy, Justin E; Hoggett, James G

2004-01-30

A single-molecule transcription assay has been developed that allows, for the first time, the direct observation of promoter binding, initiation, and elongation by a single RNA polymerase (RNAP) molecule in real-time. To promote DNA binding and transcription initiation, a DNA molecule tethered between two optically trapped beads was held near a third immobile surface bead sparsely coated with RNAP. By driving the optical trap holding the upstream bead with a triangular oscillation while measuring the position of both trapped beads, we observed the onset of promoter binding, promoter escape (productive initiation), and processive elongation by individual RNAP molecules. After DNA template release, transcription re-initiation on the same DNA template is possible; thus, multiple enzymatic turnovers by an individual RNAP molecule can be observed. Using bacteriophage T7 RNAP, a commonly used RNAP paradigm, we observed the association and dissociation (k(off)= 2.9 s(-1)) of T7 RNAP and promoter DNA, the transition to the elongation mode (k(for) = 0.36 s(-1)), and the processive synthesis (k(pol) = 43 nt s(-1)) and release of a gene-length RNA transcript ( approximately 1200 nt). The transition from initiation to elongation is much longer than the mean lifetime of the binary T7 RNAP-promoter DNA complex (k(off) > k(for)), identifying a rate-limiting step between promoter DNA binding and promoter escape.

GARN2: coarse-grained prediction of 3D structure of large RNA molecules by regret minimization.

Science.gov (United States)

Boudard, Mélanie; Barth, Dominique; Bernauer, Julie; Denise, Alain; Cohen, Johanne

2017-08-15

Predicting the 3D structure of RNA molecules is a key feature towards predicting their functions. Methods which work at atomic or nucleotide level are not suitable for large molecules. In these cases, coarse-grained prediction methods aim to predict a shape which could be refined later by using more precise methods on smaller parts of the molecule. We developed a complete method for sampling 3D RNA structure at a coarse-grained model, taking a secondary structure as input. One of the novelties of our method is that a second step extracts two best possible structures close to the native, from a set of possible structures. Although our method benefits from the first version of GARN, some of the main features on GARN2 are very different. GARN2 is much faster than the previous version and than the well-known methods of the state-of-art. Our experiments show that GARN2 can also provide better structures than the other state-of-the-art methods. GARN2 is written in Java. It is freely distributed and available at http://garn.lri.fr/. melanie.boudard@lri.fr or johanne.cohen@lri.fr. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

RNAiFold 2.0: a web server and software to design custom and Rfam-based RNA molecules.

Science.gov (United States)

Garcia-Martin, Juan Antonio; Dotu, Ivan; Clote, Peter

2015-07-01

Several algorithms for RNA inverse folding have been used to design synthetic riboswitches, ribozymes and thermoswitches, whose activity has been experimentally validated. The RNAiFold software is unique among approaches for inverse folding in that (exhaustive) constraint programming is used instead of heuristic methods. For that reason, RNAiFold can generate all sequences that fold into the target structure or determine that there is no solution. RNAiFold 2.0 is a complete overhaul of RNAiFold 1.0, rewritten from the now defunct COMET language to C++. The new code properly extends the capabilities of its predecessor by providing a user-friendly pipeline to design synthetic constructs having the functionality of given Rfam families. In addition, the new software supports amino acid constraints, even for proteins translated in different reading frames from overlapping coding sequences; moreover, structure compatibility/incompatibility constraints have been expanded. With these features, RNAiFold 2.0 allows the user to design single RNA molecules as well as hybridization complexes of two RNA molecules. the web server, source code and linux binaries are publicly accessible at http://bioinformatics.bc.edu/clotelab/RNAiFold2.0. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

A Systems’ Biology Approach to Study MicroRNA-Mediated Gene Regulatory Networks

Directory of Open Access Journals (Sweden)

Xin Lai

2013-01-01

Full Text Available MicroRNAs (miRNAs are potent effectors in gene regulatory networks where aberrant miRNA expression can contribute to human diseases such as cancer. For a better understanding of the regulatory role of miRNAs in coordinating gene expression, we here present a systems biology approach combining data-driven modeling and model-driven experiments. Such an approach is characterized by an iterative process, including biological data acquisition and integration, network construction, mathematical modeling and experimental validation. To demonstrate the application of this approach, we adopt it to investigate mechanisms of collective repression on p21 by multiple miRNAs. We first construct a p21 regulatory network based on data from the literature and further expand it using algorithms that predict molecular interactions. Based on the network structure, a detailed mechanistic model is established and its parameter values are determined using data. Finally, the calibrated model is used to study the effect of different miRNA expression profiles and cooperative target regulation on p21 expression levels in different biological contexts.

MicroRNA Biomarkers in Neurodegenerative Diseases and Emerging Nano-Sensors Technology

DEFF Research Database (Denmark)

Shah, Pratik; Cho, Seok Keun; Thulstrup, Peter Waaben

2017-01-01

MicroRNAs (miRNAs) are essential small RNA molecules (20–24 nt) that negatively regulate the expression of target genes at the post-transcriptional level. Due to their roles in a variety of biological processes, the aberrant expression profiles of miRNAs have been identified as biomarkers for man...

The specificity of long noncoding RNA expression.

Science.gov (United States)

Gloss, Brian S; Dinger, Marcel E

2016-01-01

Over the last decade, long noncoding RNAs (lncRNAs) have emerged as a fundamental molecular class whose members play pivotal roles in the regulation of the genome. The observation of pervasive transcription of mammalian genomes in the early 2000s sparked a revolution in the understanding of information flow in eukaryotic cells and the incredible flexibility and dynamic nature of the transcriptome. As a molecular class, distinct loci yielding lncRNAs are set to outnumber those yielding mRNAs. However, like many important discoveries, the road leading to uncovering this diverse class of molecules that act through a remarkable repertoire of mechanisms, was not a straight one. The same characteristic that most distinguishes lncRNAs from mRNAs, i.e. their developmental-stage, tissue-, and cell-specific expression, was one of the major impediments to their discovery and recognition as potentially functional regulatory molecules. With growing numbers of lncRNAs being assigned to biological functions, the specificity of lncRNA expression is now increasingly recognized as a characteristic that imbues lncRNAs with great potential as biomarkers and for the development of highly targeted therapeutics. Here we review the history of lncRNA research and how technological advances and insight into biological complexity have gone hand-in-hand in shaping this revolution. We anticipate that as increasing numbers of these molecules, often described as the dark matter of the genome, are characterized and the structure-function relationship of lncRNAs becomes better understood, it may ultimately be feasible to decipher what these non-(protein)-coding genes encode. This article is part of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa. Copyright © 2015 Elsevier B.V. All rights reserved.

Towards Antiviral shRNAs Based on the AgoshRNA Design.

Directory of Open Access Journals (Sweden)

Ying Poi Liu

Full Text Available RNA interference (RNAi can be induced by intracellular expression of a short hairpin RNA (shRNA. Processing of the shRNA requires the RNaseIII-like Dicer enzyme to remove the loop and to release the biologically active small interfering RNA (siRNA. Dicer is also involved in microRNA (miRNA processing to liberate the mature miRNA duplex, but recent studies indicate that miR-451 is not processed by Dicer. Instead, this miRNA is processed by the Argonaute 2 (Ago2 protein, which also executes the subsequent cleavage of a complementary mRNA target. Interestingly, shRNAs that structurally resemble miR-451 can also be processed by Ago2 instead of Dicer. The key determinant of these "AgoshRNA" molecules is a relatively short basepaired stem, which avoids Dicer recognition and consequently allows alternative processing by Ago2. AgoshRNA processing yields a single active RNA strand, whereas standard shRNAs produce a duplex with guide and passenger strands and the latter may cause adverse off-target effects. In this study, we converted previously tested active anti-HIV-1 shRNA molecules into AgoshRNA. We tested several designs that could potentially improve AgoshRNA activity, including extension of the complementarity between the guide strand and the mRNA target and reduction of the thermodynamic stability of the hairpins. We demonstrate that active AgoshRNAs can be generated. However, the RNAi activity is reduced compared to the matching shRNAs. Despite reduced RNAi activity, comparison of an active AgoshRNA and the matching shRNA in a sensitive cell toxicity assay revealed that the AgoshRNA is much less toxic.

From Molecules to Living Organisms : an Interplay between Biology and Physics : Lecture Notes of the Les Houches School of Physics

CERN Document Server

Nury, Hughes; Parcy, François; Ruigrok, Rob W H; Ziegler, Christine; Cugliandolo, Leticia F; Session CII

2016-01-01

The aim of this book is to provide new ideas for studying living matter by a simultaneous understanding of behavior from molecules to the cell, to the whole organism in the light of physical concepts. Indeed, forces guide most biological phenomena. In some cases these forces can be well-described and thus used to model a particular biological phenomenon. This is exemplified here by the study of membranes, where their shapes and curvatures can be modeled using a limited number of parameters that are measured experimentally. The growth of plants is another example where the combination of physics, biology and mathematics leads to a predictive model. The laws of thermodynamics are essential, as they dictate the behavior of proteins, or more generally biological molecules, in an aqueous environment. Integrated studies from the molecule to a larger scale need a combination of cutting-edge approaches, such as the use of new X-ray sources, in-cell NMR, cryo-electron microscopy or single-molecule microscopy. Some are...

dPORE-miRNA: Polymorphic regulation of microRNA genes

KAUST Repository

Schmeier, Sebastian; Schaefer, Ulf; MacPherson, Cameron R.; Bajic, Vladimir B.

2011-01-01

Background: MicroRNAs (miRNAs) are short non-coding RNA molecules that act as post-transcriptional regulators and affect the regulation of protein-coding genes. Mostly transcribed by PolII, miRNA genes are regulated at the transcriptional level similarly to protein-coding genes. In this study we focus on human miRNAs. These miRNAs are involved in a variety of pathways and can affect many diseases. Our interest is on possible deregulation of the transcription initiation of the miRNA encoding genes, which is facilitated by variations in the genomic sequence of transcriptional control regions (promoters). Methodology: Our aim is to provide an online resource to facilitate the investigation of the potential effects of single nucleotide polymorphisms (SNPs) on miRNA gene regulation. We analyzed SNPs overlapped with predicted transcription factor binding sites (TFBSs) in promoters of miRNA genes. We also accounted for the creation of novel TFBSs due to polymorphisms not present in the reference genome. The resulting changes in the original TFBSs and potential creation of new TFBSs were incorporated into the Dragon Database of Polymorphic Regulation of miRNA genes (dPORE-miRNA). Conclusions: The dPORE-miRNA database enables researchers to explore potential effects of SNPs on the regulation of miRNAs. dPORE-miRNA can be interrogated with regards to: a/miRNAs (their targets, or involvement in diseases, or biological pathways), b/SNPs, or c/transcription factors. dPORE-miRNA can be accessed at http://cbrc.kaust.edu.sa/dpore and http://apps.sanbi.ac.za/dpore/. Its use is free for academic and non-profit users. © 2011 Schmeier et al.

dPORE-miRNA: Polymorphic regulation of microRNA genes

KAUST Repository

Schmeier, Sebastian

2011-02-04

Background: MicroRNAs (miRNAs) are short non-coding RNA molecules that act as post-transcriptional regulators and affect the regulation of protein-coding genes. Mostly transcribed by PolII, miRNA genes are regulated at the transcriptional level similarly to protein-coding genes. In this study we focus on human miRNAs. These miRNAs are involved in a variety of pathways and can affect many diseases. Our interest is on possible deregulation of the transcription initiation of the miRNA encoding genes, which is facilitated by variations in the genomic sequence of transcriptional control regions (promoters). Methodology: Our aim is to provide an online resource to facilitate the investigation of the potential effects of single nucleotide polymorphisms (SNPs) on miRNA gene regulation. We analyzed SNPs overlapped with predicted transcription factor binding sites (TFBSs) in promoters of miRNA genes. We also accounted for the creation of novel TFBSs due to polymorphisms not present in the reference genome. The resulting changes in the original TFBSs and potential creation of new TFBSs were incorporated into the Dragon Database of Polymorphic Regulation of miRNA genes (dPORE-miRNA). Conclusions: The dPORE-miRNA database enables researchers to explore potential effects of SNPs on the regulation of miRNAs. dPORE-miRNA can be interrogated with regards to: a/miRNAs (their targets, or involvement in diseases, or biological pathways), b/SNPs, or c/transcription factors. dPORE-miRNA can be accessed at http://cbrc.kaust.edu.sa/dpore and http://apps.sanbi.ac.za/dpore/. Its use is free for academic and non-profit users. © 2011 Schmeier et al.

RNA interference for performance enhancement and detection in doping control.

Science.gov (United States)

Kohler, Maxie; Schänzer, Wilhelm; Thevis, Mario

2011-10-01

RNA interference represents a comparably new route of regulating and manipulating specific gene expression. Promising results were obtained in experimental therapies aim at the treatment of different kinds of diseases including cancer, diabetes mellitus or Dychenne muscular dystrophy. While studies on down-regulation efficiency are often performed by analyzing the regulated protein, the direct detection of small, interfering RNA molecules and antisense oligonucleotides is of great interest for the investigation of the metabolism and degradation and also for the detection of a putative misuse of these molecules in sports. Myostatin down-regulation was shown to result in increased performance and muscle growth and the regulation of several other proteins could be relevant for performance enhancement. This mini-review summarizes current approaches for the mass spectrometric analysis of siRNA and antisense oligonucleotides from biological matrices and the available data on biodistribution, metabolism, and half-life of relevant substances are discussed. Copyright © 2011 John Wiley & Sons, Ltd.

Drawbacks of the ancient RNA-based life-like system under primitive earth conditions.

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Kawamura, Kunio

2012-07-01

Following the discovery of ribozymes, the "RNA world" hypothesis has become the most accepted hypothesis concerning the origin of life and genetic information. However, this hypothesis has several drawbacks. Verification of the hypothesis from different viewpoints led us to proposals from the viewpoint of the hydrothermal origin of life, solubility of RNA and related biopolymers, and the possibility of creating an evolutionary system comparable to the in vitro selection technique for functional RNA molecules based on molecular biology. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

In vitro studies. Contribution of radioactive marking to molecular biology development

International Nuclear Information System (INIS)

Sentenac, A.

1997-01-01

The spectacular and rapid development of molecular biology is essentially related to the utilization of marked molecules which leads to quantitative and qualitative information; the use of radioactive tracers allowed for the observation of the biosynthesis of biological polymers, and thus, for example, the formation of DNA, RNA or proteins. A historical review of the great discoveries in this field, is presented

"Life-bearing molecules" versus "life-embodying systems": Two contrasting views on the what-is-life (WIL) problem persisting from the early days of molecular biology to the post-genomic cell- and organism-level biology.

Science.gov (United States)

Sato, Naoki

2018-05-01

"What is life?" is an ultimate biological quest for the principle that makes organisms alive. This 'WIL problem' is not, however, a simple one that we have a straightforward strategy to attack. From the beginning, molecular biology tried to identify molecules that bear the essence of life: the double helical DNA represented replication, and enzymes were micro-actuators of biological activities. A dominating idea behind these mainstream biological studies relies on the identification of life-bearing molecules, which themselves are models of life. Another, prevalent idea emphasizes that life resides in the whole system of an organism, but not in some particular molecules. The behavior of a complex system may be considered to embody the essence of life. The thermodynamic view of life system in the early 20th century was remodeled as physics of complex systems and systems biology. The two views contrast with each other, but they are no longer heritage of the historical dualism in biology, such as mechanism/materialism versus vitalism, or reductionism versus holism. These two views are both materialistic and mechanistic, and act as driving forces of modern biology. In reality, molecules function in a context of systems, whereas systems presuppose functional molecules. A key notion to reconcile this conflict is that subjects of biological studies are given before we start to study them. Cell- or organism-level biology is destined to the dialectic of molecules and systems, but this antagonism can be resolved by dynamic thinking involving biological evolution. Copyright © 2018 Elsevier B.V. All rights reserved.

miRSponge: a manually curated database for experimentally supported miRNA sponges and ceRNAs.

Science.gov (United States)

Wang, Peng; Zhi, Hui; Zhang, Yunpeng; Liu, Yue; Zhang, Jizhou; Gao, Yue; Guo, Maoni; Ning, Shangwei; Li, Xia

2015-01-01

In this study, we describe miRSponge, a manually curated database, which aims at providing an experimentally supported resource for microRNA (miRNA) sponges. Recent evidence suggests that miRNAs are themselves regulated by competing endogenous RNAs (ceRNAs) or 'miRNA sponges' that contain miRNA binding sites. These competitive molecules can sequester miRNAs to prevent them interacting with their natural targets to play critical roles in various biological and pathological processes. It has become increasingly important to develop a high quality database to record and store ceRNA data to support future studies. To this end, we have established the experimentally supported miRSponge database that contains data on 599 miRNA-sponge interactions and 463 ceRNA relationships from 11 species following manual curating from nearly 1200 published articles. Database classes include endogenously generated molecules including coding genes, pseudogenes, long non-coding RNAs and circular RNAs, along with exogenously introduced molecules including viral RNAs and artificial engineered sponges. Approximately 70% of the interactions were identified experimentally in disease states. miRSponge provides a user-friendly interface for convenient browsing, retrieval and downloading of dataset. A submission page is also included to allow researchers to submit newly validated miRNA sponge data. Database URL: http://www.bio-bigdata.net/miRSponge. © The Author(s) 2015. Published by Oxford University Press.

Switching off small RNA regulation with trap-mRNA

DEFF Research Database (Denmark)

Overgaard, Martin; Johansen, Jesper; Møller-Jensen, Jakob

2009-01-01

to operate at the level of transcription initiation. By employing a highly sensitive genetic screen we uncovered a novel RNA-based regulatory principle in which induction of a trap-mRNA leads to selective degradation of a small regulatory RNA molecule, thereby abolishing the sRNA-based silencing of its...

Nanomechanical microcantilever operated in vibration modes with use of RNA aptamer as receptor molecules for label-free detection of HCV helicase.

Science.gov (United States)

Hwang, Kyo Seon; Lee, Sang-Myung; Eom, Kilho; Lee, Jeong Hoon; Lee, Yoon-Sik; Park, Jung Ho; Yoon, Dae Sung; Kim, Tae Song

2007-11-30

We report the nanomechanical microcantilevers operated in vibration modes (oscillation) with use of RNA aptamers as receptor molecules for label-free detection of hepatitis C virus (HCV) helicase. The nanomechanical detection principle is that the ligand-receptor binding on the microcantilever surface induces the dynamic response change of microcantilevers. We implemented the label-free detection of HCV helicase in the low concentration as much as 100 pg/ml from measuring the dynamic response change of microcantilevers. Moreover, from the recent studies showing that the ligand-receptor binding generates the surface stress on the microcantilever, we estimate the surface stress, on the oscillating microcantilevers, induced by ligand-receptor binding, i.e. binding between HCV helicase and RNA aptamer. In this article, it is suggested that the oscillating microcantilevers with use of RNA aptamers as receptor molecules may enable one to implement the sensitive label-free detection of very small amount of small-scale proteins.

Development of a new method to identify aminoacylated RNA

Directory of Open Access Journals (Sweden)

Wang Ji

2014-02-01

Full Text Available A RT-PCR method is developed to isolate RNA aminoacylated on their 3’ end from large pools of RNA. The method is being applied in two separate projects. We are interested in isolating a new class of ribozymes that could successively catalyze the two chemical reactions leading to their own 3’ aminoacylation (ATP activation of an amino acid followed by 3' esterification of the RNA. The catalysis of each of the two reactions has independently been demonstrated for some RNA isolated with the SELEX methodology [1-2]. However, the coupling of both reactions on a same molecule has not been achieved yet. The identification of these still hypothetical ribozymes may help understand how the former translation system started in the absence of the aminoacyltRNA Synthetase, which catalyzes the above two reactions on tRNA in modern cells. In another project, we would like to identify the whole repertoire of aminoacylated RNA (the “aminoacylome” in cells. There are strong indications that other RNA besides tRNA and tmRNA may be aminoacylated for biological purposes [3-4].

Rapid PCR-mediated synthesis of competitor molecules for accurate quantification of beta(2) GABA(A) receptor subunit mRNA.

Science.gov (United States)

Vela, J; Vitorica, J; Ruano, D

2001-12-01

We describe a fast and easy method for the synthesis of competitor molecules based on non-specific conditions of PCR. RT-competitive PCR is a sensitive technique that allows quantification of very low quantities of mRNA molecules in small tissue samples. This technique is based on the competition established between the native and standard templates for nucleotides, primers or other factors during PCR. Thus, the most critical parameter is the use of good internal standards to generate a standard curve from which the amount of native sequences can be properly estimated. At the present time different types of internal standards and methods for their synthesis have been described. Normally, most of these methods are time-consuming and require the use of different sets of primers, different rounds of PCR or specific modifications, such as site-directed mutagenesis, that need subsequent analysis of the PCR products. Using our method, we obtained in a single round of PCR and with the same primer pair, competitor molecules that were successfully used in RT-competitive PCR experiments. The principal advantage of this method is high versatility and economy. Theoretically it is possible to synthesize a specific competitor molecule for each primer pair used. Finally, using this method we have been able to quantify the increase in the expression of the beta(2) GABA(A) receptor subunit mRNA that occurs during rat hippocampus development.

Regulation of drug-metabolizing enzymes in infectious and inflammatory disease: implications for biologics-small molecule drug interactions.

Science.gov (United States)

Mallick, Pankajini; Taneja, Guncha; Moorthy, Bhagavatula; Ghose, Romi

2017-06-01

Drug-metabolizing enzymes (DMEs) are primarily down-regulated during infectious and inflammatory diseases, leading to disruption in the metabolism of small molecule drugs (smds), which are increasingly being prescribed therapeutically in combination with biologics for a number of chronic diseases. The biologics may exert pro- or anti-inflammatory effect, which may in turn affect the expression/activity of DMEs. Thus, patients with infectious/inflammatory diseases undergoing biologic/smd treatment can have complex changes in DMEs due to combined effects of the disease and treatment. Areas covered: We will discuss clinical biologics-SMD interaction and regulation of DMEs during infection and inflammatory diseases. Mechanistic studies will be discussed and consequences on biologic-small molecule combination therapy on disease outcome due to changes in drug metabolism will be highlighted. Expert opinion: The involvement of immunomodulatory mediators in biologic-SMDs is well known. Regulatory guidelines recommend appropriate in vitro or in vivo assessments for possible interactions. The role of cytokines in biologic-SMDs has been documented. However, the mechanisms of drug-drug interactions is much more complex, and is probably multi-factorial. Studies aimed at understanding the mechanism by which biologics effect the DMEs during inflammation/infection are clinically important.

The Non-Coding RNA Ontology (NCRO): a comprehensive resource for the unification of non-coding RNA biology.

Science.gov (United States)

Huang, Jingshan; Eilbeck, Karen; Smith, Barry; Blake, Judith A; Dou, Dejing; Huang, Weili; Natale, Darren A; Ruttenberg, Alan; Huan, Jun; Zimmermann, Michael T; Jiang, Guoqian; Lin, Yu; Wu, Bin; Strachan, Harrison J; He, Yongqun; Zhang, Shaojie; Wang, Xiaowei; Liu, Zixing; Borchert, Glen M; Tan, Ming

2016-01-01

In recent years, sequencing technologies have enabled the identification of a wide range of non-coding RNAs (ncRNAs). Unfortunately, annotation and integration of ncRNA data has lagged behind their identification. Given the large quantity of information being obtained in this area, there emerges an urgent need to integrate what is being discovered by a broad range of relevant communities. To this end, the Non-Coding RNA Ontology (NCRO) is being developed to provide a systematically structured and precisely defined controlled vocabulary for the domain of ncRNAs, thereby facilitating the discovery, curation, analysis, exchange, and reasoning of data about structures of ncRNAs, their molecular and cellular functions, and their impacts upon phenotypes. The goal of NCRO is to serve as a common resource for annotations of diverse research in a way that will significantly enhance integrative and comparative analysis of the myriad resources currently housed in disparate sources. It is our belief that the NCRO ontology can perform an important role in the comprehensive unification of ncRNA biology and, indeed, fill a critical gap in both the Open Biological and Biomedical Ontologies (OBO) Library and the National Center for Biomedical Ontology (NCBO) BioPortal. Our initial focus is on the ontological representation of small regulatory ncRNAs, which we see as the first step in providing a resource for the annotation of data about all forms of ncRNAs. The NCRO ontology is free and open to all users, accessible at: http://purl.obolibrary.org/obo/ncro.owl.

Computer-Aided Design of RNA Origami Structures.

Science.gov (United States)

Sparvath, Steffen L; Geary, Cody W; Andersen, Ebbe S

2017-01-01

RNA nanostructures can be used as scaffolds to organize, combine, and control molecular functionalities, with great potential for applications in nanomedicine and synthetic biology. The single-stranded RNA origami method allows RNA nanostructures to be folded as they are transcribed by the RNA polymerase. RNA origami structures provide a stable framework that can be decorated with functional RNA elements such as riboswitches, ribozymes, interaction sites, and aptamers for binding small molecules or protein targets. The rich library of RNA structural and functional elements combined with the possibility to attach proteins through aptamer-based binding creates virtually limitless possibilities for constructing advanced RNA-based nanodevices.In this chapter we provide a detailed protocol for the single-stranded RNA origami design method using a simple 2-helix tall structure as an example. The first step involves 3D modeling of a double-crossover between two RNA double helices, followed by decoration with tertiary motifs. The second step deals with the construction of a 2D blueprint describing the secondary structure and sequence constraints that serves as the input for computer programs. In the third step, computer programs are used to design RNA sequences that are compatible with the structure, and the resulting outputs are evaluated and converted into DNA sequences to order.

Combinatorics of RNA-RNA interaction

DEFF Research Database (Denmark)

Li, Thomas J X; Reidys, Christian

2012-01-01

RNA-RNA binding is an important phenomenon observed for many classes of non-coding RNAs and plays a crucial role in a number of regulatory processes. Recently several MFE folding algorithms for predicting the joint structure of two interacting RNA molecules have been proposed. Here joint structure...... means that in a diagram representation the intramolecular bonds of each partner are pseudoknot-free, that the intermolecular binding pairs are noncrossing, and that there is no so-called "zigzag" configuration. This paper presents the combinatorics of RNA interaction structures including...

NOBAI: a web server for character coding of geometrical and statistical features in RNA structure

Science.gov (United States)

Knudsen, Vegeir; Caetano-Anollés, Gustavo

2008-01-01

The Numeration of Objects in Biology: Alignment Inferences (NOBAI) web server provides a web interface to the applications in the NOBAI software package. This software codes topological and thermodynamic information related to the secondary structure of RNA molecules as multi-state phylogenetic characters, builds character matrices directly in NEXUS format and provides sequence randomization options. The web server is an effective tool that facilitates the search for evolutionary history embedded in the structure of functional RNA molecules. The NOBAI web server is accessible at ‘http://www.manet.uiuc.edu/nobai/nobai.php’. This web site is free and open to all users and there is no login requirement. PMID:18448469

Disregarded Effect of Biological Fluids in siRNA Delivery: Human Ascites Fluid Severely Restricts Cellular Uptake of Nanoparticles.

Science.gov (United States)

Dakwar, George R; Braeckmans, Kevin; Demeester, Joseph; Ceelen, Wim; De Smedt, Stefaan C; Remaut, Katrien

2015-11-04

Small interfering RNA (siRNA) offers a great potential for the treatment of various diseases and disorders. Nevertheless, inefficient in vivo siRNA delivery hampers its translation into the clinic. While numerous successful in vitro siRNA delivery stories exist in reduced-protein conditions, most studies so far overlook the influence of the biological fluids present in the in vivo environment. In this study, we compared the transfection efficiency of liposomal formulations in Opti-MEM (low protein content, routinely used for in vitro screening) and human undiluted ascites fluid obtained from a peritoneal carcinomatosis patient (high protein content, representing the in vivo situation). In Opti-MEM, all formulations are biologically active. In ascites fluid, however, the biological activity of all lipoplexes is lost except for lipofectamine RNAiMAX. The drop in transfection efficiency was not correlated to the physicochemical properties of the nanoparticles, such as premature siRNA release and aggregation of the nanoparticles in the human ascites fluid. Remarkably, however, all of the formulations except for lipofectamine RNAiMAX lost their ability to be taken up by cells following incubation in ascites fluid. To take into account the possible effects of a protein corona formed around the nanoparticles, we recommend always using undiluted biological fluids for the in vitro optimization of nanosized siRNA formulations next to conventional screening in low-protein content media. This should tighten the gap between in vitro and in vivo performance of nanoparticles and ensure the optimal selection of nanoparticles for further in vivo studies.

A new method to study the change of miRNA-mRNA interactions due to environmental exposures.

Science.gov (United States)

Petralia, Francesca; Aushev, Vasily N; Gopalakrishnan, Kalpana; Kappil, Maya; W Khin, Nyan; Chen, Jia; Teitelbaum, Susan L; Wang, Pei

2017-07-15

Integrative approaches characterizing the interactions among different types of biological molecules have been demonstrated to be useful for revealing informative biological mechanisms. One such example is the interaction between microRNA (miRNA) and messenger RNA (mRNA), whose deregulation may be sensitive to environmental insult leading to altered phenotypes. The goal of this work is to develop an effective data integration method to characterize deregulation between miRNA and mRNA due to environmental toxicant exposures. We will use data from an animal experiment designed to investigate the effect of low-dose environmental chemical exposure on normal mammary gland development in rats to motivate and evaluate the proposed method. We propose a new network approach-integrative Joint Random Forest (iJRF), which characterizes the regulatory system between miRNAs and mRNAs using a network model. iJRF is designed to work under the high-dimension low-sample-size regime, and can borrow information across different treatment conditions to achieve more accurate network inference. It also effectively takes into account prior information of miRNA-mRNA regulatory relationships from existing databases. When iJRF is applied to the data from the environmental chemical exposure study, we detected a few important miRNAs that regulated a large number of mRNAs in the control group but not in the exposed groups, suggesting the disruption of miRNA activity due to chemical exposure. Effects of chemical exposure on two affected miRNAs were further validated using breast cancer human cell lines. R package iJRF is available at CRAN. pei.wang@mssm.edu or susan.teitelbaum@mssm.edu. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Study of radionuclides speciation with biological molecules of interest by spectrometric techniques

International Nuclear Information System (INIS)

Lourenco, V.

2007-07-01

Mechanisms of complexation and accumulation of the radionuclides at the cellular and molecular level are complex and poorly known because the studies on these subjects are scarce. Within the framework of this thesis, we studied the interactions of europium (analogue of trivalent actinides) and uranium (VI) (actinide) with biological molecules of interest: phyto-chelatins. Their role is to protect cells against intrusions from nonessential heavy metals (thus toxic). These proteins are likely to be implied in the mechanisms of sequestration of radionuclides in living organisms. However, their structure is complex, this is why, in order to better understand their reactivity, we extended our studies to lower entities which constitute them (amino acids and glutathione). We determined solution speciation (stoichiometry, structure) as well as the complexing constants associated with the formation of these species. These studies were undertaken by Time Resolved Laser induced Fluorescence (TRLIF), Electro-Spray Mass Spectrometry (ES-MS), Nuclear Magnetic Resonance (NMR), Fourier Transform Infra-Rouge spectroscopy (FTIR) and Extended X-ray Absorption Fine Structure Spectroscopy (EXAFS). The determination of the complexation constants enabled us to conclude that the complexing capacity of these molecules with respect to radionuclides was moderate (log 10 K 1 < 3, pH 3 or 6), the formed species are mononuclear with only one ligand molecule (1:1). The interaction is performed via oxygenated (hard) groups. The direct complexation of europium with phyto-chelatins at acidic pH was studied jointly by TRLIF and ES-MS. The complexing capacity of these molecules is much higher than that of GSH from which they result. In addition to studies undertaken on synthetic solutions reproducing the 'biological' conditions (pH close to neutrality, ionic strength 0.1 mol/L, etc), tests of cellular contamination were realized. The quantification of integrated europium showed that those are able to

Non-coding RNA networks in cancer.

Science.gov (United States)

Anastasiadou, Eleni; Jacob, Leni S; Slack, Frank J

2018-01-01

Thousands of unique non-coding RNA (ncRNA) sequences exist within cells. Work from the past decade has altered our perception of ncRNAs from 'junk' transcriptional products to functional regulatory molecules that mediate cellular processes including chromatin remodelling, transcription, post-transcriptional modifications and signal transduction. The networks in which ncRNAs engage can influence numerous molecular targets to drive specific cell biological responses and fates. Consequently, ncRNAs act as key regulators of physiological programmes in developmental and disease contexts. Particularly relevant in cancer, ncRNAs have been identified as oncogenic drivers and tumour suppressors in every major cancer type. Thus, a deeper understanding of the complex networks of interactions that ncRNAs coordinate would provide a unique opportunity to design better therapeutic interventions.

Computational prediction of miRNA genes from small RNA sequencing data

Directory of Open Access Journals (Sweden)

Wenjing eKang

2015-01-01

Full Text Available Next-generation sequencing now for the first time allows researchers to gauge the depth and variation of entire transcriptomes. However, now as rare transcripts can be detected that are present in cells at single copies, more advanced computational tools are needed to accurately annotate and profile them. miRNAs are 22 nucleotide small RNAs (sRNAs that post-transcriptionally reduce the output of protein coding genes. They have established roles in numerous biological processes, including cancers and other diseases. During miRNA biogenesis, the sRNAs are sequentially cleaved from precursor molecules that have a characteristic hairpin RNA structure. The vast majority of new miRNA genes that are discovered are mined from small RNA sequencing (sRNA-seq, which can detect more than a billion RNAs in a single run. However, given that many of the detected RNAs are degradation products from all types of transcripts, the accurate identification of miRNAs remain a non-trivial computational problem. Here we review the tools available to predict animal miRNAs from sRNA sequencing data. We present tools for generalist and specialist use cases, including prediction from massively pooled data or in species without reference genome. We also present wet-lab methods used to validate predicted miRNAs, and approaches to computationally benchmark prediction accuracy. For each tool, we reference validation experiments and benchmarking efforts. Last, we discuss the future of the field.

RNA-based, transient modulation of gene expression in human haematopoietic stem and progenitor cells

Science.gov (United States)

Diener, Yvonne; Jurk, Marion; Kandil, Britta; Choi, Yeong-Hoon; Wild, Stefan; Bissels, Ute; Bosio, Andreas

2015-01-01

Modulation of gene expression is a useful tool to study the biology of haematopoietic stem and progenitor cells (HSPCs) and might also be instrumental to expand these cells for therapeutic approaches. Most of the studies so far have employed stable gene modification by viral vectors that are burdensome when translating protocols into clinical settings. Our study aimed at exploring new ways to transiently modify HSPC gene expression using non-integrating, RNA-based molecules. First, we tested different methods to deliver these molecules into HSPCs. The delivery of siRNAs with chemical transfection methods such as lipofection or cationic polymers did not lead to target knockdown, although we observed more than 90% fluorescent cells using a fluorochrome-coupled siRNA. Confocal microscopic analysis revealed that despite extensive washing, siRNA stuck to or in the cell surface, thereby mimicking a transfection event. In contrast, electroporation resulted in efficient, siRNA-mediated protein knockdown. For transient overexpression of proteins, we used optimised mRNA molecules with modified 5′- and 3′-UTRs. Electroporation of mRNA encoding GFP resulted in fast, efficient and persistent protein expression for at least seven days. Our data provide a broad-ranging comparison of transfection methods for hard-to-transfect cells and offer new opportunities for DNA-free, non-integrating gene modulation in HSPCs. PMID:26599627

Biomarker MicroRNAs for Diagnosis of Oral Squamous Cell Carcinoma Identified Based on Gene Expression Data and MicroRNA-mRNA Network Analysis

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Zhang, Hui; Li, Tangxin; Zheng, Linqing

2017-01-01

Oral squamous cell carcinoma is one of the most malignant tumors with high mortality rate worldwide. Biomarker discovery is critical for early diagnosis and precision treatment of this disease. MicroRNAs are small noncoding RNA molecules which often regulate essential biological processes and are good candidates for biomarkers. By integrative analysis of both the cancer-associated gene expression data and microRNA-mRNA network, miR-148b-3p, miR-629-3p, miR-27a-3p, and miR-142-3p were screened as novel diagnostic biomarkers for oral squamous cell carcinoma based on their unique regulatory abilities in the network structure of the conditional microRNA-mRNA network and their important functions. These findings were confirmed by literature verification and functional enrichment analysis. Future experimental validation is expected for the further investigation of their molecular mechanisms. PMID:29098014

The MicroRNA Interaction Network of Lipid Diseases

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Kandhro, Abdul H.; Shoombuatong, Watshara; Nantasenamat, Chanin; Prachayasittikul, Virapong; Nuchnoi, Pornlada

2017-01-01

Background: Dyslipidemia is one of the major forms of lipid disorder, characterized by increased triglycerides (TGs), increased low-density lipoprotein-cholesterol (LDL-C), and decreased high-density lipoprotein-cholesterol (HDL-C) levels in blood. Recently, MicroRNAs (miRNAs) have been reported to involve in various biological processes; their potential usage being a biomarkers and in diagnosis of various diseases. Computational approaches including text mining have been used recently to analyze abstracts from the public databases to observe the relationships/associations between the biological molecules, miRNAs, and disease phenotypes. Materials and Methods: In the present study, significance of text mined extracted pair associations (miRNA-lipid disease) were estimated by one-sided Fisher's exact test. The top 20 significant miRNA-disease associations were visualized on Cytoscape. The CyTargetLinker plug-in tool on Cytoscape was used to extend the network and predicts new miRNA target genes. The Biological Networks Gene Ontology (BiNGO) plug-in tool on Cytoscape was used to retrieve gene ontology (GO) annotations for the targeted genes. Results: We retrieved 227 miRNA-lipid disease associations including 148 miRNAs. The top 20 significant miRNAs analysis on CyTargetLinker provides defined, predicted and validated gene targets, further targeted genes analyzed by BiNGO showed targeted genes were significantly associated with lipid, cholesterol, apolipoprotein, and fatty acids GO terms. Conclusion: We are the first to provide a reliable miRNA-lipid disease association network based on text mining. This could help future experimental studies that aim to validate predicted gene targets. PMID:29018475

The Juxtaposition of Ribose Hydroxyl Groups: The Root of Biological Catalysis and the RNA World?

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Bernhardt, Harold S.

2015-06-01

We normally think of enzymes as being proteins; however, the RNA world hypothesis suggests that the earliest biological catalysts may have been composed of RNA. One of the oldest surviving RNA enzymes we are aware of is the peptidyl transferase centre (PTC) of the large ribosomal RNA, which joins amino acids together to form proteins. Recent evidence indicates that the enzymatic activity of the PTC is principally due to ribose 2 '-OHs. Many other reactions catalyzed by RNA and/or in which RNA is a substrate similarly utilize ribose 2 '-OHs, including phosphoryl transfer reactions that involve the cleavage and/or ligation of the ribose-phosphate backbone. It has recently been proposed by Yakhnin (2013) that phosphoryl transfer reactions were important in the prebiotic chemical evolution of RNA, by enabling macromolecules composed of polyols joined by phosphodiester linkages to undergo recombination reactions, with the reaction energy supplied by the phosphodiester bond itself. The almost unique juxtaposition of the ribose 2'-hydroxyl and 3'-oxygen in ribose-containing polymers such as RNA, which gives ribose the ability to catalyze such reactions, may have been an important factor in the selection of ribose as a component of the first biopolymer. In addition, the juxtaposition of hydroxyl groups in free ribose: (i) allows coordination of borate ions, which could have provided significant and preferential stabilization of ribose in a prebiotic environment; and (ii) enhances the rate of permeation by ribose into a variety of lipid membrane systems, possibly favouring its incorporation into early metabolic pathways and an ancestral ribose-phosphate polymer. Somewhat more speculatively, hydrogen bonds formed by juxtaposed ribose hydroxyl groups may have stabilized an ancestral ribose-phosphate polymer against degradation (Bernhardt and Sandwick 2014). I propose that the almost unique juxtaposition of ribose hydroxyl groups constitutes the root of both biological

The microRNA-132/212 family fine-tunes multiple targets in Angiotensin II signalling in cardiac fibroblasts

DEFF Research Database (Denmark)

Eskildsen, Tilde V; Schneider, Mikael; Sandberg, Maria B

2015-01-01

INTRODUCTION: MicroRNAs (miRNAs) are emerging as key regulators of cardiovascular development and disease; however, the cardiac miRNA target molecules are not well understood. We and others have described the Angiotensin II (AngII)-induced miR-132/212 family as novel regulators of cardiovascular...... in silico and in vitro experiments to identify miR-132/212 molecular targets in primary rat cardiac fibroblasts. RESULTS: MiR-132/212 overexpression increased fibroblast cell size and mRNA arrays detected several hundred genes that were differentially expressed, including a wide panel of receptors...... pathways that fine-tuned by miR-132/212, suggesting a role for this miRNA family as master signalling switches in cardiac fibroblasts. Our data underscore the potential for miRNA tools to manipulate a large array of molecules and thereby control biological function....

TmiRUSite and TmiROSite scripts: searching for mRNA fragments with miRNA binding sites with encoded amino acid residues.

Science.gov (United States)

Berillo, Olga; Régnier, Mireille; Ivashchenko, Anatoly

2014-01-01

microRNAs are small RNA molecules that inhibit the translation of target genes. microRNA binding sites are located in the untranslated regions as well as in the coding domains. We describe TmiRUSite and TmiROSite scripts developed using python as tools for the extraction of nucleotide sequences for miRNA binding sites with their encoded amino acid residue sequences. The scripts allow for retrieving a set of additional sequences at left and at right from the binding site. The scripts presents all received data in table formats that are easy to analyse further. The predicted data finds utility in molecular and evolutionary biology studies. They find use in studying miRNA binding sites in animals and plants. TmiRUSite and TmiROSite scripts are available for free from authors upon request and at https: //sites.google.com/site/malaheenee/downloads for download.

An RNA Phage Lab: MS2 in Walter Fiers' laboratory of molecular biology in Ghent, from genetic code to gene and genome, 1963-1976.

Science.gov (United States)

Pierrel, Jérôme

2012-01-01

The importance of viruses as model organisms is well-established in molecular biology and Max Delbrück's phage group set standards in the DNA phage field. In this paper, I argue that RNA phages, discovered in the 1960s, were also instrumental in the making of molecular biology. As part of experimental systems, RNA phages stood for messenger RNA (mRNA), genes and genome. RNA was thought to mediate information transfers between DNA and proteins. Furthermore, RNA was more manageable at the bench than DNA due to the availability of specific RNases, enzymes used as chemical tools to analyse RNA. Finally, RNA phages provided scientists with a pure source of mRNA to investigate the genetic code, genes and even a genome sequence. This paper focuses on Walter Fiers' laboratory at Ghent University (Belgium) and their work on the RNA phage MS2. When setting up his Laboratory of Molecular Biology, Fiers planned a comprehensive study of the virus with a strong emphasis on the issue of structure. In his lab, RNA sequencing, now a little-known technique, evolved gradually from a means to solve the genetic code, to a tool for completing the first genome sequence. Thus, I follow the research pathway of Fiers and his 'RNA phage lab' with their evolving experimental system from 1960 to the late 1970s. This study illuminates two decisive shifts in post-war biology: the emergence of molecular biology as a discipline in the 1960s in Europe and of genomics in the 1990s.

Laser desorption/ionization mass spectrometry for direct profiling and imaging of small molecules from raw biological materials

Energy Technology Data Exchange (ETDEWEB)

Cha, Sangwon [Iowa State Univ., Ames, IA (United States)

2008-01-01

Matrix-assisted laser desorption/ionization(MALDI) mass spectrometry(MS) has been widely used for analysis of biological molecules, especially macromolecules such as proteins. However, MALDI MS has a problem in small molecule (less than 1 kDa) analysis because of the signal saturation by organic matrixes in the low mass region. In imaging MS (IMS), inhomogeneous surface formation due to the co-crystallization process by organic MALDI matrixes limits the spatial resolution of the mass spectral image. Therefore, to make laser desorption/ionization (LDI) MS more suitable for mass spectral profiling and imaging of small molecules directly from raw biological tissues, LDI MS protocols with various alternative assisting materials were developed and applied to many biological systems of interest. Colloidal graphite was used as a matrix for IMS of small molecules for the first time and methodologies for analyses of small metabolites in rat brain tissues, fruits, and plant tissues were developed. With rat brain tissues, the signal enhancement for cerebroside species by colloidal graphite was observed and images of cerebrosides were successfully generated by IMS. In addition, separation of isobaric lipid ions was performed by imaging tandem MS. Directly from Arabidopsis flowers, flavonoids were successfully profiled and heterogeneous distribution of flavonoids in petals was observed for the first time by graphite-assisted LDI(GALDI) IMS.

Evolutionary game theory: molecules as players.

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Bohl, Katrin; Hummert, Sabine; Werner, Sarah; Basanta, David; Deutsch, Andreas; Schuster, Stefan; Theissen, Günter; Schroeter, Anja

2014-12-01

In this and an accompanying paper we review the use of game theoretical concepts in cell biology and molecular biology. This review focuses on the subcellular level by considering viruses, genes, and molecules as players. We discuss in which way catalytic RNA can be treated by game theory. Moreover, genes can compete for success in replication and can have different strategies in interactions with other genetic elements. Also transposable elements, or "jumping genes", can act as players because they usually bear different traits or strategies. Viruses compete in the case of co-infecting a host cell. Proteins interact in a game theoretical sense when forming heterodimers. Finally, we describe how the Shapley value can be applied to enzymes in metabolic pathways. We show that game theory can be successfully applied to describe and analyse scenarios at the molecular level resulting in counterintuitive conclusions.

In vitro synthesis of biologically active transcripts of tomato black ring virus satellite RNA.

Science.gov (United States)

Greif, C; Hemmer, O; Demangeat, G; Fritsch, C

1990-04-01

Synthetic transcripts of tomato black ring virus satellite RNA (TBRV satRNA), isolate L, were prepared from cDNA cloned in the Bluescribe transcription vector. Transcripts with 49 (T49L) or two (T2GL) extra nucleotides at their 5' ends and 42 extra nucleotides at their 3' ends were able to induce, but to different extents, the synthesis in vitro of the satRNA-encoded 48K protein. However, when inoculated into Chenopodium quinoa together with TBRV L genomic RNAs, only T2GL was biologically active, in the presence or absence of a 5' cap analogue in the transcription reactions. Analysis of the 5' and 3' termini of the satRNA isolated from plants showed that nonviral extensions were not maintained in the transcript progeny.

[Satellite RNA (RNA3) of tomato black ring virus is found with one of the 2 major RNAs (RNA2) in a new capsid nucleoprotein].

Science.gov (United States)

Doz, B; Dunez, J; Bove, J M

1977-12-19

Tomato Black Ring Virus (TBRV) like other NEPOviruses posseses two nucleoproteins M and B and two major RNAs, RNA1 and RNA2 respectively distributed in B and M. A new nucleoprotein has just been discovered and comprises one molecule of RNA2 associated with one molecule of RNA3. RNA3 is a small RNA of molecular weight 500,000 d considered to be a satellite RNA. Its level appears to depend on the infection stage, local or systemic. RNA3 is able to modify the relative proportions of nucleoproteins M and B and their respective RNAs. The satellite RNA, might be part of the genome and represent a monocistronic mRNA for protein capsid synthesis. However it seems perhaps more tempting to correlate TBRV-RNA3 with satellite RNA5 of certain strains of Cucumber mosaic virus.

Platinum Interference with siRNA Non-seed Regions Fine-Tunes Silencing Capacity

DEFF Research Database (Denmark)

Hedman, Hanna K; Kirpekar, Finn; Elmroth, Sofi K C

2011-01-01

expression, and the other one focused on the function of endogenous miRNAs. In both cases, the active molecule consists of a ∼20-nucleotide-long RNA duplex. In the siRNA case, improved systemic stability is of central interest for its further development toward clinical applications. With respect to mi......RNA processing and function, understanding its influence on mRNA targeting and the silencing ability of individual miRNAs, e.g., under pathological conditions, remains a scientific challenge. In the present study, a model system is presented where the influence of the two clinically used anticancer drugs......, cisplatin and oxaliplatin, on siRNA's silencing capacity has been evaluated. More specifically, siRNAs targeting the 3' UTR region of Wnt-5a mRNA (NM_003352) were constructed, and the biologically active antisense RNA strand was pre-platinated. Platinum adducts were detected and characterized...

Polymerase-free measurement of microRNA-122 with single base specificity using single molecule arrays: Detection of drug-induced liver injury.

Directory of Open Access Journals (Sweden)

David M Rissin

Full Text Available We have developed a single probe method for detecting microRNA from human serum using single molecule arrays, with sequence specificity down to a single base, and without the use of amplification by polymerases. An abasic peptide nucleic acid (PNA probe-containing a reactive amine instead of a nucleotide at a specific position in the sequence-for detecting a microRNA was conjugated to superparamagnetic beads. These beads were incubated with a sample containing microRNA, a biotinylated reactive nucleobase-containing an aldehyde group-that was complementary to the missing base in the probe sequence, and a reducing agent. When a target molecule with an exact match in sequence hybridized to the capture probe, the reactive nucleobase was covalently attached to the backbone of the probe by a dynamic covalent chemical reaction. Single molecules of the biotin-labeled probe were then labeled with streptavidin-β-galactosidase (SβG, the beads were resuspended in a fluorogenic enzyme substrate, loaded into an array of femtoliter wells, and sealed with oil. The array was imaged fluorescently to determine which beads were associated with single enzymes, and the average number of enzymes per bead was determined. The assay had a limit of detection of 500 fM, approximately 500 times more sensitive than a corresponding analog bead-based assay, with target specificity down to a single base mis-match. This assay was used to measure microRNA-122 (miR-122-an established biomarker of liver toxicity-extracted from the serum of patients who had acute liver injury due to acetaminophen, and control healthy patients. All patients with liver injury had higher levels of miR-122 in their serum compared to controls, and the concentrations measured correlated well with those determined using RT-qPCR. This approach allows rapid quantification of circulating microRNA with single-based specificity and a limit of quantification suitable for clinical use.

Single-Molecule View of Small RNA-Guided Target Search and Recognition.

Science.gov (United States)

Globyte, Viktorija; Kim, Sung Hyun; Joo, Chirlmin

2018-05-20

Most everyday processes in life involve a necessity for an entity to locate its target. On a cellular level, many proteins have to find their target to perform their function. From gene-expression regulation to DNA repair to host defense, numerous nucleic acid-interacting proteins use distinct target search mechanisms. Several proteins achieve that with the help of short RNA strands known as guides. This review focuses on single-molecule advances studying the target search and recognition mechanism of Argonaute and CRISPR (clustered regularly interspaced short palindromic repeats) systems. We discuss different steps involved in search and recognition, from the initial complex prearrangement into the target-search competent state to the final proofreading steps. We focus on target search mechanisms that range from weak interactions, to one- and three-dimensional diffusion, to conformational proofreading. We compare the mechanisms of Argonaute and CRISPR with a well-studied target search system, RecA.

RNA modifications by oxidation

DEFF Research Database (Denmark)

Poulsen, Henrik E; Specht, Elisabeth; Broedbaek, Kasper

2012-01-01

to encompass various classes of novel regulatory RNAs, including, e.g., microRNAs. It is well known that DNA is constantly oxidized and repaired by complex genome maintenance mechanisms. Analogously, RNA also undergoes significant oxidation, and there are now convincing data suggesting that oxidation......The past decade has provided exciting insights into a novel class of central (small) RNA molecules intimately involved in gene regulation. Only a small percentage of our DNA is translated into proteins by mRNA, yet 80% or more of the DNA is transcribed into RNA, and this RNA has been found......, and the consequent loss of integrity of RNA, is a mechanism for disease development. Oxidized RNA is found in a large variety of diseases, and interest has been especially devoted to degenerative brain diseases such as Alzheimer disease, in which up to 50-70% of specific mRNA molecules are reported oxidized, whereas...

Comprehensive characterization of lncRNA-mRNA related ceRNA network across 12 major cancers

Science.gov (United States)

Feng, Li; Li, Feng; Sun, Zeguo; Wu, Tan; Shi, Xinrui; Li, Jing; Li, Xia

2016-01-01

Recent studies indicate that long noncoding RNAs (lncRNAs) can act as competing endogenous RNAs (ceRNAs) to indirectly regulate mRNAs through shared microRNAs, which represents a novel layer of RNA crosstalk and plays critical roles in the development of tumor. However, the global regulation landscape and characterization of these lncRNA related ceRNA crosstalk in cancers is still largely unknown. Here, we systematically characterized the lncRNA related ceRNA interactions across 12 major cancers and the normal physiological states by integrating multidimensional molecule profiles of more than 5000 samples. Our study suggest the large difference of ceRNA regulation between normal and tumor states and the higher similarity across similar tissue origin of tumors. The ceRNA related molecules have more conserved features in tumor networks and they play critical roles in both the normal and tumorigenesis processes. Besides, lncRNAs in the pan-cancer ceRNA network may be potential biomarkers of tumor. By exploring hub lncRNAs, we found that these conserved key lncRNAs dominate variable tumor hallmark processes across pan-cancers. Network dynamic analysis highlights the critical roles of ceRNA regulation in tumorigenesis. By analyzing conserved ceRNA interactions, we found that miRNA mediate ceRNA regulation showed different patterns across pan-cancer; while analyzing the cancer specific ceRNA interactions reveal that lncRNAs synergistically regulated tumor driver genes of cancer hallmarks. Finally, we found that ceRNA modules have the potential to predict patient survival. Overall, our study systematically dissected the lncRNA related ceRNA networks in pan-cancer that shed new light on understanding the molecular mechanism of tumorigenesis. PMID:27580177

New Insights into the Biological Role of Mammalian ADARs; the RNA Editing Proteins

Directory of Open Access Journals (Sweden)

Niamh Mannion

2015-09-01

Full Text Available The ADAR proteins deaminate adenosine to inosine in double-stranded RNA which is one of the most abundant modifications present in mammalian RNA. Inosine can have a profound effect on the RNAs that are edited, not only changing the base-pairing properties, but can also result in recoding, as inosine behaves as if it were guanosine. In mammals there are three ADAR proteins and two ADAR-related proteins (ADAD expressed. All have a very similar modular structure; however, both their expression and biological function differ significantly. Only two of the ADAR proteins have enzymatic activity. However, both ADAR and ADAD proteins possess the ability to bind double-strand RNA. Mutations in ADARs have been associated with many diseases ranging from cancer, innate immunity to neurological disorders. Here, we will discuss in detail the domain structure of mammalian ADARs, the effects of RNA editing, and the role of ADARs in human diseases.

New Insights into the Biological Role of Mammalian ADARs; the RNA Editing Proteins

Science.gov (United States)

Mannion, Niamh; Arieti, Fabiana; Gallo, Angela; Keegan, Liam P.; O’Connell, Mary A.

2015-01-01

The ADAR proteins deaminate adenosine to inosine in double-stranded RNA which is one of the most abundant modifications present in mammalian RNA. Inosine can have a profound effect on the RNAs that are edited, not only changing the base-pairing properties, but can also result in recoding, as inosine behaves as if it were guanosine. In mammals there are three ADAR proteins and two ADAR-related proteins (ADAD) expressed. All have a very similar modular structure; however, both their expression and biological function differ significantly. Only two of the ADAR proteins have enzymatic activity. However, both ADAR and ADAD proteins possess the ability to bind double-strand RNA. Mutations in ADARs have been associated with many diseases ranging from cancer, innate immunity to neurological disorders. Here, we will discuss in detail the domain structure of mammalian ADARs, the effects of RNA editing, and the role of ADARs in human diseases. PMID:26437436

Sequence analysis of RNase MRP RNA reveals its origination from eukaryotic RNase P RNA

Science.gov (United States)

Zhu, Yanglong; Stribinskis, Vilius; Ramos, Kenneth S.; Li, Yong

2006-01-01

RNase MRP is a eukaryote-specific endoribonuclease that generates RNA primers for mitochondrial DNA replication and processes precursor rRNA. RNase P is a ubiquitous endoribonuclease that cleaves precursor tRNA transcripts to produce their mature 5′ termini. We found extensive sequence homology of catalytic domains and specificity domains between their RNA subunits in many organisms. In Candida glabrata, the internal loop of helix P3 is 100% conserved between MRP and P RNAs. The helix P8 of MRP RNA from microsporidia Encephalitozoon cuniculi is identical to that of P RNA. Sequence homology can be widely spread over the whole molecule of MRP RNA and P RNA, such as those from Dictyostelium discoideum. These conserved nucleotides between the MRP and P RNAs strongly support the hypothesis that the MRP RNA is derived from the P RNA molecule in early eukaryote evolution. PMID:16540690

Single-molecule fluorescence measurements reveal the reaction mechanisms of the core RISC, composed of human Argonaute 2 and a guide RNA.

Science.gov (United States)

Jo, Myung Hyun; Song, Ji-Joon; Hohng, Sungchul

2015-12-01

In eukaryotes, small RNAs play important roles in both gene regulation and resistance to viral infection. Argonaute proteins have been identified as a key component of the effector complexes of various RNA-silencing pathways, but the mechanistic roles of Argonaute proteins in these pathways are not clearly understood. To address this question, we performed single-molecule fluorescence experiments using an RNA-induced silencing complex (core-RISC) composed of a small RNA and human Argonaute 2. We found that target binding of core-RISC starts at the seed region of the guide RNA. After target binding, four distinct reactions followed: target cleavage, transient binding, stable binding, and Argonaute unloading. Target cleavage required extensive sequence complementarity and accelerated core-RISC dissociation for recycling. In contrast, the stable binding of core-RISC to target RNAs required seed-match only, suggesting a potential explanation for the seed-match rule of microRNA (miRNA) target selection.

Synthetic biology: engineering molecular computers

CERN Multimedia

CERN. Geneva

2018-01-01

Complicated systems cannot survive the rigors of a chaotic environment, without balancing mechanisms that sense, decide upon and counteract the exerted disturbances. Especially so with living organisms, forced by competition to incredible complexities, escalating also their self-controlling plight. Therefore, they compute. Can we harness biological mechanisms to create artificial computing systems? Biology offers several levels of design abstraction: molecular machines, cells, organisms... ranging from the more easily-defined to the more inherently complex. At the bottom of this stack we find the nucleic acids, RNA and DNA, with their digital structure and relatively precise interactions. They are central enablers of designing artificial biological systems, in the confluence of engineering and biology, that we call Synthetic biology. In the first part, let us follow their trail towards an overview of building computing machines with molecules -- and in the second part, take the case study of iGEM Greece 201...

Small Molecule Modulator of p53 Signaling Pathway: Application for Radiosensitizing or Radioprotection Agents

International Nuclear Information System (INIS)

Oh, Sang Taek; Cho, Mun Ju; Gwak, Jung Sug; Ryu, Min Jung; Song, Jie Young; Yun, Yeon Sook

2009-01-01

The tumor suppressor p53 is key molecule to protect the cell against genotoxic stress and..the most frequently mutated..protein..in cancer cells. Lack of functional p53..is accompanied by high rate of genomic instability, rapid tumor progression, resistance to anticancer therapy, and increased angiogenesis. In response to DNA damage, p53 protein rapidly accumulated through attenuated proteolysis and is also activated as transcription factor. Activated p53 up-regulates target genes involved in cell cycle arrest and/or apoptosis and then lead to suppression of malignant transformation and the maintenance of genomic integrity. Chemical genetics is a new technology to uncover the signaling networks that regulated biological phenotype using exogenous reagents such as small molecules. Analogous to classical forward genetic screens in model organism, this approach makes use of high throughput, phenotypic assay to identify small molecules that disrupt gene product function in a way that alters a phenotype of interest. Recently, interesting small molecules were identified from cell based high throughput screening and its target protein or mechanism of action were identified by various methods including affinity chromatography, protein array profiling, mRNA or phage display, transcription profiling, and RNA interference

Small Molecule Modulator of p53 Signaling Pathway: Application for Radiosensitizing or Radioprotection Agents

Energy Technology Data Exchange (ETDEWEB)

Oh, Sang Taek; Cho, Mun Ju; Gwak, Jung Sug; Ryu, Min Jung [PharmacoGenomics Research Center, Inje University, Busan (Korea, Republic of); Song, Jie Young; Yun, Yeon Sook [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

2009-05-15

The tumor suppressor p53 is key molecule to protect the cell against genotoxic stress and..the most frequently mutated..protein..in cancer cells. Lack of functional p53..is accompanied by high rate of genomic instability, rapid tumor progression, resistance to anticancer therapy, and increased angiogenesis. In response to DNA damage, p53 protein rapidly accumulated through attenuated proteolysis and is also activated as transcription factor. Activated p53 up-regulates target genes involved in cell cycle arrest and/or apoptosis and then lead to suppression of malignant transformation and the maintenance of genomic integrity. Chemical genetics is a new technology to uncover the signaling networks that regulated biological phenotype using exogenous reagents such as small molecules. Analogous to classical forward genetic screens in model organism, this approach makes use of high throughput, phenotypic assay to identify small molecules that disrupt gene product function in a way that alters a phenotype of interest. Recently, interesting small molecules were identified from cell based high throughput screening and its target protein or mechanism of action were identified by various methods including affinity chromatography, protein array profiling, mRNA or phage display, transcription profiling, and RNA interference.

Targeting MicroRNA Function in Respiratory Diseases: Mini-review

Directory of Open Access Journals (Sweden)

Steven eMaltby

2016-02-01

Full Text Available MicroRNAs (miRNAs are small non-coding RNA molecules that modulate expression of the majority of genes by inhibiting protein translation. Growing literature has identified functional roles for miRNAs across a broad range of biological processes. As such, miRNAs are recognised as potential disease biomarkers and novel targets for therapies. While several miRNA-targeted therapies are currently in clinical trials (e.g. for the treatment of hepatitis C virus infection and cancer, no therapies have targeted miRNAs in respiratory diseases in the clinic. In this mini-review, we review the current knowledge on miRNA expression and function in respiratory diseases, intervention strategies to target miRNA function and considerations specific to respiratory diseases. Altered miRNA expression profiles have been reported in a number of respiratory diseases, including asthma, chronic obstructive pulmonary disease, cystic fibrosis and idiopathic pulmonary fibrosis. These include alterations in isolated lung tissue, as well as sputum, bronchoalveolar lavage fluids and peripheral blood or serum. The observed alterations in easily accessible body fluids (e.g. serum have been proposed as new biomarkers that may inform disease diagnosis and patient management. In a subset of studies, miRNA-targeted interventions also improved disease outcomes, indicating functional roles for altered miRNA expression in disease pathogenesis. In fact, direct administration of miRNA-targeting molecules to the lung has yielded promising results in a number of animal models. The ability to directly administer compounds to the lung holds considerable promise and may limit potential off-target effects and side effects caused by the systemic administration required to treat other diseases.

Direct, rapid RNA sequence analysis

International Nuclear Information System (INIS)

Peattie, D.A.

1987-01-01

The original methods of RNA sequence analysis were based on enzymatic production and chromatographic separation of overlapping oligonucleotide fragments from within an RNA molecule followed by identification of the mononucleotides comprising the oligomer. Over the past decade the field of nucleic acid sequencing has changed dramatically, however, and RNA molecules now can be sequenced in a variety of more streamlined fashions. Most of the more recent advances in RNA sequencing have involved one-dimensional electrophoretic separation of 32 P-end-labeled oligoribonucleotides on polyacrylamide gels. In this chapter the author discusses two of these methods for determining the nucleotide sequences of RNA molecules rapidly: the chemical method and the enzymatic method. Both methods are direct and degradative, i.e., they rely on fragmatic and chemical approaches should be utilized. The single-strand-specific ribonucleases (A, T 1 , T 2 , and S 1 ) provide an efficient means to locate double-helical regions rapidly, and the chemical reactions provide a means to determine the RNA sequence within these regions. In addition, the chemical reactions allow one to assign interactions to specific atoms and to distinguish secondary interactions from tertiary ones. If the RNA molecule is small enough to be sequenced directly by the enzymatic or chemical method, the probing reactions can be done easily at the same time as sequencing reactions

Ab Initio Calculations of the Electronic Structures and Biological Functions of Protein Molecules

Science.gov (United States)

Zheng, Haoping

2003-04-01

The self-consistent cluster-embedding (SCCE) calculation method reduces the computational effort from M3 to about M1 (M is the number of atoms in the system) with unchanged calculation precision. So the ab initio, all-electron calculation of the electronic structure and biological function of protein molecule becomes a reality, which will promote new proteomics considerably. The calculated results of two real protein molecules, the trypsin inhibitor from the seeds of squash Cucurbita maxima (CMTI-I, 436 atoms) and the Ascaris trypsin inhibitor (912 atoms, two three-dimensional structures), are presented. The reactive sites of the inhibitors are determined and explained. The precision of structure determination of inhibitors are tested theoretically.

Biological significance of 5S rRNA import into human mitochondria: role of ribosomal protein MRP-L18

Science.gov (United States)

Smirnov, Alexandre; Entelis, Nina; Martin, Robert P.; Tarassov, Ivan

2011-01-01

5S rRNA is an essential component of ribosomes of all living organisms, the only known exceptions being mitochondrial ribosomes of fungi, animals, and some protists. An intriguing situation distinguishes mammalian cells: Although the mitochondrial genome contains no 5S rRNA genes, abundant import of the nuclear DNA-encoded 5S rRNA into mitochondria was reported. Neither the detailed mechanism of this pathway nor its rationale was clarified to date. In this study, we describe an elegant molecular conveyor composed of a previously identified human 5S rRNA import factor, rhodanese, and mitochondrial ribosomal protein L18, thanks to which 5S rRNA molecules can be specifically withdrawn from the cytosolic pool and redirected to mitochondria, bypassing the classic nucleolar reimport pathway. Inside mitochondria, the cytosolic 5S rRNA is shown to be associated with mitochondrial ribosomes. PMID:21685364

Ambient pollutants, polymorphisms associated with microRNA processing and adhesion molecules: the Normative Aging Study

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Vokonas Pantel S

2011-05-01

Full Text Available Abstract Background Particulate air pollution has been associated with cardiovascular morbidity and mortality, but it remains unclear which time windows and pollutant sources are most critical. MicroRNA (miRNA is thought to be involved in cardiovascular regulation. However, little is known about whether polymorphisms in genes that process microRNAs influence response to pollutant exposure. We hypothesized that averaging times longer than routinely measured one or two day moving averages are associated with higher soluble intercellular adhesion molecule-1 (sICAM-1 and vascular cell adhesion molecule-1 (sVCAM-1 levels, and that stationary and mobile sources contribute differently to these effects. We also investigated whether single nucleotide polymorphisms (SNPs in miRNA-processing genes modify these associations. Methods sICAM-1 and sVCAM-1 were measured from 1999-2008 and matched to air pollution monitoring for fine particulate matter (PM2.5 black carbon, and sulfates (SO42-. We selected 17 SNPs in five miRNA-processing genes. Mixed-effects models were used to assess effects of pollutants, SNPs, and interactions under recessive inheritance models using repeated measures. Results 723 participants with 1652 observations and 1-5 visits were included in our analyses for black carbon and PM2.5. Sulfate data was available for 672 participants with 1390 observations. An interquartile range change in seven day moving average of PM2.5 (4.27 μg/m3 was associated with 3.1% (95%CI: 1.6, 4.6 and 2.5% (95%CI: 0.6, 4.5 higher sICAM-1 and sVCAM-1. Interquartile range changes in sulfates (1.39 μg/m3 were associated with 1.4% higher (95%CI: 0.04, 2.7 and 1.6% (95%CI: -0.4, 3.7 higher sICAM-1 and sVCAM-1 respectively. No significant associations were observed for black carbon. In interaction models with PM2.5, both sICAM-1 and sVCAM-1 levels were lower in rs1062923 homozygous carriers. These interactions remained significant after multiple comparisons

Fluorescence Correlation Spectroscopy to find the critical balance between extracellular association and intracellular dissociation of mRNA-complexes.

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Zhang, Heyang; De Smedt, Stefaan C; Remaut, Katrien

2018-05-10

Fluorescence Correlation Spectroscopy (FCS) is a promising tool to study interactions on a single molecule level. The diffusion of fluorescent molecules in and out of the excitation volume of a confocal microscope leads to the fluorescence fluctuations that give information on the average number of fluorescent molecules present in the excitation volume and their diffusion coefficients. In this context, we complexed mRNA into lipoplexes and polyplexes and explored the association/dissociation degree of complexes by using gel electrophoresis and FCS. FCS enabled us to measure the association and dissociation degree of mRNA-based complexes both in buffer and protein-rich biological fluids such as human serum and ascitic fluid, which is a clear advantage over gel electrophoresis that was only applicable in protein-free buffer solutions. Furthermore, following the complex stability in buffer and biological fluids by FCS assisted to understand how complex characteristics, such as charge ratio and strength of mRNA binding, correlated to the transfection efficiency. We found that linear polyethyleneimine prevented efficient translation of mRNA, most likely due to a too strong mRNA binding, whereas the lipid based carrier Lipofectamine ® messengerMAX did succeed in efficient release and subsequent translation of mRNA in the cytoplasm of the cells. Overall, FCS is a reliable tool for the in depth characterization of mRNA complexes and can help us to find the critical balance keeping mRNA bound in complexes in the extracellular environment and efficient intracellular mRNA release leading to protein production. The delivery of messenger RNA (mRNA) to cells is promising to treat a variety of diseases. Therefore, the mRNA is typically packed in small lipid particles or polymer particles that help the mRNA to reach the cytoplasm of the cells. These particles should bind and carry the mRNA in the extracellular environment (e.g. blood, peritoneal fluid, ...), but should release

The early history of tRNA recognition by aminoacyl-tRNA synthetases

Indian Academy of Sciences (India)

Madhu

2006-10-04

Oct 4, 2006 ... Discovery of aminoacyl-tRNA synthetases and importance ... The pioneering work of Fritz Lipmann on the high-energy ... the peculiar structural and functional relationships tRNAs ... a bulk of only 20 families of tRNA molecules in contrast ...... balance of tRNA and aminoacyl-tRNA synthetase; Science 242.

Single Molecule Science for Personalized Nanomedicine: Atomic Force Microscopy of Biopolymer-Protein Interactions

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Hsueh, Carlin

Nanotechnology has a unique and relatively untapped utility in the fields of medicine and dentistry at the level of single-biopolymer and -molecule diagnostics. In recent years atomic force microscopy (AFM) has garnered much interest due to its ability to obtain atomic-resolution of molecular structures and probe biophysical behaviors of biopolymers and proteins in a variety of biologically significant environments. The work presented in this thesis focuses on the nanoscale manipulation and observation of biopolymers to develop an innovative technology for personalized medicine while understanding complex biological systems. These studies described here primarily use AFM to observe biopolymer interactions with proteins and its surroundings with unprecedented resolution, providing a better understanding of these systems and interactions at the nanoscale. Transcriptional profiling, the measure of messenger RNA (mRNA) abundance in a single cell, is a powerful technique that detects "behavior" or "symptoms" at the tissue and cellular level. We have sought to develop an alternative approach, using our expertise in AFM and single molecule nanotechnology, to achieve a cost-effective high throughput method for sensitive detection and profiling of subtle changes in transcript abundance. The technique does not require amplification of the mRNA sample because the AFM provides three-dimensional views of molecules with unprecedented resolution, requires minimal sample preparation, and utilizes a simple tagging chemistry on cDNA molecules. AFM images showed collagen polymers in teeth and of Drebrin-A remodeling of filamentous actin structure and mechanics. AFM was used to image collagen on exposed dentine tubules and confirmed tubule occlusion with a desensitizing prophylaxis paste by Colgate-Palmolive. The AFM also superseded other microscopy tools in resolving F-actin helix remodeling and possible cooperative binding by a neuronal actin binding protein---Drebrin-A, an

Synthetic biology expands chemical control of microorganisms.

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Ford, Tyler J; Silver, Pamela A

2015-10-01

The tools of synthetic biology allow researchers to change the ways engineered organisms respond to chemical stimuli. Decades of basic biology research and new efforts in computational protein and RNA design have led to the development of small molecule sensors that can be used to alter organism function. These new functions leap beyond the natural propensities of the engineered organisms. They can range from simple fluorescence or growth reporting to pathogen killing, and can involve metabolic coordination among multiple cells or organisms. Herein, we discuss how synthetic biology alters microorganisms' responses to chemical stimuli resulting in the development of microbes as toxicity sensors, disease treatments, and chemical factories. Copyright © 2015 Elsevier Ltd. All rights reserved.

Detection of a high-molecular-weight LHRH precursor by cell-free translation of mRNA from human, rat, and mouse hypothalamus

International Nuclear Information System (INIS)

Curtis, A.; Szelke, M.; Fink, G.

1986-01-01

Large precursors have also been predicted using the immunoprecipitation technique which relies upon the identification of large immunoreactive molecules following in vitro translation of mRNA. The mRNA is presumed to represent the largest form of a nascent precursor polypeptide molecule irrespective of the number of biosynthetic cleavage steps which are necessary to liberate the active peptide. However, as has been shown for somatostatin, nonprotein modifications may be made which apparently increase molecular weight, such as glycosylation or phosphorylation of the molecule. The authors employed the immunoprecipitation technique to confirm earlier chromatographic studies that the hypothalamic decapeptide, luteinizing hormone releasing hormone (LHRH) is also synthesized by way of a large precursor form. The authors' finding show that the translation of hypothalamic mRNA produces a primary translation product with an apparent molecule weight of 28,000 which contains an amino acid sequence immunologically similar to that of biologically active LHRH. The procedure involved the incorporation of a radioactive amino acid into polypeptides synthesized by in vitro translation of hypothalamic messenger RNA. The resulting complex protein mixture was immunoprecipitated with a specific anti-LHRH serum, and the immunoprecipitate was identified by polyacrylamide gel electrophoresis and autoradiography

Riboswitches for the alarmone ppGpp expand the collection of RNA-based signaling systems.

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Sherlock, Madeline E; Sudarsan, Narasimhan; Breaker, Ronald R

2018-05-21

Riboswitches are noncoding portions of certain mRNAs that bind metabolite, coenzyme, signaling molecule, or inorganic ion ligands and regulate gene expression. Most known riboswitches sense derivatives of RNA monomers. This bias in ligand chemical composition is consistent with the hypothesis that widespread riboswitch classes first emerged during the RNA World, which is proposed to have existed before proteins were present. Here we report the discovery and biochemical validation of a natural riboswitch class that selectively binds guanosine tetraphosphate (ppGpp), a widespread signaling molecule and bacterial "alarmone" derived from the ribonucleotide GTP. Riboswitches for ppGpp are predicted to regulate genes involved in branched-chain amino acid biosynthesis and transport, as well as other gene classes that previously had not been implicated to be part of its signaling network. This newfound riboswitch-alarmone partnership supports the hypothesis that prominent RNA World signaling pathways have been retained by modern cells to control key biological processes. Copyright © 2018 the Author(s). Published by PNAS.

RNA STRAND: The RNA Secondary Structure and Statistical Analysis Database

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Andronescu Mirela

2008-08-01

Full Text Available Abstract Background The ability to access, search and analyse secondary structures of a large set of known RNA molecules is very important for deriving improved RNA energy models, for evaluating computational predictions of RNA secondary structures and for a better understanding of RNA folding. Currently there is no database that can easily provide these capabilities for almost all RNA molecules with known secondary structures. Results In this paper we describe RNA STRAND – the RNA secondary STRucture and statistical ANalysis Database, a curated database containing known secondary structures of any type and organism. Our new database provides a wide collection of known RNA secondary structures drawn from public databases, searchable and downloadable in a common format. Comprehensive statistical information on the secondary structures in our database is provided using the RNA Secondary Structure Analyser, a new tool we have developed to analyse RNA secondary structures. The information thus obtained is valuable for understanding to which extent and with which probability certain structural motifs can appear. We outline several ways in which the data provided in RNA STRAND can facilitate research on RNA structure, including the improvement of RNA energy models and evaluation of secondary structure prediction programs. In order to keep up-to-date with new RNA secondary structure experiments, we offer the necessary tools to add solved RNA secondary structures to our database and invite researchers to contribute to RNA STRAND. Conclusion RNA STRAND is a carefully assembled database of trusted RNA secondary structures, with easy on-line tools for searching, analyzing and downloading user selected entries, and is publicly available at http://www.rnasoft.ca/strand.

Physics of Complex Polymeric Molecules

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Kelly, Joshua Walter

The statistical physics of complex polymers with branches and circuits is the topic of this dissertation. An important motivation are large, single-stranded (ss) RNA molecules. Such molecules form complex ``secondary" and ``tertiary" structures that can be represented as branched polymers with circuits. Such structures are in part directly determined by the nucleotide sequence and in part subject to thermal fluctuations. The polymer physics literature on molecules in this class has mostly focused on randomly branched polymers without circuits while there has been minimal research on polymers with specific structures and on polymers that contain circuits. The dissertation is composed of three parts: Part I studies branched polymers with thermally fluctuating structure confined to a potential well as a simple model for the encapsidation of viral RNA. Excluded volume interactions were ignored. In Part II, I apply Flory theory to the study of the encapsidation of viral ss RNA molecules with specific branched structures, but without circuits, in the presence of excluded volume interaction. In Part III, I expand on Part II and consider complex polymers with specific structure including both branching and circuits. I introduce a method based on the mathematics of Laplacian matrices that allows me to calculate density profiles for such molecules, which was not possible within Flory theory.

The synthesis and biological evaluation of integrin receptor targeting molecules as potential radiopharmaceuticals

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Pellegrini, Paul

This thesis reports on the synthesis, characterisation and biological evaluation of a number of metal complexes designed to interact with the alphavbeta3 integrin receptor, an important biological target that is heavily involved in angiogenesis, and thus cancer related processes. Two approaches were used to synthesise the integrin-avid targets. The first was to attach a variety of bifunctional chelators (BFC's) for the incorporation of different metal centres to a known integrin antagonist, L-748,415, developed by Merck. The BFC's used were the hydrazinonicotinamide (HYNIC) and monoamine monoamide dithiol (MAMA) systems for coordination to Tc-99m and rhenium of which was used as a characterization surrogate for the unstable Tc core. The 1,4,7,10-tetraazacyclotridecanetetraacetic acid (TRITA) BFC was attached for the inclusion of copper and lutetium. This 'conjugate' approach was designed to yield information on how the BFC and the linker length would affect the affinity for the integrin receptor. The second approach was an 'integrated' method where the chelation moiety was integral to the biologically relevant part of the molecule, which in the case of the alphavbeta3 integrin receptor, is the arginine-glycine-aspartic acid (RGD) mimicking sequence. Two complexes were created with a modified MAMA derivative placed between a benzimidazole moiety (arginine mimick) and the aspartic acid mimicking terminal carboxylic acid to see how it would affect binding while keeping the molecular weight relatively low. The molecules were tested in vitro against purified human alphavbeta3 integrin receptor protein in a solid phase receptor binding assay to evaluate their inhibition constants against a molecule of known high affinity and selectivity in [I125]L-775,219, the I125 labelled alphavbeta3 integrin antagonist. The radiolabelled analogues were also tested in vivo against the A375 human melanoma cell line transplanted into balb/c nude mice as well as Fischer rats implanted

Single molecule approaches for quantifying transcription and degradation rates in intact mammalian tissues.

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Bahar Halpern, Keren; Itzkovitz, Shalev

2016-04-01

A key challenge in mammalian biology is to understand how rates of transcription and mRNA degradation jointly shape cellular gene expression. Powerful techniques have been developed for measuring these rates either genome-wide or at the single-molecule level, however these techniques are not applicable to assessment of cells within their native tissue microenvironment. Here we describe a technique based on single molecule Fluorescence in-situ Hybridization (smFISH) to measure transcription and degradation rates in intact mammalian tissues. The technique is based on dual-color libraries targeting the introns and exons of the genes of interest, enabling visualization and quantification of both nascent and mature mRNA. We present a software, TransQuant, that facilitates quantifying these rates from smFISH images. Our approach enables assessment of both transcription and degradation rates of any gene of interest while controlling for the inherent heterogeneity of intact tissues. Copyright © 2015 Elsevier Inc. All rights reserved.

Myricetin: A Dietary Molecule with Diverse Biological Activities

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Deepak Kumar Semwal

2016-02-01

Full Text Available Myricetin is a common plant-derived flavonoid and is well recognised for its nutraceuticals value. It is one of the key ingredients of various foods and beverages. The compound exhibits a wide range of activities that include strong anti-oxidant, anticancer, antidiabetic and anti-inflammatory activities. It displays several activities that are related to the central nervous system and numerous studies have suggested that the compound may be beneficial to protect against diseases such as Parkinson’s and Alzheimer’s. The use of myricetin as a preserving agent to extend the shelf life of foods containing oils and fats is attributed to the compound’s ability to protect lipids against oxidation. A detailed search of existing literature revealed that there is currently no comprehensive review available on this important molecule. Hence, the present work includes the history, synthesis, pharmaceutical applications and toxicity studies of myricetin. This report also highlights structure-activity relationships and mechanisms of action for various biological activities.

Prion-like nanofibrils of small molecules (PriSM): A new frontier at the intersection of supramolecular chemistry and cell biology.

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Zhou, Jie; Du, Xuewen; Xu, Bing

2015-01-01

Formed by non-covalent interactions and not defined at genetic level, the assemblies of small molecules in biology are complicated and less explored. A common morphology of the supramolecular assemblies of small molecules is nanofibrils, which coincidentally resembles the nanofibrils formed by proteins such as prions. So these supramolecular assemblies are termed as prion-like nanofibrils of small molecules (PriSM). Emerging evidence from several unrelated fields over the past decade implies the significance of PriSM in biology and medicine. This perspective aims to highlight some recent advances of the research on PriSM. This paper starts with description of the intriguing similarities between PriSM and prions, discusses the paradoxical features of PriSM, introduces the methods for elucidating the biological functions of PriSM, illustrates several examples of beneficial aspects of PriSM, and finishes with the promises and current challenges in the research of PriSM. We anticipate that the research of PriSM will contribute to the fundamental understanding at the intersection of supramolecular chemistry and cell biology and ultimately lead to a new paradigm of molecular (or supramolecular) therapeutics for biomedicine.

Chess games: a model for RNA based computation.

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Cukras, A R; Faulhammer, D; Lipton, R J; Landweber, L F

1999-10-01

Here we develop the theory of RNA computing and a method for solving the 'knight problem' as an instance of a satisfiability (SAT) problem. Using only biological molecules and enzymes as tools, we developed an algorithm for solving the knight problem (3 x 3 chess board) using a 10-bit combinatorial pool and sequential RNase H digestions. The results of preliminary experiments presented here reveal that the protocol recovers far more correct solutions than expected at random, but the persistence of errors still presents the greatest challenge.

Cancer-Related Triplets of mRNA-lncRNA-miRNA Revealed by Integrative Network in Uterine Corpus Endometrial Carcinoma

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Chenglin Liu

2017-01-01

Full Text Available The regulation of transcriptome expression level is a complex process involving multiple-level interactions among molecules such as protein coding RNA (mRNA, long noncoding RNA (lncRNA, and microRNA (miRNA, which are essential for the transcriptome stability and maintenance and regulation of body homeostasis. The availability of multilevel expression data enables a comprehensive view of the regulatory network. In this study, we analyzed the coding and noncoding gene expression profiles of 301 patients with uterine corpus endometrial carcinoma (UCEC. A new method was proposed to construct a genome-wide integrative network based on variance inflation factor (VIF regression method. The cross-regulation relations of mRNA, lncRNA, and miRNA were then selected based on clique-searching algorithm from the network, when any two molecules of the three were shown as interacting according to the integrative network. Such relation, which we call the mRNA-lncRNA-miRNA triplet, demonstrated the complexity in transcriptome regulation process. Finally, six UCEC-related triplets were selected in which the mRNA participates in endometrial carcinoma pathway, such as CDH1 and TP53. The multi-type RNAs are proved to be cross-regulated as to each of the six triplets according to literature. All the triplets demonstrated the association with the initiation and progression of UCEC. Our method provides a comprehensive strategy for the investigation of transcriptome regulation mechanism.

Identifying microRNA/mRNA dysregulations in ovarian cancer.

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Miles, Gregory D; Seiler, Michael; Rodriguez, Lorna; Rajagopal, Gunaretnam; Bhanot, Gyan

2012-03-27

MicroRNAs are a class of noncoding RNA molecules that co-regulate the expression of multiple genes via mRNA transcript degradation or translation inhibition. Since they often target entire pathways, they may be better drug targets than genes or proteins. MicroRNAs are known to be dysregulated in many tumours and associated with aggressive or poor prognosis phenotypes. Since they regulate mRNA in a tissue specific manner, their functional mRNA targets are poorly understood. In previous work, we developed a method to identify direct mRNA targets of microRNA using patient matched microRNA/mRNA expression data using an anti-correlation signature. This method, applied to clear cell Renal Cell Carcinoma (ccRCC), revealed many new regulatory pathways compromised in ccRCC. In the present paper, we apply this method to identify dysregulated microRNA/mRNA mechanisms in ovarian cancer using data from The Cancer Genome Atlas (TCGA). TCGA Microarray data was normalized and samples whose class labels (tumour or normal) were ambiguous with respect to consensus ensemble K-Means clustering were removed. Significantly anti-correlated and correlated genes/microRNA differentially expressed between tumour and normal samples were identified. TargetScan was used to identify gene targets of microRNA. We identified novel microRNA/mRNA mechanisms in ovarian cancer. For example, the expression level of RAD51AP1 was found to be strongly anti-correlated with the expression of hsa-miR-140-3p, which was significantly down-regulated in the tumour samples. The anti-correlation signature was present separately in the tumour and normal samples, suggesting a direct causal dysregulation of RAD51AP1 by hsa-miR-140-3p in the ovary. Other pairs of potentially biological relevance include: hsa-miR-145/E2F3, hsa-miR-139-5p/TOP2A, and hsa-miR-133a/GCLC. We also identified sets of positively correlated microRNA/mRNA pairs that are most likely result from indirect regulatory mechanisms. Our findings identify

X-ray structure analyses of biological molecules and particles in Japan. A brief history and future prospect

International Nuclear Information System (INIS)

Nakasako, Masayoshi; Yamamoto, Masaki

2015-01-01

In Japan, X-ray structure analyses of molecules and particles from biology started in the 1970s. The structure analysis methods have been developed through the innovation of various techniques in advance, and have contributed for understanding the elementary and microscopic processes in life. Here we summarize briefly the history of X-ray structure analyses for structural biology in Japan and think about the prospect. (author)

Anti-replicative recombinant 5S rRNA molecules can modulate the mtDNA heteroplasmy in a glucose-dependent manner.

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Loutre, Romuald; Heckel, Anne-Marie; Jeandard, Damien; Tarassov, Ivan; Entelis, Nina

2018-01-01

Mutations in mitochondrial DNA are an important source of severe and incurable human diseases. The vast majority of these mutations are heteroplasmic, meaning that mutant and wild-type genomes are present simultaneously in the same cell. Only a very high proportion of mutant mitochondrial DNA (heteroplasmy level) leads to pathological consequences. We previously demonstrated that mitochondrial targeting of small RNAs designed to anneal with mutant mtDNA can decrease the heteroplasmy level by specific inhibition of mutant mtDNA replication, thus representing a potential therapy. We have also shown that 5S ribosomal RNA, partially imported into human mitochondria, can be used as a vector to deliver anti-replicative oligoribonucleotides into human mitochondria. So far, the efficiency of cellular expression of recombinant 5S rRNA molecules bearing therapeutic insertions remained very low. In the present study, we designed new versions of anti-replicative recombinant 5S rRNA targeting a large deletion in mitochondrial DNA which causes the KSS syndrome, analyzed their specific annealing to KSS mitochondrial DNA and demonstrated their import into mitochondria of cultured human cells. To obtain an increased level of the recombinant 5S rRNA stable expression, we created transmitochondrial cybrid cell line bearing a site for Flp-recombinase and used this system for the recombinase-mediated integration of genes coding for the anti-replicative recombinant 5S rRNAs into nuclear genome. We demonstrated that stable expression of anti-replicative 5S rRNA versions in human transmitochondrial cybrid cells can induce a shift in heteroplasmy level of KSS mutation in mtDNA. This shift was directly dependent on the level of the recombinant 5S rRNA expression and the sequence of the anti-replicative insertion. Quantification of mtDNA copy number in transfected cells revealed the absence of a non-specific effect on wild type mtDNA replication, indicating that the decreased proportion

Identification of Subtype Specific miRNA-mRNA Functional Regulatory Modules in Matched miRNA-mRNA Expression Data: Multiple Myeloma as a Case

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Yunpeng Zhang

2015-01-01

Full Text Available Identification of miRNA-mRNA modules is an important step to elucidate their combinatorial effect on the pathogenesis and mechanisms underlying complex diseases. Current identification methods primarily are based upon miRNA-target information and matched miRNA and mRNA expression profiles. However, for heterogeneous diseases, the miRNA-mRNA regulatory mechanisms may differ between subtypes, leading to differences in clinical behavior. In order to explore the pathogenesis of each subtype, it is important to identify subtype specific miRNA-mRNA modules. In this study, we integrated the Ping-Pong algorithm and multiobjective genetic algorithm to identify subtype specific miRNA-mRNA functional regulatory modules (MFRMs through integrative analysis of three biological data sets: GO biological processes, miRNA target information, and matched miRNA and mRNA expression data. We applied our method on a heterogeneous disease, multiple myeloma (MM, to identify MM subtype specific MFRMs. The constructed miRNA-mRNA regulatory networks provide modular outlook at subtype specific miRNA-mRNA interactions. Furthermore, clustering analysis demonstrated that heterogeneous MFRMs were able to separate corresponding MM subtypes. These subtype specific MFRMs may aid in the further elucidation of the pathogenesis of each subtype and may serve to guide MM subtype diagnosis and treatment.

An automated procedure for covariation-based detection of RNA structure

International Nuclear Information System (INIS)

Winker, S.; Overbeek, R.; Woese, C.R.; Olsen, G.J.; Pfluger, N.

1989-12-01

This paper summarizes our investigations into the computational detection of secondary and tertiary structure of ribosomal RNA. We have developed a new automated procedure that not only identifies potential bondings of secondary and tertiary structure, but also provides the covariation evidence that supports the proposed bondings, and any counter-evidence that can be detected in the known sequences. A small number of previously unknown bondings have been detected in individual RNA molecules (16S rRNA and 7S RNA) through the use of our automated procedure. Currently, we are systematically studying mitochondrial rRNA. Our goal is to detect tertiary structure within 16S rRNA and quaternary structure between 16S and 23S rRNA. Our ultimate hope is that automated covariation analysis will contribute significantly to a refined picture of ribosome structure. Our colleagues in biology have begun experiments to test certain hypotheses suggested by an examination of our program's output. These experiments involve sequencing key portions of the 23S ribosomal RNA for species in which the known 16S ribosomal RNA exhibits variation (from the dominant pattern) at the site of a proposed bonding. The hope is that the 23S ribosomal RNA of these species will exhibit corresponding complementary variation or generalized covariation. 24 refs

An automated procedure for covariation-based detection of RNA structure

Energy Technology Data Exchange (ETDEWEB)

Winker, S.; Overbeek, R.; Woese, C.R.; Olsen, G.J.; Pfluger, N.

1989-12-01

This paper summarizes our investigations into the computational detection of secondary and tertiary structure of ribosomal RNA. We have developed a new automated procedure that not only identifies potential bondings of secondary and tertiary structure, but also provides the covariation evidence that supports the proposed bondings, and any counter-evidence that can be detected in the known sequences. A small number of previously unknown bondings have been detected in individual RNA molecules (16S rRNA and 7S RNA) through the use of our automated procedure. Currently, we are systematically studying mitochondrial rRNA. Our goal is to detect tertiary structure within 16S rRNA and quaternary structure between 16S and 23S rRNA. Our ultimate hope is that automated covariation analysis will contribute significantly to a refined picture of ribosome structure. Our colleagues in biology have begun experiments to test certain hypotheses suggested by an examination of our program's output. These experiments involve sequencing key portions of the 23S ribosomal RNA for species in which the known 16S ribosomal RNA exhibits variation (from the dominant pattern) at the site of a proposed bonding. The hope is that the 23S ribosomal RNA of these species will exhibit corresponding complementary variation or generalized covariation. 24 refs.

Exploring matter through photons and neutrons: from biological molecules to designer materials

International Nuclear Information System (INIS)

Chidambaram, R.; Hosur, M.V.; Ramanadham, M.; Godwal, B.K.

2000-01-01

Understanding structure-property relationships of naturally occurring materials has been the aim of scientific research for centuries. The discovery of short wavelength x-rays and neutrons in the 20th century provided a means of studying molecular structure. The methodology of x-ray and neutron diffraction has been successfully applied to determine structures of molecules across disciplines of physics, chemistry, biology, biochemistry and medicine. Typical applications in physics include study of phase transformations, elasticity measurements, magnetic structure, surface scattering etc. In chemistry, the applications have ranged from routine structure determinations of reaction intermediates or natural products to refinement of quantum chemical parameters of atomic and molecular charge densities. The science of crystallography has had a profound effect on the disciplines of biology and medicine. A whole new discipline and industry was created when the structure of DNA was discovered through x-ray diffraction

De-repressing LncRNA-Targeted Genes to Upregulate Gene Expression: Focus on Small Molecule Therapeutics

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Roya Pedram Fatemi

2014-01-01

Full Text Available Non-protein coding RNAs (ncRNAs make up the overwhelming majority of transcripts in the genome and have recently gained attention for their complex regulatory role in cells, including the regulation of protein-coding genes. Furthermore, ncRNAs play an important role in normal development and their expression levels are dysregulated in several diseases. Recently, several long noncoding RNAs (lncRNAs have been shown to alter the epigenetic status of genomic loci and suppress the expression of target genes. This review will present examples of such a mechanism and focus on the potential to target lncRNAs for achieving therapeutic gene upregulation by de-repressing genes that are epigenetically silenced in various diseases. Finally, the potential to target lncRNAs, through their interactions with epigenetic enzymes, using various tools, such as small molecules, viral vectors and antisense oligonucleotides, will be discussed. We suggest that small molecule modulators of a novel class of drug targets, lncRNA-protein interactions, have great potential to treat some cancers, cardiovascular disease, and neurological disorders.

Synthetic biology era: Improving antibiotic's world.

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Guzmán-Trampe, Silvia; Ceapa, Corina D; Manzo-Ruiz, Monserrat; Sánchez, Sergio

2017-06-15

The emergence of antibiotic-resistant pathogen microorganisms is problematic in the context of the current spectrum of available medication. The poor specificity and the high toxicity of some available molecules have made imperative the search for new strategies to improve the specificity and to pursue the discovery of novel compounds with increased bioactivity. Using living cells as platforms, synthetic biology has counteracted this problem by offering novel pathways to create synthetic systems with improved and desired functions. Among many other biotechnological approaches, the advances in synthetic biology have made it possible to design and construct novel biological systems in order to look for new drugs with increased bioactivity. Advancements have also been made in the redesigning of RNA and DNA molecules in order to engineer antibiotic clusters for antibiotic overexpression. As for the production of these antibacterial compounds, yeasts and filamentous fungi as well as gene therapy are utilized to enhance protein solubility. Specific delivery is achieved by creating chimeras using plant genes into bacterial hosts. Some of these synthetic systems are currently in clinical trials, proving the proficiency of synthetic biology in terms of both pharmacological activities as well as an increase in the biosafety of treatments. It is possible that we may just be seeing the tip of the iceberg, and synthetic biology applications will overpass expectations beyond our present knowledge. Copyright © 2017. Published by Elsevier Inc.

Discovery of Protein–lncRNA Interactions by Integrating Large-Scale CLIP-Seq and RNA-Seq Datasets

Energy Technology Data Exchange (ETDEWEB)

Li, Jun-Hao; Liu, Shun; Zheng, Ling-Ling; Wu, Jie; Sun, Wen-Ju; Wang, Ze-Lin; Zhou, Hui; Qu, Liang-Hu, E-mail: lssqlh@mail.sysu.edu.cn; Yang, Jian-Hua, E-mail: lssqlh@mail.sysu.edu.cn [RNA Information Center, Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory for Biocontrol, Sun Yat-sen University, Guangzhou (China)

2015-01-14

Long non-coding RNAs (lncRNAs) are emerging as important regulatory molecules in developmental, physiological, and pathological processes. However, the precise mechanism and functions of most of lncRNAs remain largely unknown. Recent advances in high-throughput sequencing of immunoprecipitated RNAs after cross-linking (CLIP-Seq) provide powerful ways to identify biologically relevant protein–lncRNA interactions. In this study, by analyzing millions of RNA-binding protein (RBP) binding sites from 117 CLIP-Seq datasets generated by 50 independent studies, we identified 22,735 RBP–lncRNA regulatory relationships. We found that one single lncRNA will generally be bound and regulated by one or multiple RBPs, the combination of which may coordinately regulate gene expression. We also revealed the expression correlation of these interaction networks by mining expression profiles of over 6000 normal and tumor samples from 14 cancer types. Our combined analysis of CLIP-Seq data and genome-wide association studies data discovered hundreds of disease-related single nucleotide polymorphisms resided in the RBP binding sites of lncRNAs. Finally, we developed interactive web implementations to provide visualization, analysis, and downloading of the aforementioned large-scale datasets. Our study represented an important step in identification and analysis of RBP–lncRNA interactions and showed that these interactions may play crucial roles in cancer and genetic diseases.

Discovery of Protein–lncRNA Interactions by Integrating Large-Scale CLIP-Seq and RNA-Seq Datasets

International Nuclear Information System (INIS)

Li, Jun-Hao; Liu, Shun; Zheng, Ling-Ling; Wu, Jie; Sun, Wen-Ju; Wang, Ze-Lin; Zhou, Hui; Qu, Liang-Hu; Yang, Jian-Hua

2015-01-01

Long non-coding RNAs (lncRNAs) are emerging as important regulatory molecules in developmental, physiological, and pathological processes. However, the precise mechanism and functions of most of lncRNAs remain largely unknown. Recent advances in high-throughput sequencing of immunoprecipitated RNAs after cross-linking (CLIP-Seq) provide powerful ways to identify biologically relevant protein–lncRNA interactions. In this study, by analyzing millions of RNA-binding protein (RBP) binding sites from 117 CLIP-Seq datasets generated by 50 independent studies, we identified 22,735 RBP–lncRNA regulatory relationships. We found that one single lncRNA will generally be bound and regulated by one or multiple RBPs, the combination of which may coordinately regulate gene expression. We also revealed the expression correlation of these interaction networks by mining expression profiles of over 6000 normal and tumor samples from 14 cancer types. Our combined analysis of CLIP-Seq data and genome-wide association studies data discovered hundreds of disease-related single nucleotide polymorphisms resided in the RBP binding sites of lncRNAs. Finally, we developed interactive web implementations to provide visualization, analysis, and downloading of the aforementioned large-scale datasets. Our study represented an important step in identification and analysis of RBP–lncRNA interactions and showed that these interactions may play crucial roles in cancer and genetic diseases.

Interactions of electrons with biologically important molecules

International Nuclear Information System (INIS)

Pisklova, K.; Papp, P.; Stano, M.

2012-01-01

For the study of interactions of low-energy electrons with the molecules in the gas phase, the authors used electron-molecule cross-beam apparatus. The experiment is carried out in high vacuum, where molecules of the tested compound are inducted through a capillary. For purposes of this experiment the sample was electrically heated to 180 Deg C., giving a bundle of GlyGly molecules into the gas phase. The resulting signals can be evaluated in two different modes: mass spectrum - at continuous electron energy (e.g. 100 eV) they obtained the signal of intensity of the ions according to their mass to charge ratio; ionization and resonance spectra - for selected ion mass when the authors received the signal of intensity of the ions, depending on the energy of interacting electron.

A Synthetic Biology Project - Developing a single-molecule device for screening drug-target interactions.

Science.gov (United States)

Firman, Keith; Evans, Luke; Youell, James

2012-07-16

This review describes a European-funded project in the area of Synthetic Biology. The project seeks to demonstrate the application of engineering techniques and methodologies to the design and construction of a biosensor for detecting drug-target interactions at the single-molecule level. Production of the proteins required for the system followed the principle of previously described "bioparts" concepts (a system where a database of biological parts - promoters, genes, terminators, linking tags and cleavage sequences - is used to construct novel gene assemblies) and cassette-type assembly of gene expression systems (the concept of linking different "bioparts" to produce functional "cassettes"), but problems were quickly identified with these approaches. DNA substrates for the device were also constructed using a cassette-system. Finally, micro-engineering was used to build a magnetoresistive Magnetic Tweezer device for detection of single molecule DNA modifying enzymes (motors), while the possibility of constructing a Hall Effect version of this device was explored. The device is currently being used to study helicases from Plasmodium as potential targets for anti-malarial drugs, but we also suggest other potential uses for the device. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Independent alignment of RNA for dynamic studies using residual dipolar couplings

Energy Technology Data Exchange (ETDEWEB)

Bardaro, Michael F.; Varani, Gabriele, E-mail: varani@chem.washington.edu [University of Washington, Department of Chemistry (United States)

2012-09-15

Molecular motion and dynamics play an essential role in the biological function of many RNAs. An important source of information on biomolecular motion can be found in residual dipolar couplings which contain dynamics information over the entire ms-ps timescale. However, these methods are not fully applicable to RNA because nucleic acid molecules tend to align in a highly collinear manner in different alignment media. As a consequence, information on dynamics that can be obtained with this method is limited. In order to overcome this limitation, we have generated a chimeric RNA containing both the wild type TAR RNA, the target of our investigation of dynamics, as well as the binding site for U1A protein. When U1A protein was bound to the portion of the chimeric RNA containing its binding site, we obtained independent alignment of TAR by exploiting the physical chemical characteristics of this protein. This technique can allow the extraction of new information on RNA dynamics, which is particularly important for time scales not covered by relaxation methods where important RNA motions occur.

Cell-killing efficiency and number of platinum atoms binding to DNA, RNA and protein molecules of HeLa cells treated with combinations of hyperthermia and carboplatin

International Nuclear Information System (INIS)

Akaboshi, M.; Kawai, K.; Tanaka, Y.; Takada, J.; Sumino, T.

1999-01-01

The effect of hyperthermia on the cell killing efficiency of Pt atoms binding to DNA, RNA and protein molecules of HeLa cells treated with cis-diamine(1,1-cyclobutanedicarboxylato)platinum(II) (CBDCA) was examined. HeLa S-3 cells were treated with 195m Pt-radiolabeled CBDCA for 60 minutes at various temperatures, and the relationship between the lethal effect and the number of Pt atoms binding to DNA, RNA and proteins was examined. The mean lethal concentration (D 0 ) of carboplatin for a 60 min-treatment at 0, 25, 37, 40, 42 and 44 deg C was 671.2, 201.5, 67.3, 33.4, 20.2 and 15.6 μM, respectively. By using identically treated cells, the number of Pt-atoms combined with DNA, RNA and protein molecules were determined in the subcellular fractions. Thus, the D 0 's given as the drug concentrations were replaced with the number of Pt-atoms combined in each fraction. Then, the cell-killing efficiency of the Pt atom was expressed as the reciprocal of the number of Pt-atoms combined and was calculated for each molecule. The efficiency for DNA molecules was 0.699, 1.42, 2.65, 4.84, 7.74 and 8.28x10 4 nucleotides, respectively, for the conditions described above. From 0 to 44 deg C, the cell-killing efficiency of Pt atoms increased by a factor of 11.9. (author)

RNA versatility, flexibility, and thermostability for practice in RNA nanotechnology and biomedical applications.

Science.gov (United States)

Haque, Farzin; Pi, Fengmei; Zhao, Zhengyi; Gu, Shanqing; Hu, Haibo; Yu, Hang; Guo, Peixuan

2018-01-01

In recent years, RNA has attracted widespread attention as a unique biomaterial with distinct biophysical properties for designing sophisticated architectures in the nanometer scale. RNA is much more versatile in structure and function with higher thermodynamic stability compared to its nucleic acid counterpart DNA. Larger RNA molecules can be viewed as a modular structure built from a combination of many 'Lego' building blocks connected via different linker sequences. By exploiting the diversity of RNA motifs and flexibility of structure, varieties of RNA architectures can be fabricated with precise control of shape, size, and stoichiometry. Many structural motifs have been discovered and characterized over the years and the crystal structures of many of these motifs are available for nanoparticle construction. For example, using the flexibility and versatility of RNA structure, RNA triangles, squares, pentagons, and hexagons can be constructed from phi29 pRNA three-way-junction (3WJ) building block. This review will focus on 2D RNA triangles, squares, and hexamers; 3D and 4D structures built from basic RNA building blocks; and their prospective applications in vivo as imaging or therapeutic agents via specific delivery and targeting. Methods for intracellular cloning and expression of RNA molecules and the in vivo assembly of RNA nanoparticles will also be reviewed. WIREs RNA 2018, 9:e1452. doi: 10.1002/wrna.1452 This article is categorized under: RNA Methods > RNA Nanotechnology RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA in Disease and Development > RNA in Disease Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs. © 2017 Wiley Periodicals, Inc.

Free energy minimization to predict RNA secondary structures and computational RNA design.

Science.gov (United States)

Churkin, Alexander; Weinbrand, Lina; Barash, Danny

2015-01-01

Determining the RNA secondary structure from sequence data by computational predictions is a long-standing problem. Its solution has been approached in two distinctive ways. If a multiple sequence alignment of a collection of homologous sequences is available, the comparative method uses phylogeny to determine conserved base pairs that are more likely to form as a result of billions of years of evolution than by chance. In the case of single sequences, recursive algorithms that compute free energy structures by using empirically derived energy parameters have been developed. This latter approach of RNA folding prediction by energy minimization is widely used to predict RNA secondary structure from sequence. For a significant number of RNA molecules, the secondary structure of the RNA molecule is indicative of its function and its computational prediction by minimizing its free energy is important for its functional analysis. A general method for free energy minimization to predict RNA secondary structures is dynamic programming, although other optimization methods have been developed as well along with empirically derived energy parameters. In this chapter, we introduce and illustrate by examples the approach of free energy minimization to predict RNA secondary structures.

[Detection and clinical value of epithelial cellular adhesion molecule (EpCAM) mRNA positive circulating tumor cells in metastatic breast cancer].

Science.gov (United States)

Yan, Ying; Cheng, Jian-ping; Di, Li-jun; Song, Guo-hong; Ren, Jun

2012-04-18

To test for circulating tumor cells (CTCs) relying on epithelial cellular adhesion molecule (EpCAM) expression in metastatic breast cancer by quantitative real-time reverse transcription-PCR. In the study,47 metastatic breast cancer patients were evaluated by quantitative real-time PCR for detecting EpCAM mRNA. In addition, analyses were carried out for their correlation with patients' clinicopathologic features, response, and the time to progression (TTP). The sensitivity of EpCAM mRNA in the metastatic breast cancer patients was about 40%. However, the specificity of EpCAM mRNA for 20 healthy controls was 100%. TTP was calculated, and compared with that between EpCAM mRNA-positive and EpCAM mRNA-negative groups. For the retrospective study, the median TTP was 7.1 months and 11.1 months (P=0.013) for patients with EpCAM mRNA-positive and EpCAM mRNA-negative, respectively, after the first cycle chemotherapy. Moreover, a statistically significant correlation was demonstrated between EpCAM mRNA and TTP in patients who underwent the first or the second-line chemotherapy (P=0.018), but there was no significance in the patients pretreated with two or more previous chemotherapy lines (P=0.471). This study provides evidence of the presence of EpCAM mRNA in approximately 40% of patients with metastatic breast cancer. There is a strong correlation between EpCAM mRNA results after the first cycle therapy and TTP in metastatic breast cancer patients, and EpCAM mRNA positive after chemotherapy may predict shorter TTP.

In situ single molecule imaging of cell membranes: linking basic nanotechniques to cell biology, immunology and medicine

Science.gov (United States)

Pi, Jiang; Jin, Hua; Yang, Fen; Chen, Zheng W.; Cai, Jiye

2014-10-01

The cell membrane, which consists of a viscous phospholipid bilayer, different kinds of proteins and various nano/micrometer-sized domains, plays a very important role in ensuring the stability of the intracellular environment and the order of cellular signal transductions. Exploring the precise cell membrane structure and detailed functions of the biomolecules in a cell membrane would be helpful to understand the underlying mechanisms involved in cell membrane signal transductions, which could further benefit research into cell biology, immunology and medicine. The detection of membrane biomolecules at the single molecule level can provide some subtle information about the molecular structure and the functions of the cell membrane. In particular, information obtained about the molecular mechanisms and other information at the single molecule level are significantly different from that detected from a large amount of biomolecules at the large-scale through traditional techniques, and can thus provide a novel perspective for the study of cell membrane structures and functions. However, the precise investigations of membrane biomolecules prompts researchers to explore cell membranes at the single molecule level by the use of in situ imaging methods, as the exact conformation and functions of biomolecules are highly controlled by the native cellular environment. Recently, the in situ single molecule imaging of cell membranes has attracted increasing attention from cell biologists and immunologists. The size of biomolecules and their clusters on the cell surface are set at the nanoscale, which makes it mandatory to use high- and super-resolution imaging techniques to realize the in situ single molecule imaging of cell membranes. In the past few decades, some amazing imaging techniques and instruments with super resolution have been widely developed for molecule imaging, which can also be further employed for the in situ single molecule imaging of cell membranes. In

Small molecule probes finely differentiate between various ds- and ss-DNA and RNA by fluorescence, CD and NMR response

Energy Technology Data Exchange (ETDEWEB)

Crnolatac, Ivo; Rogan, Iva; Majić, Boris; Tomić, Sanja [Division of Organic Chemistry and Biochemistry, Division of Physical Chemistry, Ruđer Bošković Institute, P.O. Box 180, 10002 Zagreb (Croatia); Deligeorgiev, Todor [Faculty of Chemistry and Pharmacy, University of Sofia (Bulgaria); Horvat, Gordan [Department of Physical Chemistry, Faculty of Science/Chemistry, Horvatovac 102A, HR-10000 Zagreb (Croatia); Makuc, Damjan; Plavec, Janez [Slovenian NMR Centre, National Institute of Chemistry, Hajdrihova 19, Ljubljana (Slovenia); EN-FIST Centre of Excellence, Trg Osvobodilne Fronte 13, Ljubljana (Slovenia); Pescitelli, Gennaro [Department of Chemistry, University of Pisa, Via Moruzzi 13, Pisa (Italy); Piantanida, Ivo, E-mail: pianta@irb.hr [Division of Organic Chemistry and Biochemistry, Division of Physical Chemistry, Ruđer Bošković Institute, P.O. Box 180, 10002 Zagreb (Croatia)

2016-10-12

Two small molecules showed intriguing properties of analytical multipurpose probes, whereby one chromophore gives different signal for many different DNA/RNA by application of several highly sensitive spectroscopic methods. Dyes revealed pronounced fluorescence ratiomeric differentiation between ds-AU-RNA, AT-DNA and GC-DNA in approximate order 10:8:1. Particularly interesting, dyes showed specific fluorimetric response for poly rA even at 10-fold excess of any other ss-RNA, and moreover such emission selectivity is preserved in multicomponent ss-RNA mixtures. The dyes also showed specific chiral recognition of poly rU in respect to the other ss-RNA by induced CD (ICD) pattern in visible range (400–500 nm), which was attributed to the dye-side-chain contribution to binding (confirmed by absence of any ICD band for reference compound lacking side-chain). Most intriguingly, minor difference in the side-chain attached to dye chromophore resulted in opposite sign of dye-ICD pattern, whereby differences in NMR NOESY contacts and proton chemical shifts between two dye/oligo rU complexes combined with MD simulations and CD calculations attributed observed bisignate ICD to the dimeric dye aggregate within oligo rU. - Highlights: • Novel dyes emit fluorescence only for poly rA even at high excess of all other ss-RNA. • Fluorescence response for AT-DNA is 8 times stronger than for GC-DNA. • Florescence induced by ds-RNA is 20% stronger that emission induced by ds-DNA. • Intrinsically non-chiral, dyes show strong and characteristic ICD response for poly rU.

Expression profiles of mRNA and long noncoding RNA in the ovaries of letrozole-induced polycystic ovary syndrome rat model through deep sequencing.

Science.gov (United States)

Fu, Lu-Lu; Xu, Ying; Li, Dan-Dan; Dai, Xiao-Wei; Xu, Xin; Zhang, Jing-Shun; Ming, Hao; Zhang, Xue-Ying; Zhang, Guo-Qing; Ma, Ya-Lan; Zheng, Lian-Wen

2018-05-30

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in reproductive-aged women. However, the exact pathophysiology of PCOS remains largely unclear. We performed deep sequencing to investigate the mRNA and long noncoding RNA (lncRNA) expression profiles in the ovarian tissues of letrozole-induced PCOS rat model and control rats. A total of 2147 mRNAs and 158 lncRNAs were differentially expressed between the PCOS models and control. Gene ontology analysis indicated that differentially expressed mRNAs were associated with biological adhesion, reproduction, and metabolic process. Pathway analysis results indicated that these aberrantly expressed mRNAs were related to several specific signaling pathways, including insulin resistance, steroid hormone biosynthesis, PPAR signaling pathway, cell adhesion molecules, autoimmune thyroid disease, and AMPK signaling pathway. The relative expression levels of mRNAs and lncRNAs were validated through qRT-PCR. LncRNA-miRNA-mRNA network was constructed to explore ceRNAs involved in the PCOS model and were also verified by qRTPCR experiment. These findings may provide insight into the pathogenesis of PCOS and clues to find key diagnostic and therapeutic roles of lncRNA in PCOS. Copyright © 2018 Elsevier B.V. All rights reserved.

MicroRNA Functions in Osteogenesis and Dysfunctions in Osteoporosis

Science.gov (United States)

van Wijnen, Andre J.; van de Peppel, Jeroen; van Leeuwen, Johannes P.; Lian, Jane B.; Stein, Gary S.; Westendorf, Jennifer J.; Oursler, Merry-Jo; Sampen, Hee-Jeong Im; Taipaleenmaki, Hanna; Hesse, Eric; Riester, Scott; Kakar, Sanjeev

2013-01-01

MicroRNAs (miRNAs) are critical post-transcriptional regulators of gene expression that control osteoblast mediated bone formation and osteoclast-related bone remodelling. Deregulation of miRNA mediated mechanisms is emerging as an important pathological factor in bone degeneration (e.g., osteoporosis) and other bone-related diseases. MiRNAs are intriguing regulatory molecules that are networked with cell signaling pathways and intricate transcriptional programs through ingenuous circuits with remarkably simple logic. This overview examines key principles by which miRNAs control differentiation of osteoblasts as they evolve from mesenchymal stromal cells during osteogenesis, or of osteoclasts as they originate from monocytic precursors in the hematopoietic lineage during osteoclastogenesis. Of particular note are miRNAs that are temporally up-regulated during osteoblastogenesis (e.g., miR-218) or osteoclastogenesis (e.g., miR-148a). Each miRNA stimulates differentiation by suppressing inhibitory signalling pathways (‘double-negative’ regulation). The excitement surrounding miRNAs in bone biology stems from the prominent effects that individual miRNAs can have on biological transitions during differentiation of skeletal cells and correlations of miRNA dysfunction with bone diseases. MiRNAs have significant clinical potential which is reflected by their versatility as disease-specific biomarkers and their promise as therapeutic agents to ameliorate or reverse bone tissue degeneration. PMID:23605904

Footprints of a trypanosomatid RNA world: pre-small subunit rRNA processing by spliced leader addition trans-splicing

Directory of Open Access Journals (Sweden)

Mario Gustavo Mayer

2012-06-01

Full Text Available The addition of a capped mini-exon [spliced leader (SL] through trans-splicing is essential for the maturation of RNA polymerase (pol II-transcribed polycistronic pre-mRNAs in all members of the Trypanosomatidae family. This process is an inter-molecular splicing reaction that follows the same basic rules of cis-splicing reactions. In this study, we demonstrated that mini-exons were added to precursor ribosomal RNA (pre-rRNA are transcribed by RNA pol I, including the 5' external transcribed spacer (ETS region. Additionally, we detected the SL-5'ETS molecule using three distinct methods and located the acceptor site between two known 5'ETS rRNA processing sites (A' and A1 in four different trypanosomatids. Moreover, we detected a polyadenylated 5'ETS upstream of the trans-splicing acceptor site, which also occurs in pre-mRNA trans-splicing. After treatment with an indirect trans-splicing inhibitor (sinefungin, we observed SL-5'ETS decay. However, treatment with 5-fluorouracil (a precursor of RNA synthesis that inhibits the degradation of pre-rRNA led to the accumulation of SL-5'ETS, suggesting that the molecule may play a role in rRNA degradation. The detection of trans-splicing in these molecules may indicate broad RNA-joining properties, regardless of the polymerase used for transcription.

Graphene as a transparent conductive support for studying biological molecules by transmission electron microscopy

International Nuclear Information System (INIS)

Nair, R. R.; Anissimova, S.; Novoselov, K. S.; Blake, P.; Blake, J. R.; Geim, A. K.; Zan, R.; Bangert, U.; Golovanov, A. P.; Morozov, S. V.; Latychevskaia, T.

2010-01-01

We demonstrate the application of graphene as a support for imaging individual biological molecules in transmission electron microscope (TEM). A simple procedure to produce free-standing graphene membranes has been designed. Such membranes are extremely robust and can support practically any submicrometer object. Tobacco mosaic virus has been deposited on graphene samples and observed in a TEM. High contrast has been achieved even though no staining has been applied.

RNA SURVEILLANCE– AN EMERGING ROLE FOR RNA REGULATORY NETWORKS IN AGING

OpenAIRE

Montano, Monty; Long, Kimberly

2010-01-01

In this review, we describe recent advances in the field of RNA regulatory biology and relate these advances to aging science. We introduce a new term, RNA surveillance, an RNA regulatory process that is conserved in metazoans, and describe how RNA surveillance represents molecular cross-talk between two emerging RNA regulatory systems – RNA interference and RNA editing. We discuss how RNA surveillance mechanisms influence mRNA and microRNA expression and activity during lifespan. Additionall...

Bacterial Diversity Studies Using the 16S rRNA Gene Provide a Powerful Research-Based Curriculum for Molecular Biology Laboratory

Directory of Open Access Journals (Sweden)

Bryan E. Dutton

2002-12-01

Full Text Available We have developed a ten-week curriculum for molecular biology that uses 16S ribosomal RNA genes to characterize and compare novel bacteria from hot spring communities in Yellowstone National Park. The 16S rRNA approach bypasses selective culture-based methods. Our molecular biology course offered the opportunity for students to learn broadly applicable methods while contributing to a long-term research project. Specifically, students isolated and characterized clones that contained novel 16S rRNA inserts using restriction enzyme, DNA sequencing, and computer-based phylogenetic methods. In both classes, students retrieved novel bacterial 16S rRNA genes, several of which were most similar to Green Nonsulfur bacterial isolates. During class, we evaluated student performance and mastery of skills and concepts using quizzes, formal lab notebooks, and a broad project assignment. For this report, we also assessed student performance alongside data quality and discussed the significance, our goal being to improve both research and teaching methods.

A Deep Learning Framework for Robust and Accurate Prediction of ncRNA-Protein Interactions Using Evolutionary Information.

Science.gov (United States)

Yi, Hai-Cheng; You, Zhu-Hong; Huang, De-Shuang; Li, Xiao; Jiang, Tong-Hai; Li, Li-Ping

2018-06-01

The interactions between non-coding RNAs (ncRNAs) and proteins play an important role in many biological processes, and their biological functions are primarily achieved by binding with a variety of proteins. High-throughput biological techniques are used to identify protein molecules bound with specific ncRNA, but they are usually expensive and time consuming. Deep learning provides a powerful solution to computationally predict RNA-protein interactions. In this work, we propose the RPI-SAN model by using the deep-learning stacked auto-encoder network to mine the hidden high-level features from RNA and protein sequences and feed them into a random forest (RF) model to predict ncRNA binding proteins. Stacked assembling is further used to improve the accuracy of the proposed method. Four benchmark datasets, including RPI2241, RPI488, RPI1807, and NPInter v2.0, were employed for the unbiased evaluation of five established prediction tools: RPI-Pred, IPMiner, RPISeq-RF, lncPro, and RPI-SAN. The experimental results show that our RPI-SAN model achieves much better performance than other methods, with accuracies of 90.77%, 89.7%, 96.1%, and 99.33%, respectively. It is anticipated that RPI-SAN can be used as an effective computational tool for future biomedical researches and can accurately predict the potential ncRNA-protein interacted pairs, which provides reliable guidance for biological research. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Generation of miRNA sponge constructs

NARCIS (Netherlands)

Kluiver, Joost; Slezak-Prochazka, Izabella; Smigielska-Czepiel, Katarzyna; Halsema, Nancy; Kroesen, Bart-Jan; van den Berg, Anke

2012-01-01

MicroRNA (miRNA) sponges are RNA molecules with repeated miRNA antisense sequences that can sequester miRNAs from their endogenous targets and thus serve as a decoy. Stably expressed miRNA sponges are especially valuable for long-term loss-of-function studies and can be used in vitro and in vivo. We

Applications of Engineered DNA-Binding Molecules Such as TAL Proteins and the CRISPR/Cas System in Biology Research

Directory of Open Access Journals (Sweden)

Toshitsugu Fujita

2015-09-01

Full Text Available Engineered DNA-binding molecules such as transcription activator-like effector (TAL or TALE proteins and the clustered regularly interspaced short palindromic repeats (CRISPR and CRISPR-associated proteins (Cas (CRISPR/Cas system have been used extensively for genome editing in cells of various types and species. The sequence-specific DNA-binding activities of these engineered DNA-binding molecules can also be utilized for other purposes, such as transcriptional activation, transcriptional repression, chromatin modification, visualization of genomic regions, and isolation of chromatin in a locus-specific manner. In this review, we describe applications of these engineered DNA-binding molecules for biological purposes other than genome editing.

Application of terahertz spectroscopy for characterization of biologically active organic molecules in natural environment

Science.gov (United States)

Karaliūnas, Mindaugas; Jakštas, Vytautas; Nasser, Kinan E.; Venckevičius, Rimvydas; Urbanowicz, Andrzej; Kašalynas, Irmantas; Valušis, Gintaras

2016-09-01

In this work, a comparative research of biologically active organic molecules in its natural environment using the terahertz (THz) time domain spectroscopy (TDS) and Fourier transform spectroscopy (FTS) systems is carried out. Absorption coefficient and refractive index of Nicotiana tabacum L. leaves containing nicotine, Cannabis sativa L. leaves containing tetrahydrocannabinol, and Humulu lupulus L. leaves containing α-acids, active organic molecules that obtain in natural environment, were measured in broad frequency range from 0.1 to 13 THz at room temperature. In the spectra of absorption coefficient the features were found to be unique for N. tabacum, C. sativa and H. lupulus. Moreover, those features can be exploited for identification of C. sativa sex and N. tabacum origin. The refractive index can be also used to characterize different species.

Raman Optical Activity of Biological Molecules

Science.gov (United States)

Blanch, Ewan W.; Barron, Laurence D.

Now an incisive probe of biomolecular structure, Raman optical activity (ROA) measures a small difference in Raman scattering from chiral molecules in right- and left-circularly polarized light. As ROA spectra measure vibrational optical activity, they contain highly informative band structures sensitive to the secondary and tertiary structures of proteins, nucleic acids, viruses and carbohydrates as well as the absolute configurations of small molecules. In this review we present a survey of recent studies on biomolecular structure and dynamics using ROA and also a discussion of future applications of this powerful new technique in biomedical research.

Concepts and introduction to RNA bioinformatics

DEFF Research Database (Denmark)

Gorodkin, Jan; Hofacker, Ivo L.; Ruzzo, Walter L.

2014-01-01

RNA bioinformatics and computational RNA biology have emerged from implementing methods for predicting the secondary structure of single sequences. The field has evolved to exploit multiple sequences to take evolutionary information into account, such as compensating (and structure preserving) base...... for interactions between RNA and proteins.Here, we introduce the basic concepts of predicting RNA secondary structure relevant to the further analyses of RNA sequences. We also provide pointers to methods addressing various aspects of RNA bioinformatics and computational RNA biology....

Transcriptome Analysis of mRNA and miRNA in Somatic Embryos of Larix leptolepis Subjected to Hydrogen Treatment

Directory of Open Access Journals (Sweden)

Yali Liu

2016-11-01

Full Text Available Hydrogen is a therapeutic antioxidant that has been used extensively in clinical trials. It also acts as a bioactive molecule that can alleviate abiotic stress in plants. However, the biological effects of hydrogen in somatic embryos and the underlying molecular basis remain largely unknown. In this study, the morphological and physiological influence of exogenous H2 treatment during somatic embryogenesis was characterized in Larix leptolepis Gordon. The results showed that exposure to hydrogen increased the proportions of active pro-embryogenic cells and normal somatic embryos. We sequenced mRNA and microRNA (miRNA libraries to identify global transcriptome changes at different time points during H2 treatment of larch pro-embryogenic masses (PEMs. A total of 45,393 mRNAs and 315 miRNAs were obtained. Among them, 4253 genes and 96 miRNAs were differentially expressed in the hydrogen-treated libraries compared with the control. Further, a large number of the differentially expressed mRNAs and miRNAs were related to reactive oxygen species (ROS homeostasis and cell cycle regulation. We also identified 4399 potential target genes for 285 of the miRNAs. The differential expression data and the mRNA-miRNA interaction network described here provide new insights into the molecular mechanisms that determine the performance of PEMs exposed to H2 during somatic embryogenesis.

The Piwi pathway: from piRNA methylation to histone methylation

NARCIS (Netherlands)

Luteijn, M.J.

2013-01-01

Piwi-interacting RNAs (piRNAs) are germ line-specific small RNA molecules that have a function in genome defense and germ cell development. They associate with a specific class of Argonaute proteins, named Piwi, and function through an RNA interference-like mechanism. These small RNA molecules play

RNA deep sequencing reveals differential microRNA expression during development of sea urchin and sea star.

Directory of Open Access Journals (Sweden)

Sabah Kadri

Full Text Available microRNAs (miRNAs are small (20-23 nt, non-coding single stranded RNA molecules that act as post-transcriptional regulators of mRNA gene expression. They have been implicated in regulation of developmental processes in diverse organisms. The echinoderms, Strongylocentrotus purpuratus (sea urchin and Patiria miniata (sea star are excellent model organisms for studying development with well-characterized transcriptional networks. However, to date, nothing is known about the role of miRNAs during development in these organisms, except that the genes that are involved in the miRNA biogenesis pathway are expressed during their developmental stages. In this paper, we used Illumina Genome Analyzer (Illumina, Inc. to sequence small RNA libraries in mixed stage population of embryos from one to three days after fertilization of sea urchin and sea star (total of 22,670,000 reads. Analysis of these data revealed the miRNA populations in these two species. We found that 47 and 38 known miRNAs are expressed in sea urchin and sea star, respectively, during early development (32 in common. We also found 13 potentially novel miRNAs in the sea urchin embryonic library. miRNA expression is generally conserved between the two species during development, but 7 miRNAs are highly expressed in only one species. We expect that our two datasets will be a valuable resource for everyone working in the field of developmental biology and the regulatory networks that affect it. The computational pipeline to analyze Illumina reads is available at http://www.benoslab.pitt.edu/services.html.

RNA Deep Sequencing Reveals Differential MicroRNA Expression during Development of Sea Urchin and Sea Star

Science.gov (United States)

Kadri, Sabah; Hinman, Veronica F.; Benos, Panayiotis V.

2011-01-01

microRNAs (miRNAs) are small (20–23 nt), non-coding single stranded RNA molecules that act as post-transcriptional regulators of mRNA gene expression. They have been implicated in regulation of developmental processes in diverse organisms. The echinoderms, Strongylocentrotus purpuratus (sea urchin) and Patiria miniata (sea star) are excellent model organisms for studying development with well-characterized transcriptional networks. However, to date, nothing is known about the role of miRNAs during development in these organisms, except that the genes that are involved in the miRNA biogenesis pathway are expressed during their developmental stages. In this paper, we used Illumina Genome Analyzer (Illumina, Inc.) to sequence small RNA libraries in mixed stage population of embryos from one to three days after fertilization of sea urchin and sea star (total of 22,670,000 reads). Analysis of these data revealed the miRNA populations in these two species. We found that 47 and 38 known miRNAs are expressed in sea urchin and sea star, respectively, during early development (32 in common). We also found 13 potentially novel miRNAs in the sea urchin embryonic library. miRNA expression is generally conserved between the two species during development, but 7 miRNAs are highly expressed in only one species. We expect that our two datasets will be a valuable resource for everyone working in the field of developmental biology and the regulatory networks that affect it. The computational pipeline to analyze Illumina reads is available at http://www.benoslab.pitt.edu/services.html. PMID:22216218

Automated insertion of sequences into a ribosomal RNA alignment: An application of computational linguistics in molecular biology

Energy Technology Data Exchange (ETDEWEB)

Taylor, Ronald C. [Case Western Reserve Univ., Cleveland, OH (United States)

1991-11-01

This thesis involved the construction of (1) a grammar that incorporates knowledge on base invariancy and secondary structure in a molecule and (2) a parser engine that uses the grammar to position bases into the structural subunits of the molecule. These concepts were combined with a novel pinning technique to form a tool that semi-automates insertion of a new species into the alignment for the 16S rRNA molecule (a component of the ribosome) maintained by Dr. Carl Woese`s group at the University of Illinois at Urbana. The tool was tested on species extracted from the alignment and on a group of entirely new species. The results were very encouraging, and the tool should be substantial aid to the curators of the 16S alignment. The construction of the grammar was itself automated, allowing application of the tool to alignments for other molecules. The logic programming language Prolog was used to construct all programs involved. The computational linguistics approach used here was found to be a useful way to attach the problem of insertion into an alignment.

Automated insertion of sequences into a ribosomal RNA alignment: An application of computational linguistics in molecular biology

Energy Technology Data Exchange (ETDEWEB)

Taylor, R.C.

1991-11-01

This thesis involved the construction of (1) a grammar that incorporates knowledge on base invariancy and secondary structure in a molecule and (2) a parser engine that uses the grammar to position bases into the structural subunits of the molecule. These concepts were combined with a novel pinning technique to form a tool that semi-automates insertion of a new species into the alignment for the 16S rRNA molecule (a component of the ribosome) maintained by Dr. Carl Woese's group at the University of Illinois at Urbana. The tool was tested on species extracted from the alignment and on a group of entirely new species. The results were very encouraging, and the tool should be substantial aid to the curators of the 16S alignment. The construction of the grammar was itself automated, allowing application of the tool to alignments for other molecules. The logic programming language Prolog was used to construct all programs involved. The computational linguistics approach used here was found to be a useful way to attach the problem of insertion into an alignment.

Bioinformatics resource manager v2.3: an integrated software environment for systems biology with microRNA and cross-species analysis tools

Directory of Open Access Journals (Sweden)

Tilton Susan C

2012-11-01

Full Text Available Abstract Background MicroRNAs (miRNAs are noncoding RNAs that direct post-transcriptional regulation of protein coding genes. Recent studies have shown miRNAs are important for controlling many biological processes, including nervous system development, and are highly conserved across species. Given their importance, computational tools are necessary for analysis, interpretation and integration of high-throughput (HTP miRNA data in an increasing number of model species. The Bioinformatics Resource Manager (BRM v2.3 is a software environment for data management, mining, integration and functional annotation of HTP biological data. In this study, we report recent updates to BRM for miRNA data analysis and cross-species comparisons across datasets. Results BRM v2.3 has the capability to query predicted miRNA targets from multiple databases, retrieve potential regulatory miRNAs for known genes, integrate experimentally derived miRNA and mRNA datasets, perform ortholog mapping across species, and retrieve annotation and cross-reference identifiers for an expanded number of species. Here we use BRM to show that developmental exposure of zebrafish to 30 uM nicotine from 6–48 hours post fertilization (hpf results in behavioral hyperactivity in larval zebrafish and alteration of putative miRNA gene targets in whole embryos at developmental stages that encompass early neurogenesis. We show typical workflows for using BRM to integrate experimental zebrafish miRNA and mRNA microarray datasets with example retrievals for zebrafish, including pathway annotation and mapping to human ortholog. Functional analysis of differentially regulated (p Conclusions BRM provides the ability to mine complex data for identification of candidate miRNAs or pathways that drive phenotypic outcome and, therefore, is a useful hypothesis generation tool for systems biology. The miRNA workflow in BRM allows for efficient processing of multiple miRNA and mRNA datasets in a single

Bioinformatics resource manager v2.3: an integrated software environment for systems biology with microRNA and cross-species analysis tools

Science.gov (United States)

2012-01-01

Background MicroRNAs (miRNAs) are noncoding RNAs that direct post-transcriptional regulation of protein coding genes. Recent studies have shown miRNAs are important for controlling many biological processes, including nervous system development, and are highly conserved across species. Given their importance, computational tools are necessary for analysis, interpretation and integration of high-throughput (HTP) miRNA data in an increasing number of model species. The Bioinformatics Resource Manager (BRM) v2.3 is a software environment for data management, mining, integration and functional annotation of HTP biological data. In this study, we report recent updates to BRM for miRNA data analysis and cross-species comparisons across datasets. Results BRM v2.3 has the capability to query predicted miRNA targets from multiple databases, retrieve potential regulatory miRNAs for known genes, integrate experimentally derived miRNA and mRNA datasets, perform ortholog mapping across species, and retrieve annotation and cross-reference identifiers for an expanded number of species. Here we use BRM to show that developmental exposure of zebrafish to 30 uM nicotine from 6–48 hours post fertilization (hpf) results in behavioral hyperactivity in larval zebrafish and alteration of putative miRNA gene targets in whole embryos at developmental stages that encompass early neurogenesis. We show typical workflows for using BRM to integrate experimental zebrafish miRNA and mRNA microarray datasets with example retrievals for zebrafish, including pathway annotation and mapping to human ortholog. Functional analysis of differentially regulated (p<0.05) gene targets in BRM indicates that nicotine exposure disrupts genes involved in neurogenesis, possibly through misregulation of nicotine-sensitive miRNAs. Conclusions BRM provides the ability to mine complex data for identification of candidate miRNAs or pathways that drive phenotypic outcome and, therefore, is a useful hypothesis

Single molecules and nanotechnology

CERN Document Server

Vogel, Horst

2007-01-01

This book focuses on recent advances in the rapidly evolving field of single molecule research. These advances are of importance for the investigation of biopolymers and cellular biochemical reactions, and are essential to the development of quantitative biology. Written by leading experts in the field, the articles cover a broad range of topics, including: quantum photonics of organic dyes and inorganic nanoparticles their use in detecting properties of single molecules the monitoring of single molecule (enzymatic) reactions single protein (un)folding in nanometer-sized confined volumes the dynamics of molecular interactions in biological cells The book is written for advanced students and scientists who wish to survey the concepts, techniques and results of single molecule research and assess them for their own scientific activities.

Enthalpy-Driven RNA Folding: Single-Molecule Thermodynamics of Tetraloop–Receptor Tertiary Interaction†

Science.gov (United States)

Fiore, Julie L.; Kraemer, Benedikt; Koberling, Felix; Edmann, Rainer; Nesbitt, David J.

2010-01-01

RNA folding thermodynamics are crucial for structure prediction, which requires characterization of both enthalpic and entropic contributions of tertiary motifs to conformational stability. We explore the temperature dependence of RNA folding due to the ubiquitous GAAA tetraloop–receptor docking interaction, exploiting immobilized and freely diffusing single-molecule fluorescence resonance energy transfer (smFRET) methods. The equilibrium constant for intramolecular docking is obtained as a function of temperature (T = 21–47 °C), from which a van’t Hoff analysis yields the enthalpy (ΔH°) and entropy (ΔS°) of docking. Tetraloop–receptor docking is significantly exothermic and entropically unfavorable in 1 mM MgCl2 and 100 mM NaCl, with excellent agreement between immobilized (ΔH° = −17.4 ± 1.6 kcal/mol, and ΔS° = −56.2 ± 5.4 cal mol−1 K−1) and freely diffusing (ΔH° = −17.2 ± 1.6 kcal/mol, and ΔS° = −55.9 ± 5.2 cal mol−1 K−1) species. Kinetic heterogeneity in the tetraloop–receptor construct is unaffected over the temperature range investigated, indicating a large energy barrier for interconversion between the actively docking and nondocking subpopulations. Formation of the tetraloop–receptor interaction can account for ~60% of the ΔH° and ΔS° of P4–P6 domain folding in the Tetrahymena ribozyme, suggesting that it may act as a thermodynamic clamp for the domain. Comparison of the isolated tetraloop–receptor and other tertiary folding thermodynamics supports a theme that enthalpy- versus entropy-driven folding is determined by the number of hydrogen bonding and base stacking interactions. PMID:19186984

H19 RNA binds four molecules of insulin-like growth factor II mRNA-binding protein

DEFF Research Database (Denmark)

Runge, Steffen; Nielsen, Finn Cilius; Nielsen, Jacob

2000-01-01

H19 RNA is a major oncofetal 2.5-kilobase untranslated RNA of unknown function. The maternally expressed H19 gene is located 90 kilobase pairs downstream from the paternally expressed insulin-like growth factor II (IGF-II) gene on human chromosome 11 and mouse chromosome 7; and due to their recip......H19 RNA is a major oncofetal 2.5-kilobase untranslated RNA of unknown function. The maternally expressed H19 gene is located 90 kilobase pairs downstream from the paternally expressed insulin-like growth factor II (IGF-II) gene on human chromosome 11 and mouse chromosome 7; and due...

Single-Molecule Analysis for RISC Assembly and Target Cleavage.

Science.gov (United States)

Sasaki, Hiroshi M; Tadakuma, Hisashi; Tomari, Yukihide

2018-01-01

RNA-induced silencing complex (RISC) is a small RNA-protein complex that mediates silencing of complementary target RNAs. Biochemistry has been successfully used to characterize the molecular mechanism of RISC assembly and function for nearly two decades. However, further dissection of intermediate states during the reactions has been warranted to fill in the gaps in our understanding of RNA silencing mechanisms. Single-molecule analysis with total internal reflection fluorescence (TIRF) microscopy is a powerful imaging-based approach to interrogate complex formation and dynamics at the individual molecule level with high sensitivity. Combining this technique with our recently established in vitro reconstitution system of fly Ago2-RISC, we have developed a single-molecule observation system for RISC assembly. In this chapter, we summarize the detailed protocol for single-molecule analysis of chaperone-assisted assembly of fly Ago2-RISC as well as its target cleavage reaction.

Study of radionuclides speciation with biological molecules of interest by spectrometric techniques; Etude de la speciation des radionucleides avec les molecules d'interet biologique par approche spectrometrique

Energy Technology Data Exchange (ETDEWEB)

Lourenco, V

2007-07-15

Mechanisms of complexation and accumulation of the radionuclides at the cellular and molecular level are complex and poorly known because the studies on these subjects are scarce. Within the framework of this thesis, we studied the interactions of these cations with biological molecules of interest. We chose to focus on an actinide: uranium (VI) as well as europium as an analogue of trivalent actinides. The selected biological molecules are the phyto-chelatins: their role is to protect cells against intrusions from nonessential heavy metals (thus toxic). These proteins are likely to be implied in the mechanisms of sequestration of radionuclides in living organisms. However, their structure is complex, this is why, in order to better include/understand their reactivity, we extended our studies to lower entities which constitute them (amino acid: glycine, glutamic acid and cysteine; polypeptides: glutathione reduced and oxidized forms). In particular, we determined solution speciation (stoichiometry, structure) as well as the complexing constants associated with the formation with these species. These studies were undertaken by Time Resolved Laser induced Fluorescence (TRLIF), Electro-Spray-Mass Spectrometry (ES-MS), Nuclear Magnetic Resonance (NMR), Fourier Transform Infra-Rouge spectroscopy (FTIR) and Extended X-ray Absorption Fine Structure Spectroscopy (EXAFS).The determination of the complexation constants enabled us to conclude that the complexing capacity of these molecules with respect to radionuclides was moderate (log{sub 10}K{sub 1} {<=} 3, pH 3 or 6), the formed species are mononuclear with only one ligand molecule (1:1). The interaction is performed via oxygenated (hard) groups. The direct complexation of europium with phyto-chelatins at acidic pH was studied jointly by TRLIF and ES-MS. The complexing capacity of these molecules is much higher than that of GSH from which they result. The interaction of europium with metallothioneins is, on the contrary

Convergent microRNA actions coordinate neocortical development.

Science.gov (United States)

Barca-Mayo, Olga; De Pietri Tonelli, Davide

2014-08-01

Neocortical development is a complex process that, at the cellular level, involves tight control of self-renewal, cell fate commitment, survival, differentiation and delamination/migration. These processes require, at the molecular level, the precise regulation of intrinsic signaling pathways and extrinsic factors with coordinated action in a spatially and temporally specific manner. Transcriptional regulation plays an important role during corticogenesis; however, microRNAs (miRNAs) are emerging as important post-transcriptional regulators of various aspects of central nervous system development. miRNAs are a class of small, single-stranded noncoding RNA molecules that control the expression of the majority of protein coding genes (i.e., targets). How do different miRNAs achieve precise control of gene networks during neocortical development? Here, we critically review all the miRNA-target interactions validated in vivo, with relevance to the generation and migration of pyramidal-projection glutamatergic neurons, and for the initial formation of cortical layers in the embryonic development of rodent neocortex. In particular, we focus on convergent miRNA actions, which are still a poorly understood layer of complexity in miRNA signaling, but potentially one of the keys to disclosing how miRNAs achieve the precise coordination of complex biological processes such as neocortical development.

Single Molecule Detection in Living Biological Cells using Carbon Nanotube Optical Probes

Science.gov (United States)

Strano, Michael

2009-03-01

Nanoscale sensing elements offer promise for single molecule analyte detection in physically or biologically constrained environments. Molecular adsorption can be amplified via modulation of sharp singularities in the electronic density of states that arise from 1D quantum confinement [1]. Single-walled carbon nanotubes (SWNT), as single molecule optical sensors [2-3], offer unique advantages such as photostable near-infrared (n-IR) emission for prolonged detection through biological media, single-molecule sensitivity and, nearly orthogonal optical modes for signal transduction that can be used to identify distinct classes of analytes. Selective binding to the SWNT surface is difficult to engineer [4]. In this lecture, we will briefly review the immerging field of fluorescent diagnostics using band gap emission from SWNT. In recent work, we demonstrate that even a single pair of SWNT provides at least four optical modes that can be modulated to uniquely fingerprint chemical agents by the degree to which they alter either the emission band intensity or wavelength. We validate this identification method in vitro by demonstrating detection and identification of six genotoxic analytes, including chemotherapeutic drugs and reactive oxygen species (ROS), which are spectroscopically differentiated into four distinct classes. We also demonstrate single-molecule sensitivity in detecting hydrogen peroxide, one of the most common genotoxins and an important cellular signal. Finally, we employ our sensing and fingerprinting method of these analytes in real time within live 3T3 cells, demonstrating the first multiplexed optical detection from a nanoscale biosensor and the first label-free tool to optically discriminate between genotoxins. We will also discuss our recent efforts to fabricate biomedical sensors for real time detection of glucose and other important physiologically relevant analytes in-vivo. The response of embedded SWNT in a swellable hydrogel construct to

The Old and New RNA World

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Zofia Szweykowska-Kulińska

2014-12-01

Full Text Available Among the numerous hypotheses offering a scenario for the origin of life on Earth, the one called “The RNA World” has gained the most attention. According to this hypothesis RNA acted as a genetic information storage material, as a catalyst of all metabolic reactions, and as a regulator of all processes in the primordial world. Various experiments show that RNA molecules could have been synthesized abiotically, with the potential to mediate a whole repertoire of metabolic reactions. Ribozymes carrying out aminoacyl-tRNA reactions have been found in SELEX (systematic evolution of ligands by exponential enrichment approaches and the development of a ribosome from a RNA-built protoribosome is easy to imagine. Transfer RNA aminoacylation, protoribosome origin, and the availability of amino acids on early Earth allowed the genetic code to evolve. Encoded proteins most likely stabilized RNA molecules and were able to create channels across membranes. In the modern cell, DNA replaced RNA as the main depositor of genetic information and proteins carry out almost all metabolic reactions. However, RNA is still playing versatile, crucial roles in the cell. Apart from its classical functions in the cell, a huge small RNA world is controlling gene expression, chromatin condensation, response to environmental cues, and protecting the cell against the invasion of various nucleic acids forms. Long non-coding RNAs act as crucial gene expression regulators. Riboswitches act at the level of transcription, splicing or translation and mediate feedback regulation on biosynthesis and transport of the ligand they sense. Alternative splicing generates genetic variability and increases the protein repertoire in response to developmental or environmental changes. All these regulatory functions are essential in shaping cell plasticity in the changing milieu. Recent discoveries of new, unexpected and important functions of RNA molecules support the hypothesis that we

Deletions and recombinations with the RNA1 3' ends of different tobraviruses have created a multitude of tobacco rattle virus TCM-related RNA2 species in Alstroemeria and tulip.

Science.gov (United States)

Koenig, R; Lesemann, D-E; Pfeilstetter, E; Winter, S; Pleij, C W A

2011-04-01

In vegetatively propagated Alstroemeria plants that showed pronounced stunting and necrotic leaf spots, a tobravirus infection was diagnosed in which one tobacco rattle virus (TRV, strain AL) RNA1 species was associated with seven different RNA2 species. The latter differed considerably in size and in the types of their 3' RNA1-related sequences. The 5' RNA2-specific part of all these RNA2 molecules showed almost 100% sequence identity with that of RNA2 of the TRV isolate TCM from tulip, but in some of these RNA2 molecules it was shorter than in the TCM isolate, whereas in others it was longer. One of the TRV AL RNA2 molecules, i.e. TC3'PE-a, contained the full set of three full-length RNA2-specific ORFs (ORF2a, -2b and -2c), whereas the previously analysed TCM sequence contained only ORF2a and -2b. In four of these TRV AL RNA2 molecules, i.e. those that had a relatively short RNA2-specific part, the 3' end was identical to that of the cognate TRV AL RNA1, but in the other three, which had a long RNA2-specific part, it was closely related to that of pea early browning virus (PEBV) RNA1, which was not detected in the infected plants. A comparison with previously described TRV/PEBV RNA2 recombinants suggested that the various TRV AL RNA2 molecules may represent various steps and side steps in an evolutionary process, which is apt to open the wide host range of TRV also to PEBV-derived RNA2 species.

RNA three-way junctions can act as flexible RNA structural elements in large RNA molecules: a molecular simulation analysis

Czech Academy of Sciences Publication Activity Database

Beššeová, Ivana; Réblová, Kamila; Leontis, N.B.; Šponer, Jiří

2009-01-01

Roč. 26, č. 6 (2009), s. 830-831 ISSN 0739-1102. [The 17th Conversation . 16.06.2009-20.06.2009, Albany] Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : RNA three-way junctions * RNA Subject RIV: BO - Biophysics

Mass amplifying probe for sensitive fluorescence anisotropy detection of small molecules in complex biological samples.

Science.gov (United States)

Cui, Liang; Zou, Yuan; Lin, Ninghang; Zhu, Zhi; Jenkins, Gareth; Yang, Chaoyong James

2012-07-03

detection of small molecules by means of FA in complex biological samples.

Retroviral RNA Dimerization: From Structure to Functions

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Noé Dubois

2018-03-01

Full Text Available The genome of the retroviruses is a dimer composed by two homologous copies of genomic RNA (gRNA molecules of positive polarity. The dimerization process allows two gRNA molecules to be non-covalently linked together through intermolecular base-pairing. This step is critical for the viral life cycle and is highly conserved among retroviruses with the exception of spumaretroviruses. Furthermore, packaging of two gRNA copies into viral particles presents an important evolutionary advantage for immune system evasion and drug resistance. Recent studies reported RNA switches models regulating not only gRNA dimerization, but also translation and packaging, and a spatio-temporal characterization of viral gRNA dimerization within cells are now at hand. This review summarizes our current understanding on the structural features of the dimerization signals for a variety of retroviruses (HIVs, MLV, RSV, BLV, MMTV, MPMV…, the mechanisms of RNA dimer formation and functional implications in the retroviral cycle.

iDoRNA: An Interacting Domain-based Tool for Designing RNA-RNA Interaction Systems

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Jittrawan Thaiprasit

2016-03-01

Full Text Available RNA-RNA interactions play a crucial role in gene regulation in living organisms. They have gained increasing interest in the field of synthetic biology because of their potential applications in medicine and biotechnology. However, few novel regulators based on RNA-RNA interactions with desired structures and functions have been developed due to the challenges of developing design tools. Recently, we proposed a novel tool, called iDoDe, for designing RNA-RNA interacting sequences by first decomposing RNA structures into interacting domains and then designing each domain using a stochastic algorithm. However, iDoDe did not provide an optimal solution because it still lacks a mechanism to optimize the design. In this work, we have further developed the tool by incorporating a genetic algorithm (GA to find an RNA solution with maximized structural similarity and minimized hybridized RNA energy, and renamed the tool iDoRNA. A set of suitable parameters for the genetic algorithm were determined and found to be a weighting factor of 0.7, a crossover rate of 0.9, a mutation rate of 0.1, and the number of individuals per population set to 8. We demonstrated the performance of iDoRNA in comparison with iDoDe by using six RNA-RNA interaction models. It was found that iDoRNA could efficiently generate all models of interacting RNAs with far more accuracy and required far less computational time than iDoDe. Moreover, we compared the design performance of our tool against existing design tools using forty-four RNA-RNA interaction models. The results showed that the performance of iDoRNA is better than RiboMaker when considering the ensemble defect, the fitness score and computation time usage. However, it appears that iDoRNA is outperformed by NUPACK and RNAiFold 2.0 when considering the ensemble defect. Nevertheless, iDoRNA can still be an useful alternative tool for designing novel RNA-RNA interactions in synthetic biology research. The source code of iDoRNA

A brief introduction to single-molecule fluorescence methods

NARCIS (Netherlands)

Wildenberg, S.M.J.L.; Prevo, B.; Peterman, E.J.G.; Peterman, EJG; Wuite, GJL

2011-01-01

One of the more popular single-molecule approaches in biological science is single-molecule fluorescence microscopy, which is the subject of the following section of this volume. Fluorescence methods provide the sensitivity required to study biology on the single-molecule level, but they also allow

A brief introduction to single-molecule fluorescence methods

NARCIS (Netherlands)

van den Wildenberg, Siet M.J.L.; Prevo, Bram; Peterman, Erwin J.G.

2018-01-01

One of the more popular single-molecule approaches in biological science is single-molecule fluorescence microscopy, which will be the subject of the following section of this volume. Fluorescence methods provide the sensitivity required to study biology on the single-molecule level, but they also

Mg2+ -Dependent High Mechanical Anisotropy of Three-Way-Junction pRNA as Revealed by Single-Molecule Force Spectroscopy.

Science.gov (United States)

Sun, Yang; Di, Weishuai; Li, Yiran; Huang, Wenmao; Wang, Xin; Qin, Meng; Wang, Wei; Cao, Yi

2017-08-01

Mechanical anisotropy is ubiquitous in biological tissues but is hard to reproduce in synthetic biomaterials. Developing molecular building blocks with anisotropic mechanical response is the key towards engineering anisotropic biomaterials. The three-way-junction (3WJ) pRNA, derived from ϕ29 DNA packaging motor, shows strong mechanical anisotropy upon Mg 2+ binding. In the absence of Mg 2+ , 3WJ-pRNA is mechanically weak without noticeable mechanical anisotropy. In the presence of Mg 2+ , the unfolding forces can differ by more than 4-fold along different pulling directions, ranging from about 47 pN to about 219 pN. Mechanical anisotropy of 3WJ-pRNA stems from pulling direction dependent cooperativity for the rupture of two Mg 2+ binding sites, which is a novel mechanism for the mechanical anisotropy of biomacromolecules. It is anticipated that 3WJ-pRNA can be used as a key element for the construction of biomaterials with controllable mechanical anisotropy. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Design, synthesis, and evaluation of bioactive molecules; Quantification of tricyclic pyrones from pharmacokinetic studies; Nanodelivery of siRNA; and Synthesis of viral protease inhibitors

Science.gov (United States)

Weerasekara, Sahani Manjitha

Four research projects were carried out and they are described in this dissertation. Glycogen synthase kinase-3 beta (GSK3?) plays a pivotal and central role in the pathogenesis of Alzheimer's disease (AD) and protein kinase C (PKC) controls the function of other proteins via phosphorylation and involves in tumor promotion. In pursuit of identifying novel GSK3beta and/or PKC inhibitors, substituted quinoline molecules were designed and synthesized based on the structure-activity-relationship studies. Synthesized molecules were evaluated for their neural protective activities and selected molecules were further tested for inhibitory activities on GSK3beta and PKC enzymes. Among these compounds, compound 2 was found to have better GSK3beta enzyme inhibitory and MC65 cell protection activities at low nanomolar concentrations and poor PKC inhibitory activity whereas compound 3 shows better PKC inhibitory activity. This demonstrates the potential for uses of quinoline scaffold in designing novel compounds for AD and cancer. Pharmacokinetics and distribution profiles of two anti-Alzheimer molecules, CP2 and TP70, discovered in our laboratory were assessed using HPLC/MS. Plasma samples of mice and rats fed with TP70 via different routes over various times were analyzed to quantify the amounts of TP70 in plasma of both species. Distribution profiles of TP70 in various tissues of mice were studied and results show that TP70 penetrated the blood brain barrier and accumulated in the brain tissue in significant amounts. Similarly, the amount of CP2 in plasma of mice was analyzed. The HPLC analysis revealed that both compounds have good PK profiles and bioavailability, which would make them suitable candidates for further in vivo efficacy studies. Nanodelivery of specific dsRNA for suppressing the western corn rootworm (WCR, Diabrotica virgifera virgifera) genes was studied using modified chitosan or modified polyvinylpyrrolidinone (PVP) as nanocarriers. Computational

Integrating large-scale data and RNA technology to protect crops from fungal pathogens

Directory of Open Access Journals (Sweden)

Ian Joseph Girard

2016-05-01

Full Text Available With a rapidly growing human population it is expected that plant science researchers and the agricultural community will need to increase food productivity using less arable land. This challenge is complicated by fungal pathogens and diseases, many of which can severely impact crop yield. Current measures to control fungal pathogens are either ineffective or have adverse effects on the agricultural enterprise. Thus, developing new strategies through research innovation to protect plants from pathogenic fungi is necessary to overcome these hurdles. RNA sequencing technologies are increasing our understanding of the underlying genes and gene regulatory networks mediating disease outcomes. The application of invigorating next generation sequencing strategies to study plant-pathogen interactions has and will provide unprecedented insight into the complex patterns of gene activity responsible for crop protection. However, questions remain about how biological processes in both the pathogen and the host are specified in space directly at the site of infection and over the infection period. The integration of cutting edge molecular and computational tools will provide plant scientists with the arsenal required to identify genes and molecules that play a role in plant protection. Large scale RNA sequence data can then be used to protect plants by targeting genes essential for pathogen viability in the production of stably transformed lines expressing RNA interference molecules, or through foliar applications of double stranded RNA.

MiRNA-548ah, a Potential Molecule Associated with Transition from Immune Tolerance to Immune Activation of Chronic Hepatitis B

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Tong-Jing Xing

2014-08-01

Full Text Available Objective: The present study aims to identify the differently expressed microRNA (miRNA molecules and target genes of miRNA in the immune tolerance (IT and immune activation (IA stages of chronic hepatitis B (CHB. Methods: miRNA expression profiles of peripheral blood mononuclear cells (PBMCs at the IT and IA stages of CHB were screened using miRNA microarrays and authenticated using a quantitative real-time polymerase chain reaction (RT-PCR. Gene ontology (GO and the Kyoto encyclopedia of genes and genomes (KEGG were used to analyze the significant functions and pathways of possible target genes of miRNAs. Assays of the gain and loss of function of the miRNAs were performed to verify the target genes in THP-1 cell lines. The luciferase reporter test was used on 293T cells as direct targets. Results: Significantly upregulated miR-548 and miR-4804 were observed in the miRNA microarrays and confirmed by RT-PCR in PBMCs at the IT and IA stages of CHB. GO and KEGG analysis revealed that MiR-548 and miR-4804 could be involved in numerous signaling pathways and protein binding activity. IFNγR1 was predicted as a target gene and validated as the direct gene of MiR-548. Significant negative correlation was found between the miR-548ah and mRNA levels of IFN-γR1 in CHB patients. Conclusions: The abnormal expression profiles of miRNA in PBMCs could be closely associated with immune activation of chronic HBV infection. miR-548, by targeting IFN-γR1, may represent a mechanism that can facilitate viral pathogenesis and help determine new therapeutic molecular targets.

Conceptual Model-based Systems Biology: mapping knowledge and discovering gaps in the mRNA transcription cycle.

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Judith Somekh

2012-12-01

Full Text Available We propose a Conceptual Model-based Systems Biology framework for qualitative modeling, executing, and eliciting knowledge gaps in molecular biology systems. The framework is an adaptation of Object-Process Methodology (OPM, a graphical and textual executable modeling language. OPM enables concurrent representation of the system's structure-the objects that comprise the system, and behavior-how processes transform objects over time. Applying a top-down approach of recursively zooming into processes, we model a case in point-the mRNA transcription cycle. Starting with this high level cell function, we model increasingly detailed processes along with participating objects. Our modeling approach is capable of modeling molecular processes such as complex formation, localization and trafficking, molecular binding, enzymatic stimulation, and environmental intervention. At the lowest level, similar to the Gene Ontology, all biological processes boil down to three basic molecular functions: catalysis, binding/dissociation, and transporting. During modeling and execution of the mRNA transcription model, we discovered knowledge gaps, which we present and classify into various types. We also show how model execution enhances a coherent model construction. Identification and pinpointing knowledge gaps is an important feature of the framework, as it suggests where research should focus and whether conjectures about uncertain mechanisms fit into the already verified model.

Ultra-sensitive DNA assay based on single-molecule detection coupled with fluorescent quantum dot-labeling and its application to determination of messenger RNA

International Nuclear Information System (INIS)

Li Li; Li Xincang; Li Lu; Wang Jinxing; Jin Wenrui

2011-01-01

An ultra-sensitive single-molecule detection (SMD) method for quantification of DNA using total internal reflection fluorescence microscopy (TIRFM) coupled with fluorescent quantum dot (QD)-labeling was developed. In this method, the target DNA (tDNA) was captured by the capture DNA immobilized on the silanized coverslip blocked with ethanolamine and bovine serum albumin. Then, the QD-labeled probe DNA was hybridized to the tDNA. Ten fluorescent images of the QD-labeled sandwich DNA hybrids on the coverslip were taken by a high-sensitive CCD. The tDNA was quantified by counting the bright spots on the images using a calibration curve. The LOD of the method was 1 x 10 -14 mol L -1 . Several key factors, including image acquirement, fluorescence probe, substrate preparation, noise elimination from solutions and glass coverslips, and nonspecific adsorption and binding of solution-phase detection probes were discussed in detail. The method could be applied to quantify messenger RNA (mRNA) in cells. In order to determine mRNA, double-stranded RNA-DNA hybrids consisting of mRNA and corresponding cDNA were synthesized from the cellular mRNA template using reverse transcription in the presence of reverse transcriptase. After removing the mRNA in the double-stranded hybrids using ribonuclease, cDNA was quantified using the SMD-based TIRFM. Osteopontin mRNA in decidual stromal cells was chosen as the model analyte.

TruSeq Stranded mRNA and Total RNA Sample Preparation Kits

Science.gov (United States)

Total RNA-Seq enabled by ribosomal RNA (rRNA) reduction is compatible with formalin-fixed paraffin embedded (FFPE) samples, which contain potentially critical biological information. The family of TruSeq Stranded Total RNA sample preparation kits provides a unique combination of unmatched data quality for both mRNA and whole-transcriptome analyses, robust interrogation of both standard and low-quality samples and workflows compatible with a wide range of study designs.

Sequence-engineered mRNA Without Chemical Nucleoside Modifications Enables an Effective Protein Therapy in Large Animals

Science.gov (United States)

Thess, Andreas; Grund, Stefanie; Mui, Barbara L; Hope, Michael J; Baumhof, Patrick; Fotin-Mleczek, Mariola; Schlake, Thomas

2015-01-01

Being a transient carrier of genetic information, mRNA could be a versatile, flexible, and safe means for protein therapies. While recent findings highlight the enormous therapeutic potential of mRNA, evidence that mRNA-based protein therapies are feasible beyond small animals such as mice is still lacking. Previous studies imply that mRNA therapeutics require chemical nucleoside modifications to obtain sufficient protein expression and avoid activation of the innate immune system. Here we show that chemically unmodified mRNA can achieve those goals as well by applying sequence-engineered molecules. Using erythropoietin (EPO) driven production of red blood cells as the biological model, engineered Epo mRNA elicited meaningful physiological responses from mice to nonhuman primates. Even in pigs of about 20 kg in weight, a single adequate dose of engineered mRNA encapsulated in lipid nanoparticles (LNPs) induced high systemic Epo levels and strong physiological effects. Our results demonstrate that sequence-engineered mRNA has the potential to revolutionize human protein therapies. PMID:26050989

Voltammetric detection of biological molecules using chopped carbon fiber.

Science.gov (United States)

Sugawara, Kazuharu; Yugami, Asako; Kojima, Akira

2010-01-01

Voltammetric detection of biological molecules was carried out using chopped carbon fibers produced from carbon fiber reinforced plastics that are biocompatible and inexpensive. Because chopped carbon fibers normally are covered with a sizing agent, they are difficult to use as an electrode. However, when the surface of a chopped carbon fiber was treated with ethanol and hydrochloric acid, it became conductive. To evaluate the functioning of chopped carbon fibers, voltammetric measurements of [Fe(CN)(6)](3-) were carried out. Redoxes of FAD, ascorbic acid and NADH as biomolecules were recorded using cyclic voltammetry. The sizing agents used to bundle the fibers were epoxy, polyamide and polyurethane resins. The peak currents were the greatest when using the chopped carbon fibers that were created with epoxy resins. When the electrode response of the chopped carbon fibers was compared with that of a glassy carbon electrode, the peak currents and the reversibility of the electrode reaction were sufficient. Therefore, the chopped carbon fibers will be useful as disposable electrodes for the sensing of biomolecules.

The determinants of alternative RNA splicing in human cells.

Science.gov (United States)

Ramanouskaya, Tatsiana V; Grinev, Vasily V

2017-12-01

Alternative splicing represents an important level of the regulation of gene function in eukaryotic organisms. It plays a critical role in virtually every biological process within an organism, including regulation of cell division and cell death, differentiation of tissues in the embryo and the adult organism, as well as in cellular response to diverse environmental factors. In turn, studies of the last decade have shown that alternative splicing itself is controlled by different mechanisms. Unfortunately, there is no clear understanding of how these diverse mechanisms, or determinants, regulate and constrain the set of alternative RNA species produced from any particular gene in every cell of the human body. Here, we provide a consolidated overview of alternative splicing determinants including RNA-protein interactions, epigenetic regulation via chromatin remodeling, coupling of transcription-to-alternative splicing, effect of secondary structures in pre-RNA, and function of the RNA quality control systems. We also extensively and critically discuss some mechanistic insights on coordinated inclusion/exclusion of exons during the formation of mature RNA molecules. We conclude that the final structure of RNA is pre-determined by a complex interplay between cis- and trans-acting factors. Altogether, currently available empirical data significantly expand our understanding of the functioning of the alternative splicing machinery of cells in normal and pathological conditions. On the other hand, there are still many blind spots that require further deep investigations.

Working with RNA

DEFF Research Database (Denmark)

Nielsen, Henrik

2011-01-01

Working with RNA is not a special discipline in molecular biology. However, RNA is chemically and structurally different from DNA and a few simple work rules have to be implemented to maintain the integrity of the RNA. Alkaline pH, high temperatures, and heavy metal ions should be avoided when po...

Conserved and variable domains of RNase MRP RNA.

Science.gov (United States)

Dávila López, Marcela; Rosenblad, Magnus Alm; Samuelsson, Tore

2009-01-01

Ribonuclease MRP is a eukaryotic ribonucleoprotein complex consisting of one RNA molecule and 7-10 protein subunits. One important function of MRP is to catalyze an endonucleolytic cleavage during processing of rRNA precursors. RNase MRP is evolutionary related to RNase P which is critical for tRNA processing. A large number of MRP RNA sequences that now are available have been used to identify conserved primary and secondary structure features of the molecule. MRP RNA has structural features in common with P RNA such as a conserved catalytic core, but it also has unique features and is characterized by a domain highly variable between species. Information regarding primary and secondary structure features is of interest not only in basic studies of the function of MRP RNA, but also because mutations in the RNA give rise to human genetic diseases such as cartilage-hair hypoplasia.

Biological compost stability influences odor molecules production measured by electronic nose during food-waste high-rate composting

International Nuclear Information System (INIS)

D'Imporzano, Giuliana; Crivelli, Fernando; Adani, Fabrizio

2008-01-01

Composting is a technique that is used to convert organic waste into agriculturally useful products. Composting is an aerobic, solid-state biological process, which typically can be divided into two phases, a high-rate composting phase and a curing phase. High-rate composting plays an important role during the composting process, owing to the high microbial activity occurring during this phase. It requires an accurate plant design to prevent the formation of anaerobic conditions and odors. The formation of anaerobic conditions mainly depends on the rate of O 2 consumption needed to degrade the substrate, i.e., the biological stability of the substrate. In this study, we investigated the relationship between the biological activity, measured by the dynamic respiration index (DRI) and the odor molecules production, measured by an electronic nose (EN) during two food-waste high-rate composting processes. Although the O 2 concentration in the biomass free air space (FAS) was kept optimal (O 2 > 140 ml l -1 , v/v) during composting, strong anaerobic conditions developed. This was indicated by the high levels of sulfur compounds, methane, and hydrogen in the outlet air stream. Both the high level of O 2 consumption, needed to degrade the high-degradable water-soluble organic matter and the low water O 2 solubility, caused by high temperature reached in this stage (up to 60 deg. C), led to the anaerobic conditions observed in the biofilm-particle level. The application of the partial least square (PLS) analysis demonstrated a good regression between the DRI and the odor molecules produced that was detected by the EN (R 2 = 0.991; R 2 CV = 0.990), signifying the usefulness of the DRI as a parameter to estimate the potential production of odor molecules of the biomass

MicroRNA and Male Infertility: A Potential for Diagnosis

Directory of Open Access Journals (Sweden)

Yahya Khazaie

2014-07-01

Full Text Available MicroRNAs (miRNAs are small non-coding single stranded RNA molecules that are physiologically produced in eukaryotic cells to regulate or mostly down-regulate genes by pairing with their complementary base-sequence in related mRNA molecules in the cytoplasm. It has been reported that other than its function in many physiological cell processes, dysregulation of miRNAs plays a role in the development of many diseases. In this short review, the association between miRNAs and some male reproductive disorders is surveyed. Male factor Infertility is a devastating problem from which a notable percentage of couples suffer. However, the molecular mechanism of many infertility disorders has not been clearly elucidated. Since miRNAs have an important role in numerous biological cell processes and cellular dysfunctions, it is of interest to review the related literature on the role of miRNAs in the male reproductive organs. Aberrant expression of specific miRNAs is associated with certain male reproductive dysfunctions. For this reason, assessment of expression of such miRNAs may serve as a suitable molecular biomarker for diagnosis of those male infertility disorders. The presence of a single nucleotide polymorphism (SNP at the miRNAs’ binding site in its targeted mRNA has been reported to have an association with idiopathic male infertility. Also, a relation with male infertility has been shown with SNP in the genes of the factors necessary for miRNA biogenesis. Therefore, focusing on the role of miRNAs in male reproductive disorders can further elucidate the molecular mechanisms of male infertility and generate the potential for locating efficient biomarkers and therapeutic agents for these disorders.

RNA inverse folding using Monte Carlo tree search.

Science.gov (United States)

Yang, Xiufeng; Yoshizoe, Kazuki; Taneda, Akito; Tsuda, Koji

2017-11-06

Artificially synthesized RNA molecules provide important ways for creating a variety of novel functional molecules. State-of-the-art RNA inverse folding algorithms can design simple and short RNA sequences of specific GC content, that fold into the target RNA structure. However, their performance is not satisfactory in complicated cases. We present a new inverse folding algorithm called MCTS-RNA, which uses Monte Carlo tree search (MCTS), a technique that has shown exceptional performance in Computer Go recently, to represent and discover the essential part of the sequence space. To obtain high accuracy, initial sequences generated by MCTS are further improved by a series of local updates. Our algorithm has an ability to control the GC content precisely and can deal with pseudoknot structures. Using common benchmark datasets for evaluation, MCTS-RNA showed a lot of promise as a standard method of RNA inverse folding. MCTS-RNA is available at https://github.com/tsudalab/MCTS-RNA .

Expedient construction of small molecule macroarrays via sequential palladium- and copper-mediated reactions and their ex situ biological testing.

Science.gov (United States)

Frei, Reto; Breitbach, Anthony S; Blackwell, Helen E

2012-05-01

We report the highly efficient syntheses of a series of focused libraries in the small molecule macroarray format using Suzuki-Miyaura and copper-catalyzed azide-alkyne cycloaddition (or "click") reactions. The libraries were based on stilbene and triazole scaffolds, which are known to have a broad range of biological activities, including quorum-sensing (QS) modulation in bacteria. The library products were generated in parallel on the macroarray in extremely short reaction times (~10-20 min) and isolated in excellent purities. Biological testing of one macroarray library post-cleavage (ex situ) revealed several potent agonists of the QS receptor, LuxR, in Vibrio fischeri. These synthetic agonists, in contrast to others that we have reported, were only active in the presence of the native QS signal in V. fischeri, which is suggestive of a different mode of activity. Notably, the results presented herein showcase the ready compatibility of the macroarray platform with chemical reactions that are commonly utilized in small molecule probe and drug discovery today. As such, this work serves to expand the utility of the small molecule macroarray as a rapid and operationally straightforward approach toward the synthesis and screening of bioactive agents.

General enumeration of RNA secondary structures based on new ...

African Journals Online (AJOL)

akpobome

coding, transferring and retrieving genetic information, and in directing cell metabolism. The nucleic acid includes DNA and RNA molecule. RNA molecule is a single-stranded nucleic acid of four different kinds of nucleotides. The four nucleotides only differ by one part, called bases. Hence, one usually identifies nucleotides.

bcl::Cluster : A method for clustering biological molecules coupled with visualization in the Pymol Molecular Graphics System.

Science.gov (United States)

Alexander, Nathan; Woetzel, Nils; Meiler, Jens

2011-02-01

Clustering algorithms are used as data analysis tools in a wide variety of applications in Biology. Clustering has become especially important in protein structure prediction and virtual high throughput screening methods. In protein structure prediction, clustering is used to structure the conformational space of thousands of protein models. In virtual high throughput screening, databases with millions of drug-like molecules are organized by structural similarity, e.g. common scaffolds. The tree-like dendrogram structure obtained from hierarchical clustering can provide a qualitative overview of the results, which is important for focusing detailed analysis. However, in practice it is difficult to relate specific components of the dendrogram directly back to the objects of which it is comprised and to display all desired information within the two dimensions of the dendrogram. The current work presents a hierarchical agglomerative clustering method termed bcl::Cluster. bcl::Cluster utilizes the Pymol Molecular Graphics System to graphically depict dendrograms in three dimensions. This allows simultaneous display of relevant biological molecules as well as additional information about the clusters and the members comprising them.

Application of Live-Cell RNA Imaging Techniques to the Study of Retroviral RNA Trafficking

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Darrin V. Bann

2012-06-01

Full Text Available Retroviruses produce full-length RNA that serves both as a genomic RNA (gRNA, which is encapsidated into virus particles, and as an mRNA, which directs the synthesis of viral structural proteins. However, we are only beginning to understand the cellular and viral factors that influence trafficking of retroviral RNA and the selection of the RNA for encapsidation or translation. Live cell imaging studies of retroviral RNA trafficking have provided important insight into many aspects of the retrovirus life cycle including transcription dynamics, nuclear export of viral RNA, translational regulation, membrane targeting, and condensation of the gRNA during virion assembly. Here, we review cutting-edge techniques to visualize single RNA molecules in live cells and discuss the application of these systems to studying retroviral RNA trafficking.

Full-length mRNA sequencing uncovers a widespread coupling between transcription initiation and mRNA processing.

Science.gov (United States)

Anvar, Seyed Yahya; Allard, Guy; Tseng, Elizabeth; Sheynkman, Gloria M; de Klerk, Eleonora; Vermaat, Martijn; Yin, Raymund H; Johansson, Hans E; Ariyurek, Yavuz; den Dunnen, Johan T; Turner, Stephen W; 't Hoen, Peter A C

2018-03-29

The multifaceted control of gene expression requires tight coordination of regulatory mechanisms at transcriptional and post-transcriptional level. Here, we studied the interdependence of transcription initiation, splicing and polyadenylation events on single mRNA molecules by full-length mRNA sequencing. In MCF-7 breast cancer cells, we find 2700 genes with interdependent alternative transcription initiation, splicing and polyadenylation events, both in proximal and distant parts of mRNA molecules, including examples of coupling between transcription start sites and polyadenylation sites. The analysis of three human primary tissues (brain, heart and liver) reveals similar patterns of interdependency between transcription initiation and mRNA processing events. We predict thousands of novel open reading frames from full-length mRNA sequences and obtained evidence for their translation by shotgun proteomics. The mapping database rescues 358 previously unassigned peptides and improves the assignment of others. By recognizing sample-specific amino-acid changes and novel splicing patterns, full-length mRNA sequencing improves proteogenomics analysis of MCF-7 cells. Our findings demonstrate that our understanding of transcriptome complexity is far from complete and provides a basis to reveal largely unresolved mechanisms that coordinate transcription initiation and mRNA processing.

NIR-to-visible upconversion nanoparticles for fluorescent labeling and targeted delivery of siRNA

International Nuclear Information System (INIS)

Jiang Shan; Zhang Yong; Lim, Kian Meng; Sim, Eugene K W; Ye Lei

2009-01-01

Near-infrared (NIR)-to-visible upconversion fluorescent nanoparticles were synthesized and used for imaging and targeted delivery of small interfering RNA (siRNA) to cancer cells. Silica-coated NaYF 4 upconversion nanoparticles (UCNs) co-doped with lanthanide ions (Yb/Er) were synthesized. Folic acid and anti-Her2 antibody conjugated UCNs were used to fluorescently label the folate receptors of HT-29 cells and Her2 receptors of SK-BR-3 cells, respectively. The intracellular uptake of the folic acid and antibody conjugated UCNs was visualized using a confocal fluorescence microscope equipped with an NIR laser. siRNA was attached to anti-Her2 antibody conjugated UCNs and the delivery of these nanoparticles to SK-BR-3 cells was studied. Meanwhile, a luciferase assay was established to confirm the gene silencing effect of siRNA. Upconversion nanoparticles can serve as a fluorescent probe and delivery system for simultaneous imaging and delivery of biological molecules.

NIR-to-visible upconversion nanoparticles for fluorescent labeling and targeted delivery of siRNA

Science.gov (United States)

Jiang, Shan; Zhang, Yong; Lim, Kian Meng; Sim, Eugene K. W.; Ye, Lei

2009-04-01

Near-infrared (NIR)-to-visible upconversion fluorescent nanoparticles were synthesized and used for imaging and targeted delivery of small interfering RNA (siRNA) to cancer cells. Silica-coated NaYF4 upconversion nanoparticles (UCNs) co-doped with lanthanide ions (Yb/Er) were synthesized. Folic acid and anti-Her2 antibody conjugated UCNs were used to fluorescently label the folate receptors of HT-29 cells and Her2 receptors of SK-BR-3 cells, respectively. The intracellular uptake of the folic acid and antibody conjugated UCNs was visualized using a confocal fluorescence microscope equipped with an NIR laser. siRNA was attached to anti-Her2 antibody conjugated UCNs and the delivery of these nanoparticles to SK-BR-3 cells was studied. Meanwhile, a luciferase assay was established to confirm the gene silencing effect of siRNA. Upconversion nanoparticles can serve as a fluorescent probe and delivery system for simultaneous imaging and delivery of biological molecules.

Ultra-sensitive DNA assay based on single-molecule detection coupled with fluorescent quantum dot-labeling and its application to determination of messenger RNA

Energy Technology Data Exchange (ETDEWEB)

Li Li [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Li Xincang [School of Life Sciences, Shandong University, Jinan 250100 (China); Li Lu [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Wang Jinxing [School of Life Sciences, Shandong University, Jinan 250100 (China); Jin Wenrui, E-mail: jwr@sdu.edu.cn [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China)

2011-01-24

An ultra-sensitive single-molecule detection (SMD) method for quantification of DNA using total internal reflection fluorescence microscopy (TIRFM) coupled with fluorescent quantum dot (QD)-labeling was developed. In this method, the target DNA (tDNA) was captured by the capture DNA immobilized on the silanized coverslip blocked with ethanolamine and bovine serum albumin. Then, the QD-labeled probe DNA was hybridized to the tDNA. Ten fluorescent images of the QD-labeled sandwich DNA hybrids on the coverslip were taken by a high-sensitive CCD. The tDNA was quantified by counting the bright spots on the images using a calibration curve. The LOD of the method was 1 x 10{sup -14} mol L{sup -1}. Several key factors, including image acquirement, fluorescence probe, substrate preparation, noise elimination from solutions and glass coverslips, and nonspecific adsorption and binding of solution-phase detection probes were discussed in detail. The method could be applied to quantify messenger RNA (mRNA) in cells. In order to determine mRNA, double-stranded RNA-DNA hybrids consisting of mRNA and corresponding cDNA were synthesized from the cellular mRNA template using reverse transcription in the presence of reverse transcriptase. After removing the mRNA in the double-stranded hybrids using ribonuclease, cDNA was quantified using the SMD-based TIRFM. Osteopontin mRNA in decidual stromal cells was chosen as the model analyte.

A low temperature scanning force microscope for biological samples

Energy Technology Data Exchange (ETDEWEB)

Gustafsson, Mats Gustaf Lennart [Univ. of California, Berkeley, CA (United States)

1993-05-01

An SFM has been constructed capable of operating at 143 K. Two contributions to SFM technology are described: a new method of fabricating tips, and new designs of SFM springs that significantly lower the noise level. The SFM has been used to image several biological samples (including collagen, ferritin, RNA, purple membrane) at 143 K and room temperature. No improvement in resolution resulted from 143 K operation; several possible reasons for this are discussed. Possibly sharper tips may help. The 143 K SFM will allow the study of new categories of samples, such as those prepared by freeze-frame, single molecules (temperature dependence of mechanical properties), etc. The SFM was used to cut single collagen molecules into segments with a precision of {le} 10 nm.

Overview of methods in RNA nanotechnology: synthesis, purification, and characterization of RNA nanoparticles.

Science.gov (United States)

Haque, Farzin; Guo, Peixuan

2015-01-01

RNA nanotechnology encompasses the use of RNA as a construction material to build homogeneous nanostructures by bottom-up self-assembly with defined size, structure, and stoichiometry; this pioneering concept demonstrated in 1998 (Guo et al., Molecular Cell 2:149-155, 1998; featured in Cell) has emerged as a new field that also involves materials engineering and synthetic structural biology (Guo, Nature Nanotechnology 5:833-842, 2010). The field of RNA nanotechnology has skyrocketed over the last few years, as evidenced by the burst of publications in prominent journals on RNA nanostructures and their applications in nanomedicine and nanotechnology. Rapid advances in RNA chemistry, RNA biophysics, and RNA biology have created new opportunities for translating basic science into clinical practice. RNA nanotechnology holds considerable promise in this regard. Increased evidence also suggests that substantial part of the 98.5 % of human genome (Lander et al. Nature 409:860-921, 2001) that used to be called "junk DNA" actually codes for noncoding RNA. As we understand more on how RNA structures are related to function, we can fabricate synthetic RNA nanoparticles for the diagnosis and treatment of diseases. This chapter provides a brief overview of the field regarding the design, construction, purification, and characterization of RNA nanoparticles for diverse applications in nanotechnology and nanomedicince.

Application of Fourier transform infrared ellipsometry to assess the concentration of biological molecules

Science.gov (United States)

Garcia-Caurel, Enric; Drevillon, Bernard; De Martino, Antonello; Schwartz, Laurent

2002-12-01

Spectroscopic ellipsometry is a noninvasive optical characterization technique mainly used in the semiconductor field to characterize bare substrates and thin films. In particular, it allows the gathering of information concerning the physical structure of the sample, such as roughness and film thickness, as well as its optical response. In the mid-infrared (IR) range each molecule exhibits a characteristic absorption fingerprint, which makes this technique chemically selective. Phase-modulated IR ellipsometry does not require a baseline correction procedure or suppression of atmospheric CO2 and water-vapor absorption bands, thus greatly reducing the subjectivity in data analysis. We have found that ellipsometric measurements of thin films, such as the solid residuals left on a plane surface after evaporation of a liquid drop containing a given compound in solution, are particularly favorable for dosing purposes because the intensity of IR absorptions shows a linear behavior along a wide range of solution concentrations of the given compound. Our aim is to illustrate with a concrete example and to justify theoretically the linearity experimentally found between radiation absorption and molecule concentration. For the example, we prepared aqueous solutions of glycogen, a molecule of huge biological importance currently tested in biochemical analyses, at concentrations ranging from 1 mg/l to 1 g/l, which correspond to those found in physiological conditions. The results of this example are promising for the application of ellipsometry for dosing purposes in biochemistry and biomedicine.

The RNA synthesis machinery of negative-stranded RNA viruses

International Nuclear Information System (INIS)

Ortín, Juan; Martín-Benito, Jaime

2015-01-01

The group of Negative-Stranded RNA Viruses (NSVs) includes many human pathogens, like the influenza, measles, mumps, respiratory syncytial or Ebola viruses, which produce frequent epidemics of disease and occasional, high mortality outbreaks by transmission from animal reservoirs. The genome of NSVs consists of one to several single-stranded, negative-polarity RNA molecules that are always assembled into mega Dalton-sized complexes by association to many nucleoprotein monomers. These RNA-protein complexes or ribonucleoproteins function as templates for transcription and replication by action of the viral RNA polymerase and accessory proteins. Here we review our knowledge on these large RNA-synthesis machines, including the structure of their components, the interactions among them and their enzymatic activities, and we discuss models showing how they perform the virus transcription and replication programmes. - Highlights: • Overall organisation of NSV RNA synthesis machines. • Structure and function of the ribonucleoprotein components: Atomic structure of the RNA polymerase complex. • Commonalities and differences between segmented- and non-segmented NSVs. • Transcription versus replication programmes

The RNA synthesis machinery of negative-stranded RNA viruses

Energy Technology Data Exchange (ETDEWEB)

Ortín, Juan, E-mail: jortin@cnb.csic.es [Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología (CSIC) and CIBER de Enfermedades Respiratorias (ISCIII), Madrid (Spain); Martín-Benito, Jaime, E-mail: jmartinb@cnb.csic.es [Department of Macromolecular Structures, Centro Nacional de Biotecnología (CSIC), Madrid (Spain)

2015-05-15

The group of Negative-Stranded RNA Viruses (NSVs) includes many human pathogens, like the influenza, measles, mumps, respiratory syncytial or Ebola viruses, which produce frequent epidemics of disease and occasional, high mortality outbreaks by transmission from animal reservoirs. The genome of NSVs consists of one to several single-stranded, negative-polarity RNA molecules that are always assembled into mega Dalton-sized complexes by association to many nucleoprotein monomers. These RNA-protein complexes or ribonucleoproteins function as templates for transcription and replication by action of the viral RNA polymerase and accessory proteins. Here we review our knowledge on these large RNA-synthesis machines, including the structure of their components, the interactions among them and their enzymatic activities, and we discuss models showing how they perform the virus transcription and replication programmes. - Highlights: • Overall organisation of NSV RNA synthesis machines. • Structure and function of the ribonucleoprotein components: Atomic structure of the RNA polymerase complex. • Commonalities and differences between segmented- and non-segmented NSVs. • Transcription versus replication programmes.

How short RNAs impact the human ribonuclease Dicer activity: putative regulatory feedback-loops and other RNA-mediated mechanisms controlling microRNA processing.

Science.gov (United States)

Koralewska, Natalia; Hoffmann, Weronika; Pokornowska, Maria; Milewski, Marek; Lipinska, Andrea; Bienkowska-Szewczyk, Krystyna; Figlerowicz, Marek; Kurzynska-Kokorniak, Anna

2016-01-01

Ribonuclease Dicer plays a pivotal role in RNA interference pathways by processing long double-stranded RNAs and single-stranded hairpin RNA precursors into small interfering RNAs (siRNAs) and microRNAs (miRNAs), respectively. While details of Dicer regulation by a variety of proteins are being elucidated, less is known about non-protein factors, e.g. RNA molecules, that may influence this enzyme's activity. Therefore, we decided to investigate the question of whether the RNA molecules can function not only as Dicer substrates but also as its regulators. Our previous in vitro studies indicated that the activity of human Dicer can be influenced by short RNA molecules that either bind to Dicer or interact with its substrates, or both. Those studies were carried out with commercial Dicer preparations. Nevertheless, such preparations are usually not homogeneous enough to carry out more detailed RNA-binding studies. Therefore, we have established our own system for the production of human Dicer in insect cells. In this manuscript, we characterize the RNA-binding and RNA-cleavage properties of the obtained preparation. We demonstrate that Dicer can efficiently bind single-stranded RNAs that are longer than ~20-nucleotides. Consequently, we revisit possible scenarios of Dicer regulation by single-stranded RNA species ranging from ~10- to ~60-nucleotides, in the context of their binding to this enzyme. Finally, we show that siRNA/miRNA-sized RNAs may affect miRNA production either by binding to Dicer or by participating in regulatory feedback-loops. Altogether, our studies suggest a broad regulatory role of short RNAs in Dicer functioning.

Sources and basic threats of biological safety

International Nuclear Information System (INIS)

Nazarova, O.D.

2010-01-01

Full text: Biological safety of any state is connected with development of its public protection against biological weapons and opportunity to prevent bio terrorist attacks. That's why in modern social-economic and geo-political conditions, the problem of biological safety strengthening become significant, which is connected with migration process globalization, development of bio-technology and dramatically increased risk of pathogenic germ infections proliferation, which can be used as biological weapon. Despite of undertaken efforts by world community on full prohibition of biological weapon, its proliferation in the world still takes place. Biology revolution during second and third millennium lead to development not only biotechnology but new achievements in medicine, agriculture and other fields of economy, but also created scientific and research preconditions for development of advanced biological means of mass destruction, that make it more attractive for achieving superiority and assigned targets: low developments costs, opportunity to create it by one small laboratory with two-three high qualified specialists bio technologists; tremendous impact effect: one substance gram can contain from one till one hundreds quintillions (10"1"8 - 10"2"0) active pathogen molecules and in case if they belong to amplificated RNA and DNA, each molecule getting to organism, will multiply and contaminate environment (the last one is its principal difference from chemical weapon); bypass of organism immunological barriers and specific vaccinations; unusual clinic finding, hard diagnosis; weakness of traditional medications and treatment methods; lack of material destruction; opportunity of tight-lipped developments; opportunity of tight-lipped application; opportunity of delayed effect; opportunity of selective influence on specific population (by use of genetic, climatic and cultural specifications of race, nations and nationalities). Above mentioned specifications create

Cardiomyocyte microvesicles contain DNA/RNA and convey biological messages to target cells.

Directory of Open Access Journals (Sweden)

Anders Waldenström

Full Text Available BACKGROUND: Shedding microvesicles are membrane released vesicles derived directly from the plasma membrane. Exosomes are released membrane vesicles of late endosomal origin that share structural and biochemical characteristics with prostasomes. Microvesicles/exosomes can mediate messages between cells and affect various cell-related processes in their target cells. We describe newly detected microvesicles/exosomes from cardiomyocytes and depict some of their biological functions. METHODOLOGY/PRINCIPAL FINDINGS: Microvesicles/exosomes from media of cultured cardiomyocytes derived from adult mouse heart were isolated by differential centrifugation including preparative ultracentrifugation and identified by transmission electron microscopy and flow cytometry. They were surrounded by a bilayered membrane and flow cytometry revealed presence of both caveolin-3 and flotillin-1 while clathrin and annexin-2 were not detected. Microvesicle/exosome mRNA was identified and out of 1520 detected mRNA, 423 could be directly connected in a biological network. Furthermore, by a specific technique involving TDT polymerase, 343 different chromosomal DNA sequences were identified in the microvesicles/exosomes. Microvesicle/exosomal DNA transfer was possible into target fibroblasts, where exosomes stained for DNA were seen in the fibroblast cytosol and even in the nuclei. The gene expression was affected in fibroblasts transfected by microvesicles/exosomes and among 333 gene expression changes there were 175 upregulations and 158 downregulations compared with controls. CONCLUSIONS/SIGNIFICANCE: Our study suggests that microvesicles/exosomes released from cardiomyocytes, where we propose that exosomes derived from cardiomyocytes could be denoted "cardiosomes", can be involved in a metabolic course of events in target cells by facilitating an array of metabolism-related processes including gene expression changes.

Potent host-directed small-molecule inhibitors of myxovirus RNA-dependent RNA-polymerases.

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Stefanie A Krumm

Full Text Available Therapeutic targeting of host cell factors required for virus replication rather than of pathogen components opens new perspectives to counteract virus infections. Anticipated advantages of this approach include a heightened barrier against the development of viral resistance and a broadened pathogen target spectrum. Myxoviruses are predominantly associated with acute disease and thus are particularly attractive for this approach since treatment time can be kept limited. To identify inhibitor candidates, we have analyzed hit compounds that emerged from a large-scale high-throughput screen for their ability to block replication of members of both the orthomyxovirus and paramyxovirus families. This has returned a compound class with broad anti-viral activity including potent inhibition of different influenza virus and paramyxovirus strains. After hit-to-lead chemistry, inhibitory concentrations are in the nanomolar range in the context of immortalized cell lines and human PBMCs. The compound shows high metabolic stability when exposed to human S-9 hepatocyte subcellular fractions. Antiviral activity is host-cell species specific and most pronounced in cells of higher mammalian origin, supporting a host-cell target. While the compound induces a temporary cell cycle arrest, host mRNA and protein biosynthesis are largely unaffected and treated cells maintain full metabolic activity. Viral replication is blocked at a post-entry step and resembles the inhibition profile of a known inhibitor of viral RNA-dependent RNA-polymerase (RdRp activity. Direct assessment of RdRp activity in the presence of the reagent reveals strong inhibition both in the context of viral infection and in reporter-based minireplicon assays. In toto, we have identified a compound class with broad viral target range that blocks host factors required for viral RdRp activity. Viral adaptation attempts did not induce resistance after prolonged exposure, in contrast to rapid

Binding and Translocation of Termination Factor Rho Studied at the Single-Molecule Level

Science.gov (United States)

Koslover, Daniel J.; Fazal, Furqan M.; Mooney, Rachel A.; Landick, Robert; Block, Steven M.

2012-01-01

Rho termination factor is an essential hexameric helicase responsible for terminating 20–50% of all mRNA synthesis in E. coli. We used single- molecule force spectroscopy to investigate Rho-RNA binding interactions at the Rho- utilization (rut) site of the ? tR1 terminator. Our results are consistent with Rho complexes adopting two states, one that binds 57 ±2 nucleotides of RNA across all six of the Rho primary binding sites, and another that binds 85 ±2 nucleotides at the six primary sites plus a single secondary site situated at the center of the hexamer. The single-molecule data serve to establish that Rho translocates 5′-to-3′ towards RNA polymerase (RNAP) by a tethered-tracking mechanism, looping out the intervening RNA between the rut site and RNAP. These findings lead to a general model for Rho binding and translocation, and establish a novel experimental approach that should facilitate additional single- molecule studies of RNA-binding proteins. PMID:22885804

Rethinking the central dogma: noncoding RNAs are biologically relevant.

Science.gov (United States)

Robinson, Victoria L

2009-01-01

Non-coding RNAs (ncRNAs) are a large class of functional molecules with over 100 unique classes described to date. ncRNAs are diverse in terms of their function and size. A relatively new class of small ncRNA, called microRNAs (miRNA), have received a great deal of attention in the literature in recent years. miRNAs are endogenously encoded gene families that demonstrate striking evolutionary conservation. miRNAs serve essential and diverse physiological functions such as differentiation and development, proliferation, maintaining cell type phenotypes, and many others. The discovery and ongoing investigation of miRNAs is part of a revolution in biology that is changing the basic concepts of gene expression and RNA functionality. A single miRNA can participate in controlling the expression of up to several hundred protein-coding genes by interacting with mRNAs, generally in 3' untranslated regions. Our new and developing understanding of miRNAs, and other ncRNAs, promises to lead to significant contributions to medicine. Specifically, miRNAs are likely to serve as the basis for novel therapies and diagnostic tools.

Small-molecule inhibitor leads of ribosome-inactivating proteins developed using the doorstop approach.

Directory of Open Access Journals (Sweden)

Yuan-Ping Pang

2011-03-01

Full Text Available Ribosome-inactivating proteins (RIPs are toxic because they bind to 28S rRNA and depurinate a specific adenine residue from the α-sarcin/ricin loop (SRL, thereby inhibiting protein synthesis. Shiga-like toxins (Stx1 and Stx2, produced by Escherichia coli, are RIPs that cause outbreaks of foodborne diseases with significant morbidity and mortality. Ricin, produced by the castor bean plant, is another RIP lethal to mammals. Currently, no US Food and Drug Administration-approved vaccines nor therapeutics exist to protect against ricin, Shiga-like toxins, or other RIPs. Development of effective small-molecule RIP inhibitors as therapeutics is challenging because strong electrostatic interactions at the RIP•SRL interface make drug-like molecules ineffective in competing with the rRNA for binding to RIPs. Herein, we report small molecules that show up to 20% cell protection against ricin or Stx2 at a drug concentration of 300 nM. These molecules were discovered using the doorstop approach, a new approach to protein•polynucleotide inhibitors that identifies small molecules as doorstops to prevent an active-site residue of an RIP (e.g., Tyr80 of ricin or Tyr77 of Stx2 from adopting an active conformation thereby blocking the function of the protein rather than contenders in the competition for binding to the RIP. This work offers promising leads for developing RIP therapeutics. The results suggest that the doorstop approach might also be applicable in the development of other protein•polynucleotide inhibitors as antiviral agents such as inhibitors of the Z-DNA binding proteins in poxviruses. This work also calls for careful chemical and biological characterization of drug leads obtained from chemical screens to avoid the identification of irrelevant chemical structures and to avoid the interference caused by direct interactions between the chemicals being screened and the luciferase reporter used in screening assays.

MultiSETTER: web server for multiple RNA structure comparison.

Science.gov (United States)

Čech, Petr; Hoksza, David; Svozil, Daniel

2015-08-12

Understanding the architecture and function of RNA molecules requires methods for comparing and analyzing their tertiary and quaternary structures. While structural superposition of short RNAs is achievable in a reasonable time, large structures represent much bigger challenge. Therefore, we have developed a fast and accurate algorithm for RNA pairwise structure superposition called SETTER and implemented it in the SETTER web server. However, though biological relationships can be inferred by a pairwise structure alignment, key features preserved by evolution can be identified only from a multiple structure alignment. Thus, we extended the SETTER algorithm to the alignment of multiple RNA structures and developed the MultiSETTER algorithm. In this paper, we present the updated version of the SETTER web server that implements a user friendly interface to the MultiSETTER algorithm. The server accepts RNA structures either as the list of PDB IDs or as user-defined PDB files. After the superposition is computed, structures are visualized in 3D and several reports and statistics are generated. To the best of our knowledge, the MultiSETTER web server is the first publicly available tool for a multiple RNA structure alignment. The MultiSETTER server offers the visual inspection of an alignment in 3D space which may reveal structural and functional relationships not captured by other multiple alignment methods based either on a sequence or on secondary structure motifs.

RNA packaging of MRFV virus-like particles: The interplay between RNA pools and capsid coat protein

Science.gov (United States)

Virus-like particles (VLPs) can be produced through self-assembly of capsid protein (CP) into particles with discrete shapes and sizes and containing different types of RNA molecules. The general principle that governs particle assembly and RNA packaging is determined by unique interactions between ...

Leaf-specific pathogenesis-related 10 homolog, PgPR-10.3, shows in silico binding affinity with several biologically important molecules

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Jin Haeng Han

2015-10-01

Conclusion: Although ginseng PR-10.3 gene is expressed in all organs of 3-wk-old plantlets, its expression is restricted to leaves in mature 2-yr-old ginseng plants. The putative binding property of PgPR-10.3 with Re is intriguing. Further verification of binding affinity with other biologically important molecules in the large hydrophobic cavity of PgPR-10.3 may provide an insight into the biological features of PR-10 proteins.

Predictive biophysical modeling and understanding of the dynamics of mRNA translation and its evolution

Science.gov (United States)

Zur, Hadas; Tuller, Tamir

2016-01-01

mRNA translation is the fundamental process of decoding the information encoded in mRNA molecules by the ribosome for the synthesis of proteins. The centrality of this process in various biomedical disciplines such as cell biology, evolution and biotechnology, encouraged the development of dozens of mathematical and computational models of translation in recent years. These models aimed at capturing various biophysical aspects of the process. The objective of this review is to survey these models, focusing on those based and/or validated on real large-scale genomic data. We consider aspects such as the complexity of the models, the biophysical aspects they regard and the predictions they may provide. Furthermore, we survey the central systems biology discoveries reported on their basis. This review demonstrates the fundamental advantages of employing computational biophysical translation models in general, and discusses the relative advantages of the different approaches and the challenges in the field. PMID:27591251

Pulmonary microRNA profiling: implications in upper lobe predominant lung disease.

Science.gov (United States)

Armstrong, David A; Nymon, Amanda B; Ringelberg, Carol S; Lesseur, Corina; Hazlett, Haley F; Howard, Louisa; Marsit, Carmen J; Ashare, Alix

2017-01-01

Numerous pulmonary diseases manifest with upper lobe predominance including cystic fibrosis, smoking-related chronic obstructive pulmonary disease, and tuberculosis. Zonal hypoxia, characteristic of these pulmonary maladies, and oxygen stress in general is known to exert profound effects on various important aspects of cell biology. Lung macrophages are major participants in the pulmonary innate immune response and regional differences in macrophage responsiveness to hypoxia may contribute in the development of lung disease. MicroRNAs are ubiquitous regulators of human biology and emerging evidence indicates altered microRNA expression modulates respiratory disease processes. The objective of this study is to gain insight into the epigenetic and cellular mechanisms influencing regional differences in lung disease by investigating effect of hypoxia on regional microRNA expression in the lung. All studies were performed using primary alveolar macrophages ( n  = 10) or bronchoalveolar lavage fluid ( n  = 16) isolated from human subjects. MicroRNA was assayed via the NanoString nCounter microRNA assay. Divergent molecular patterns of microRNA expression were observed in alternate lung lobes, specifically noted was disparate expression of miR-93 and miR-4454 in alveolar macrophages along with altered expression of miR-451a and miR-663a in bronchoalveolar lavage fluid. Gene ontology was used to identify potential downstream targets of divergent microRNAs. Targets include cytokines and matrix metalloproteinases, molecules that could have a significant impact on pulmonary inflammation and fibrosis. Our findings show variant regional microRNA expression associated with hypoxia in alveolar macrophages and BAL fluid in the lung-upper vs lower lobe. Future studies should address whether these specific microRNAs may act intracellularly, in a paracrine/endocrine manner to direct the innate immune response or may ultimately be involved in pulmonary host-to-pathogen trans

RNA Interference and its therapeutic applications

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Srinivasa Rao T

2011-10-01

Full Text Available RNAi is a potent method, requiring only a few molecules of dsRNA per cell to silence the expression. Long molecules of double stranded RNA (dsRNA trigger the process. The dsRNA comes from virus and transposon activity in natural RNAi process, while it can be injected in the cells in experimental processes. The strand of the dsRNA that is identical in sequence to a region in target mRNA molecule is called the sense strand, and the other strand which is complimentary is termed the antisense strand. An enzyme complex called DICER thought to be similar to RNAase III then recognizes dsRNA, and cuts it into roughly 22- nucleotide long fragments. These fragments termed siRNAs for “small interfering RNAs” remain in double stranded duplexes with very short 3' overhangs. However, only one of the two strands, known as the guide strand or antisense strand binds the argonaute protein of RNA-induced silencing complex (RISC and target the complementary mRNA resulting gene silencing. The other anti-guide strand or passenger strand is degraded as a RISC substrate during the process of RISC activation. This form of RNAi is termed as post transcriptional gene silencing (PTGS; other forms are also thought to operate at the genomic or transcriptional level in some organisms. In mammals dsRNA longer than 30 base pairs induces a nonspecific antiviral response. This so-called interferon response results in a nonspecific arrest in translation and induction of apoptosis. This cascade induces a global non-specific suppression of translation, which in turn triggers apoptosis. Interestingly, dsRNAs less than 30 nt in length do not activate the antiviral response and specifically switched off genes in human cells without initiating the acute phase response. Thus these siRNAs are suitable for gene target validation and therapeutic applications in many species, including humans. [Vet. World 2011; 4(5.000: 225-229

Molecular knots in biology and chemistry

International Nuclear Information System (INIS)

Lim, Nicole C H; Jackson, Sophie E

2015-01-01

Knots and entanglements are ubiquitous. Beyond their aesthetic appeal, these fascinating topological entities can be either useful or cumbersome. In recent decades, the importance and prevalence of molecular knots have been increasingly recognised by scientists from different disciplines. In this review, we provide an overview on the various molecular knots found in naturally occurring biological systems (DNA, RNA and proteins), and those created by synthetic chemists. We discuss the current knowledge in these fields, including recent developments in experimental and, in some cases, computational studies which are beginning to shed light into the complex interplay between the structure, formation and properties of these topologically intricate molecules. (paper)

The quantitative determination of metabolites of 6-mercaptopurine in biological materials. VI. Evidence for posttranscriptional modification of 6-thioguanosine residues in RNA from L5178Y cells treated with 6-mercaptopurine.

Science.gov (United States)

Breter, H J

1985-05-24

Mammalian cells incorporate 6-thioguanosine into their nucleic acids when grown in the presence of 6-mercaptopurine. 35S-labeled total RNA was prepared from L5178Y murine lymphoma cells grown in vitro in the presence of 6-[35S]mercaptopurine. Base analyses of this RNA suggested that 6-thioguanosine residues in RNA molecules undergo posttranscriptional modification. Thus, enzymatic peak-shifting analyses using anion-exchange high-performance liquid chromatography were applied to the hydrolysis products released from total RNA preparations by digestion with nuclease P1 or nuclease P1 plus nucleotide pyrophosphatase. At least eight 35S-labeled, phosphatase-sensitive compounds structurally different from [35S]6thioGMP were found in nuclease P1 digests. Four of these compounds were susceptible to cleavage with nucleotide pyrophosphatase, thus indicating that they contained phosphoric acid anhydride bonds. Individual RNA species were not separately examined, the radiochromatographic data, however, which were obtained from digests of total RNA preparations, present evidence that 6-thioguanosine 5'-diphosphate and 6-thioguanosine 5'-triphosphate exist as 5'-terminal starting nucleotides (in tRNA and rRNA) and that 6-thioguanosine becomes incorporated into the highly modified dinucleoside triphosphate structures (caps) which commonly block the 5'-termini of eukaryotic poly(A)+ mRNA-molecules.

Cisplatin Targeting of Bacterial Ribosomal RNA Hairpins

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Gayani N. P. Dedduwa-Mudalige

2015-09-01

Full Text Available Cisplatin is a clinically important chemotherapeutic agent known to target purine bases in nucleic acids. In addition to major deoxyribonucleic acid (DNA intrastrand cross-links, cisplatin also forms stable adducts with many types of ribonucleic acid (RNA including siRNA, spliceosomal RNAs, tRNA, and rRNA. All of these RNAs play vital roles in the cell, such as catalysis of protein synthesis by rRNA, and therefore serve as potential drug targets. This work focused on platination of two highly conserved RNA hairpins from E. coli ribosomes, namely pseudouridine-modified helix 69 from 23S rRNA and the 790 loop of helix 24 from 16S rRNA. RNase T1 probing, MALDI mass spectrometry, and dimethyl sulfate mapping revealed platination at GpG sites. Chemical probing results also showed platination-induced RNA structural changes. These findings reveal solvent and structural accessibility of sites within bacterial RNA secondary structures that are functionally significant and therefore viable targets for cisplatin as well as other classes of small molecules. Identifying target preferences at the nucleotide level, as well as determining cisplatin-induced RNA conformational changes, is important for the design of more potent drug molecules. Furthermore, the knowledge gained through studies of RNA-targeting by cisplatin is applicable to a broad range of organisms from bacteria to human.

LA DUALIDAD ANALOGO DIGITAL DE LA INFORMACIÓN SE EJEMPLIFICA EN EL ESTUDIO DE LAS MOLECULAS DE RNA.

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Luis Eugenio Andrade

2011-09-01

, mundo RNA. ABSTRACT The study of RNA molecules have contributed to formalizing the physical notion of information considered in its dual nature analog and digital. The central dogma of molecular biology restricts the notion of information to its digital aspect and so to copying fidelity of symbol’s string during replication DNA-DNA and transcription DNA-RNA, and also in translation to proteins, by means of a genetic code. This vision leads to the paradox about what molecule came first, DNA or proteins, that is solved by the hypotheses that postulates the existence of prebiotic RNA prior to the emergence of genetically encoded proteins and DNA. This hypothesis is based on solid empirical findings such as the discovery of catalytic RNA and RNA structures highly conserved in evolution and found in all living organisms. Besides, experimental models inspired on virus RNA replication and computational models that deal with the relations between secondary planar structures and linear sequences, confirm the advantages of RNA molecules to understand the origin of genetic information, due to their decisive roll in the emergence of the genetic code, their functional roll in gene expression, their structural plasticity and the accessibility of functional structures from any arbitrary sequence that undergoes cycles of mutation and selection. To state that the emergence of digital information requires the prior existence of analog information, not only contributes to solve specific problems of biology, but mainly contributes to advance in the understanding the physical meaning of the different information measurements proposed by Shannon and Chaitin. Key words: RNA, DNA, Protein, digital information, analog information, central dogma, RNA world.

Studying Dynamic Features in Myocardial Infarction Progression by Integrating miRNA-Transcription Factor Co-Regulatory Networks and Time-Series RNA Expression Data from Peripheral Blood Mononuclear Cells.

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Hongbo Shi

Full Text Available Myocardial infarction (MI is a serious heart disease and a leading cause of mortality and morbidity worldwide. Although some molecules (genes, miRNAs and transcription factors (TFs associated with MI have been studied in a specific pathological context, their dynamic characteristics in gene expressions, biological functions and regulatory interactions in MI progression have not been fully elucidated to date. In the current study, we analyzed time-series RNA expression data from peripheral blood mononuclear cells. We observed that significantly differentially expressed genes were sharply up- or down-regulated in the acute phase of MI, and then changed slowly until the chronic phase. Biological functions involved at each stage of MI were identified. Additionally, dynamic miRNA-TF co-regulatory networks were constructed based on the significantly differentially expressed genes and miRNA-TF co-regulatory motifs, and the dynamic interplay of miRNAs, TFs and target genes were investigated. Finally, a new panel of candidate diagnostic biomarkers (STAT3 and ICAM1 was identified to have discriminatory capability for patients with or without MI, especially the patients with or without recurrent events. The results of the present study not only shed new light on the understanding underlying regulatory mechanisms involved in MI progression, but also contribute to the discovery of true diagnostic biomarkers for MI.

Earth mechanisms (fluid and solid), life mechanisms and stable isotope tracers. Isotopes and biology, a great project

International Nuclear Information System (INIS)

Fromageot, P.

1997-01-01

Historical and recent review of the development and use of radioactive isotopes for biological studies in France: study of the intermediate metabolism with 14 C tracers in organic molecules; study and biosynthesis of macromolecules (DNA, RNA and polynucleotides) through the use of marked nucleotides; tracer proteins for use in NMR and protein engineering, use of tritium for the study of hormonal regulation

Biophysics of DNA-Protein Interactions From Single Molecules to Biological Systems

CERN Document Server

Williams, Mark C

2011-01-01

This book presents a concise overview of current research on the biophysics of DNA-protein interactions. A wide range of new and classical methods are presented by authors investigating physical mechanisms by which proteins interact with DNA. For example, several chapters address the mechanisms by which proteins search for and recognize specific binding sites on DNA, a process critical for cellular function. Single molecule methods such as force spectroscopy as well as fluorescence imaging and tracking are described in these chapters as well as other parts of the book that address the dynamics of protein-DNA interactions. Other important topics include the mechanisms by which proteins engage DNA sequences and/or alter DNA structure. These simple but important model interactions are then placed in the broader biological context with discussion of larger protein-DNA complexes . Topics include replication forks, recombination complexes, DNA repair interactions, and ultimately, methods to understand the chromatin...

Orotidine-Containing RNA: Implications for the Hierarchical Selection (Systems Chemistry Emergence) of RNA.

Science.gov (United States)

Kim, Eun-Kyong; Martin, Vincent; Krishnamurthy, Ramanarayanan

2017-09-12

The prebiotic synthesis of canonical nucleobases from HCN is a cornerstone for the RNA world hypothesis. However, their role in the primordial pathways to RNA is still debated. The very same process starting from HCN also gives rise to orotic acid, which (via orotidine) plays a crucial role in extant biology in the de novo synthesis of uridine and cytidine, the informational base-pairs in RNA. However, orotidine itself is absent in RNA. Given the prebiotic and biological relevance of orotic acid vis-à-vis uracil, we investigated orotidine-containing RNA oligonucleotides and show that they have severely compromised base-pairing properties. While not unexpected, these results suggest that the emergence of extant RNA cannot just be a consequence of the plausible prebiotic formation of its chemical constituents/building blocks. In combination with other investigations on alternative prebiotic nucleobases, sugars, and linkers, these findings imply that the selection of the components of extant RNA occurred at a higher hierarchical level of an oligomer/polymer based on its functional properties-pointing to a systems chemistry emergence of RNA from a library of precursors. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Toxicology of organic-inorganic hybrid molecules: bio-organometallics and its toxicology.

Science.gov (United States)

Fujie, Tomoya; Hara, Takato; Kaji, Toshiyuki

2016-01-01

Bio-organometallics is a research strategy of biology that uses organic-inorganic hybrid molecules. The molecules are expected to exhibit useful bioactivities based on the unique structure formed by interaction between the organic structure and intramolecular metal(s). However, studies on both biology and toxicology of organic-inorganic hybrid molecules have been incompletely performed. There can be two types of toxicological studies of bio-organometallics; one is evaluation of organic-inorganic hybrid molecules and the other is analysis of biological systems from the viewpoint of toxicology using organic-inorganic hybrid molecules. Our recent studies indicate that cytotoxicity of hybrid molecules containing a metal that is nontoxic in inorganic forms can be more toxic than that of hybrid molecules containing a metal that is toxic in inorganic forms when the structure of the ligand is the same. Additionally, it was revealed that organic-inorganic hybrid molecules are useful for analysis of biological systems important for understanding the toxicity of chemical compounds including heavy metals.

Adapting to Biology: Maintaining Container-Closure System Compatibility with the Therapeutic Biologic Revolution.

Science.gov (United States)

Degrazio, Dominick

Many pharmaceutical companies are transitioning their research and development drug product pipeline from traditional small-molecule injectables to the dimension of evolving therapeutic biologics. Important concerns associated with this changeover are becoming forefront, as challenges develop of varying complexity uncommon with the synthesis and production of traditional drugs. Therefore, alternative measures must be established that aim to preserve the efficacy and functionality of a biologic that might not be implemented for small molecules. Conserving protein stability is relative to perpetuating a net equilibrium of both intrinsic and extrinsic factors. Key to sustaining this balance is the ability of container-closure systems to maintain their compatibility with the ever-changing dynamics of therapeutic biologics. Failure to recognize and adjust the material properties of packaging components to support compatibility with therapeutic biologics can compromise patient safety, drug productivity, and biological stability. This review will examine the differences between small-molecule drugs and therapeutic biologics, lay a basic foundation for understanding the stability of therapeutic biologics, and demonstrate potential sources of container-closure systems' incompatibilities with therapeutic biologics at a mechanistic level. Many pharmaceutical companies are transitioning their research and development drug product pipeline from traditional small-molecule injectables to recombinantly derived therapeutic biologics. Concerns associated with this transformation are becoming prominent, as therapeutic biologics are uncharacteristic to small-molecule drugs. Maintaining the stability of a therapeutic biologic is a combination of balancing intrinsic factors and external elements within the biologic's microenvironment. An important aspect of this balance is relegated to the overall compatibility of primary, parenteral container-closure systems with therapeutic biologics

Application of magnetic iron oxide nanoparticles in stabilization process of biological molecules

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Mohammad Hossien Salmani

2017-07-01

Conclusion: Co-precipitation method is an easy way to prepare magnetic nanoparticles of iron with a large surface and small particle size, which increases the ability of these particles to act as a suitable carrier for enzyme stabilization. Adequate modification of the surface of these nanoparticles enhances their ability to bind to biological molecules. The immobilized protein or enzyme on magnetic nanoparticles are more stable against structural changes, temperature and pH in comparison with un-stabilized structures, and it is widely used in various sciences, including protein isolation and purification, pharmaceutical science, and food analysis. Stabilization based on the covalent bonds and physical absorption is nonspecific, which greatly limits their functionality. The process of stabilization through bio-mediums provide a new method to overcome the selectivity problem.

Engineering Bacteria to Search for Specific Concentrations of Molecules by a Systematic Synthetic Biology Design Method.

Science.gov (United States)

Tien, Shin-Ming; Hsu, Chih-Yuan; Chen, Bor-Sen

2016-01-01

Bacteria navigate environments full of various chemicals to seek favorable places for survival by controlling the flagella's rotation using a complicated signal transduction pathway. By influencing the pathway, bacteria can be engineered to search for specific molecules, which has great potential for application to biomedicine and bioremediation. In this study, genetic circuits were constructed to make bacteria search for a specific molecule at particular concentrations in their environment through a synthetic biology method. In addition, by replacing the "brake component" in the synthetic circuit with some specific sensitivities, the bacteria can be engineered to locate areas containing specific concentrations of the molecule. Measured by the swarm assay qualitatively and microfluidic techniques quantitatively, the characteristics of each "brake component" were identified and represented by a mathematical model. Furthermore, we established another mathematical model to anticipate the characteristics of the "brake component". Based on this model, an abundant component library can be established to provide adequate component selection for different searching conditions without identifying all components individually. Finally, a systematic design procedure was proposed. Following this systematic procedure, one can design a genetic circuit for bacteria to rapidly search for and locate different concentrations of particular molecules by selecting the most adequate "brake component" in the library. Moreover, following simple procedures, one can also establish an exclusive component library suitable for other cultivated environments, promoter systems, or bacterial strains.

Engineering Bacteria to Search for Specific Concentrations of Molecules by a Systematic Synthetic Biology Design Method.

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Shin-Ming Tien

Full Text Available Bacteria navigate environments full of various chemicals to seek favorable places for survival by controlling the flagella's rotation using a complicated signal transduction pathway. By influencing the pathway, bacteria can be engineered to search for specific molecules, which has great potential for application to biomedicine and bioremediation. In this study, genetic circuits were constructed to make bacteria search for a specific molecule at particular concentrations in their environment through a synthetic biology method. In addition, by replacing the "brake component" in the synthetic circuit with some specific sensitivities, the bacteria can be engineered to locate areas containing specific concentrations of the molecule. Measured by the swarm assay qualitatively and microfluidic techniques quantitatively, the characteristics of each "brake component" were identified and represented by a mathematical model. Furthermore, we established another mathematical model to anticipate the characteristics of the "brake component". Based on this model, an abundant component library can be established to provide adequate component selection for different searching conditions without identifying all components individually. Finally, a systematic design procedure was proposed. Following this systematic procedure, one can design a genetic circuit for bacteria to rapidly search for and locate different concentrations of particular molecules by selecting the most adequate "brake component" in the library. Moreover, following simple procedures, one can also establish an exclusive component library suitable for other cultivated environments, promoter systems, or bacterial strains.

Hydration sites of unpaired RNA bases: a statistical analysis of the PDB structures

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Carugo Oliviero

2011-10-01

Full Text Available Abstract Background Hydration is crucial for RNA structure and function. X-ray crystallography is the most commonly used method to determine RNA structures and hydration and, therefore, statistical surveys are based on crystallographic results, the number of which is quickly increasing. Results A statistical analysis of the water molecule distribution in high-resolution X-ray structures of unpaired RNA nucleotides showed that: different bases have the same penchant to be surrounded by water molecules; clusters of water molecules indicate possible hydration sites, which, in some cases, match those of the major and minor grooves of RNA and DNA double helices; complex hydrogen bond networks characterize the solvation of the nucleotides, resulting in a significant rigidity of the base and its surrounding water molecules. Interestingly, the hydration sites around unpaired RNA bases do not match, in general, the positions that are occupied by the second nucleotide when the base-pair is formed. Conclusions The hydration sites around unpaired RNA bases were found. They do not replicate the atom positions of complementary bases in the Watson-Crick pairs.

Methods to Enrich Exosomes from Conditioned Media and Biological Fluids.

Science.gov (United States)

Sharma, Shayna; Scholz-Romero, Katherin; Rice, Gregory E; Salomon, Carlos

2018-01-01

Exosomes are nano-vesicles which can transport a range of molecules including but not limited to proteins and miRNA. This ability of exosomes renders them useful in cellular communication often resulting in biological changes. They have several functions in facilitating normal biological processes such as immune responses and an involvement in pregnancy. However, they have also been linked to pathological conditions including cancer and pregnancy complications such as preeclampsia. An understanding for the role of exosomes in preeclampsia is based on the ability to purify and characterize exosomes. There have been several techniques proposed for the enrichment of exosomes such as ultracentrifugation, density gradient separation, and ultrafiltration although there is no widely accepted optimized technique. Here we describe a workflow for isolating exosomes from cell-conditioned media and biological fluids using a combination of centrifugation, buoyant density, and ultrafiltration approaches.

Bioreducible poly(amido amine)s for siRNA delivery

NARCIS (Netherlands)

van der Aa, L.J.

2011-01-01

Successes in RNA interference based therapies are still limited due to the lack of efficient delivery of the mediator, small interfering RNA (siRNA), to the targeted site. The key to success can be the delivery of the siRNA molecules by polymer-based carrier systems, since they can be chemically

RNA Structural Dynamics As Captured by Molecular Simulations: A Comprehensive Overview

Science.gov (United States)

2018-01-01

With both catalytic and genetic functions, ribonucleic acid (RNA) is perhaps the most pluripotent chemical species in molecular biology, and its functions are intimately linked to its structure and dynamics. Computer simulations, and in particular atomistic molecular dynamics (MD), allow structural dynamics of biomolecular systems to be investigated with unprecedented temporal and spatial resolution. We here provide a comprehensive overview of the fast-developing field of MD simulations of RNA molecules. We begin with an in-depth, evaluatory coverage of the most fundamental methodological challenges that set the basis for the future development of the field, in particular, the current developments and inherent physical limitations of the atomistic force fields and the recent advances in a broad spectrum of enhanced sampling methods. We also survey the closely related field of coarse-grained modeling of RNA systems. After dealing with the methodological aspects, we provide an exhaustive overview of the available RNA simulation literature, ranging from studies of the smallest RNA oligonucleotides to investigations of the entire ribosome. Our review encompasses tetranucleotides, tetraloops, a number of small RNA motifs, A-helix RNA, kissing-loop complexes, the TAR RNA element, the decoding center and other important regions of the ribosome, as well as assorted others systems. Extended sections are devoted to RNA–ion interactions, ribozymes, riboswitches, and protein/RNA complexes. Our overview is written for as broad of an audience as possible, aiming to provide a much-needed interdisciplinary bridge between computation and experiment, together with a perspective on the future of the field. PMID:29297679

Oasis 2: improved online analysis of small RNA-seq data.

Science.gov (United States)

Rahman, Raza-Ur; Gautam, Abhivyakti; Bethune, Jörn; Sattar, Abdul; Fiosins, Maksims; Magruder, Daniel Sumner; Capece, Vincenzo; Shomroni, Orr; Bonn, Stefan

2018-02-14

Small RNA molecules play important roles in many biological processes and their dysregulation or dysfunction can cause disease. The current method of choice for genome-wide sRNA expression profiling is deep sequencing. Here we present Oasis 2, which is a new main release of the Oasis web application for the detection, differential expression, and classification of small RNAs in deep sequencing data. Compared to its predecessor Oasis, Oasis 2 features a novel and speed-optimized sRNA detection module that supports the identification of small RNAs in any organism with higher accuracy. Next to the improved detection of small RNAs in a target organism, the software now also recognizes potential cross-species miRNAs and viral and bacterial sRNAs in infected samples. In addition, novel miRNAs can now be queried and visualized interactively, providing essential information for over 700 high-quality miRNA predictions across 14 organisms. Robust biomarker signatures can now be obtained using the novel enhanced classification module. Oasis 2 enables biologists and medical researchers to rapidly analyze and query small RNA deep sequencing data with improved precision, recall, and speed, in an interactive and user-friendly environment. Oasis 2 is implemented in Java, J2EE, mysql, Python, R, PHP and JavaScript. It is freely available at https://oasis.dzne.de.

miRNA-34b is directly involved in the aging of macrophages.

Science.gov (United States)

Liang, Wei; Gao, Sheng; Liang, Liu; Huang, Xianing; Hu, Nan; Lu, Xiaoling; Zhao, Yongxiang

2017-08-01

MicroRNAs (miRNAs) are a class of short noncoding RNA that play important regulatory roles in living organisms. These RNA molecules are implicated in the development and progression of malignant diseases such as cancer and are closely associated with cell aging. Findings demonstrating that microRNA is associated with aging in macrophages have nevertheless rarely been reported. This study's objective was to investigate if miRNA-34 is linked to aging process of macrophages. We built a cell aging model in mouse RAW264.7 macrophages using D-galactose and determined the expression levels of miRNA-34a, miRNA-34b, and miRNA-34c in aging and normal macrophages by fluorescence quantitative polymerase chain reaction (q-PCR). We predicted a target gene of miRNA-34 using biological information techniques and constructed the recombinant plasmid pGL3-E2f3 for the putative target gene E2f3. The expression level of miRNA-34b was 5.23 times higher in aging macrophages than in normal macrophages. The luciferase activity decreased by nearly 50 % in cells transfected with miRNA-34b mimics, while no significant decrease in luciferase activity was noted in cells transfected with the miRNA-34b inhibitor or unrelated sequences. Our findings provide the groundwork for further research into the molecular mechanisms whereby miRNA-34b regulates the aging of macrophages. miRNA-34b is associated with the aging of RAW264.7 macrophages, and E2f3 is a target gene of miRNA-34b.

Stochastic processes, multiscale modeling, and numerical methods for computational cellular biology

CERN Document Server

2017-01-01

This book focuses on the modeling and mathematical analysis of stochastic dynamical systems along with their simulations. The collected chapters will review fundamental and current topics and approaches to dynamical systems in cellular biology. This text aims to develop improved mathematical and computational methods with which to study biological processes. At the scale of a single cell, stochasticity becomes important due to low copy numbers of biological molecules, such as mRNA and proteins that take part in biochemical reactions driving cellular processes. When trying to describe such biological processes, the traditional deterministic models are often inadequate, precisely because of these low copy numbers. This book presents stochastic models, which are necessary to account for small particle numbers and extrinsic noise sources. The complexity of these models depend upon whether the biochemical reactions are diffusion-limited or reaction-limited. In the former case, one needs to adopt the framework of s...

The origin and effect of small RNA signaling in plants

Directory of Open Access Journals (Sweden)

Jean-Sébastien eParent

2012-08-01

Full Text Available Given their sessile condition, land plants need to integrate environmental cues rapidly and send signal throughout the organism to modify their metabolism accordingly. Small RNA (sRNA molecules are among the messengers that plant cells use to carry such signals. These molecules originate from fold-back stem-loops transcribed from endogenous loci or from perfect double-stranded RNA produced through the action of RNA-dependent RNA polymerases. Once produced, sRNAs associate with Argonaute and other proteins to form the RNA-induced silencing complex (RISC that executes silencing of complementary RNA molecules. Depending on the nature of the RNA target and the Argonaute protein involved, RISC triggers either DNA methylation and chromatin modification (leading to transcriptional gene silencing, TGS or RNA cleavage or translational inhibition (leading to post-transcriptional gene silencing, PTGS. In some cases, sRNAs move to neighboring cells and/or to the vascular tissues for long-distance trafficking. Many genes are involved in the biogenesis of sRNAs and recent studies have shown that both their origin and their protein partners have great influence on their activity and range. Here we summarize the work done to uncover the mode of action of the different classes of small RNA with special emphasis on their movement and how plants can take advantage of their mobility. We also review the various genetic requirements needed for production, movement and perception of the silencing signal.

Selection platforms for directed evolution in synthetic biology.

Science.gov (United States)

Tizei, Pedro A G; Csibra, Eszter; Torres, Leticia; Pinheiro, Vitor B

2016-08-15

Life on Earth is incredibly diverse. Yet, underneath that diversity, there are a number of constants and highly conserved processes: all life is based on DNA and RNA; the genetic code is universal; biology is limited to a small subset of potential chemistries. A vast amount of knowledge has been accrued through describing and characterizing enzymes, biological processes and organisms. Nevertheless, much remains to be understood about the natural world. One of the goals in Synthetic Biology is to recapitulate biological complexity from simple systems made from biological molecules-gaining a deeper understanding of life in the process. Directed evolution is a powerful tool in Synthetic Biology, able to bypass gaps in knowledge and capable of engineering even the most highly conserved biological processes. It encompasses a range of methodologies to create variation in a population and to select individual variants with the desired function-be it a ligand, enzyme, pathway or even whole organisms. Here, we present some of the basic frameworks that underpin all evolution platforms and review some of the recent contributions from directed evolution to synthetic biology, in particular methods that have been used to engineer the Central Dogma and the genetic code. © 2016 The Author(s).

Pivotal role of microRNA-33 in metabolic syndrome: A systematic review

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Mojgan Gharipour

2013-11-01

Full Text Available Metabolic syndrome (MetS is a major public health concerns and increase in the incidence of MetS caused a rise in the rates of global morbidity, and mortality due to cardiovascular disease and diabetes. Lifestyle modification, a healthy diet, and pharmacological treatment and bariatric surgery are recommended in order to control this syndrome. Molecular mechanisms of metabolic disorders are essential in order to develop novel, valid therapeutic strategies. MicroRNA-33 plays imperative regulatory roles in a variety of biological processes including collaboration with sterol regulatory element-binding protein (SREBP to maintain cholesterol homeostasis, high-density lipoprotein formation, fatty acid oxidation, and insulin signaling. Investigation of these molecules and their genetic targets may potentially identify new pathways involved in complex metabolic disease processes, improve our understanding of metabolic disorders, and influence future approaches to the treatment of obesity. This article reviews the role of miRNA-33 in metabolic syndrome, and highlights the potential of using miRNA-33 as a novel biomarker and therapeutic target for this syndrome.   Keywords: MicroRNA-33, Insulin Resistance Syndrome X, Regulatory Role 

Therapeutic miRNA and siRNA: Moving from Bench to Clinic as Next Generation Medicine

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Chiranjib Chakraborty

2017-09-01

Full Text Available In the past few years, therapeutic microRNA (miRNA and small interfering RNA (siRNA are some of the most important biopharmaceuticals that are in commercial space as future medicines. This review summarizes the patents of miRNA- and siRNA-based new drugs, and also provides a snapshot about significant biopharmaceutical companies that are investing for the therapeutic development of miRNA and siRNA molecules. An insightful view about individual siRNA and miRNA drugs has been depicted with their present status, which is gaining attention in the therapeutic landscape. The efforts of the biopharmaceuticals are discussed with the status of their preclinical and/or clinical trials. Here, some of the setbacks have been highlighted during the biopharmaceutical development of miRNA and siRNA as individual therapeutics. Finally, a snapshot is illustrated about pharmacokinetics, pharmacodynamics with absorption, distribution, metabolism, and excretion (ADME, which is the fundamental development process of these therapeutics, as well as the delivery system for miRNA- and siRNA-based drugs. Keywords: miRNA, siRNA, drug development

11th IUBMB Focused Meeting on the Aminoacyl-tRNA Synthetases: Sailing a New Sea of Complex Functions in Human Biology and Disease.

Science.gov (United States)

Francklyn, Christopher; Roy, Herve; Alexander, Rebecca

2018-05-01

The 11th IUBMB Focused Meeting on Aminoacyl-tRNA Synthetases was held in Clearwater Beach, Florida from 29 October⁻2 November 2017, with the aim of presenting the latest research on these enzymes and promoting interchange among aminoacyl-tRNA synthetase (ARS) researchers. Topics covered in the meeting included many areas of investigation, including ARS evolution, mechanism, editing functions, biology in prokaryotic and eukaryotic cells and their organelles, their roles in human diseases, and their application to problems in emerging areas of synthetic biology. In this report, we provide a summary of the major themes of the meeting, citing contributions from the oral presentations in the meeting.

Parallel mRNA, proteomics and miRNA expression analysis in cell line models of the intestine.

Science.gov (United States)

O'Sullivan, Finbarr; Keenan, Joanne; Aherne, Sinead; O'Neill, Fiona; Clarke, Colin; Henry, Michael; Meleady, Paula; Breen, Laura; Barron, Niall; Clynes, Martin; Horgan, Karina; Doolan, Padraig; Murphy, Richard

2017-11-07

and cholesterol metabolic processes, small molecule transport and a range of responses to external stimuli, while similar analysis of the DE protein list identified gene expression/transcription, epigenetic mechanisms, DNA replication, differentiation and translation ontology categories. The DE protein and gene lists were found to share 15 biological processes including for example epithelial cell differentiation [ P value ≤ 1.81613E-08 (protein list); P ≤ 0.000434311 (gene list)] and actin filament bundle assembly [ P value ≤ 0.001582797 (protein list); P ≤ 0.002733714 (gene list)]. Analysis was conducted on the three data streams acquired in parallel to identify targets undergoing potential miRNA translational repression identified 34 proteins, whose respective mRNAs were detected but no change in expression was observed. Of these 34 proteins, 27 proteins downregulated in the Caco-2 cell line relative to the HT-29 cell line and predicted to be targeted by 19 unique anti-correlated/upregulated microRNAs and 7 proteins upregulated in the Caco-2 cell line relative to the HT-29 cell line and predicted to be targeted by 15 unique anti-correlated/downregulated microRNAs. This first study providing "tri-omics" analysis of the principal intestinal cell line models Caco-2 and HT-29 has identified 34 proteins potentially undergoing miRNA translational repression.

The identification and functional annotation of RNA structures conserved in vertebrates

DEFF Research Database (Denmark)

Seemann, Ernst Stefan; Mirza, Aashiq Hussain; Hansen, Claus

2017-01-01

Structured elements of RNA molecules are essential in, e.g., RNA stabilization, localization and protein interaction, and their conservation across species suggests a common functional role. We computationally screened vertebrate genomes for Conserved RNA Structures (CRSs), leveraging structure-b......-structured counterparts. Our findings of transcribed uncharacterized regulatory regions that contain CRSs support their RNA-mediated functionality.......Structured elements of RNA molecules are essential in, e.g., RNA stabilization, localization and protein interaction, and their conservation across species suggests a common functional role. We computationally screened vertebrate genomes for Conserved RNA Structures (CRSs), leveraging structure......-based, rather than sequence-based, alignments. After careful correction for sequence identity and GC content, we predict ~516k human genomic regions containing CRSs. We find that a substantial fraction of human-mouse CRS regions (i) co-localize consistently with binding sites of the same RNA binding proteins...

Pyrite footprinting of RNA

International Nuclear Information System (INIS)

Schlatterer, Jörg C.; Wieder, Matthew S.; Jones, Christopher D.; Pollack, Lois; Brenowitz, Michael

2012-01-01

Highlights: ► RNA structure is mapped by pyrite mediated · OH footprinting. ► Repetitive experiments can be done in a powdered pyrite filled cartridge. ► High · OH reactivity of nucleotides imply dynamic role in Diels–Alderase catalysis. -- Abstract: In RNA, function follows form. Mapping the surface of RNA molecules with chemical and enzymatic probes has revealed invaluable information about structure and folding. Hydroxyl radicals ( · OH) map the surface of nucleic acids by cutting the backbone where it is accessible to solvent. Recent studies showed that a microfluidic chip containing pyrite (FeS 2 ) can produce sufficient · OH to footprint DNA. The 49-nt Diels–Alder RNA enzyme catalyzes the C–C bond formation between a diene and a dienophile. A crystal structure, molecular dynamics simulation and atomic mutagenesis studies suggest that nucleotides of an asymmetric bulge participate in the dynamic architecture of the ribozyme’s active center. Of note is that residue U42 directly interacts with the product in the crystallized RNA/product complex. Here, we use powdered pyrite held in a commercially available cartridge to footprint the Diels–Alderase ribozyme with single nucleotide resolution. Residues C39 to U42 are more reactive to · OH than predicted by the solvent accessibility calculated from the crystal structure suggesting that this loop is dynamic in solution. The loop’s flexibility may contribute to substrate recruitment and product release. Our implementation of pyrite-mediated · OH footprinting is a readily accessible approach to gleaning information about the architecture of small RNA molecules.

Multi-omic data integration enables discovery of hidden biological regularities

DEFF Research Database (Denmark)

Ebrahim, Ali; Brunk, Elizabeth; Tan, Justin

2016-01-01

Rapid growth in size and complexity of biological data sets has led to the 'Big Data to Knowledge' challenge. We develop advanced data integration methods for multi- level analysis of genomic, transcriptomic, ribosomal profiling, proteomic and fluxomic data. First, we show that pairwise integration...... of primary omics data reveals regularities that tie cellular processes together in Escherichia coli: the number of protein molecules made per mRNA transcript and the number of ribosomes required per translated protein molecule. Second, we show that genome- scale models, based on genomic and bibliomic data......, enable quantitative synchronization of disparate data types. Integrating omics data with models enabled the discovery of two novel regularities: condition invariant in vivo turnover rates of enzymes and the correlation of protein structural motifs and translational pausing. These regularities can...

Small molecule probes for cellular death machines.

Science.gov (United States)

Li, Ying; Qian, Lihui; Yuan, Junying

2017-08-01

The past decade has witnessed a significant expansion of our understanding about the regulated cell death mechanisms beyond apoptosis. The application of chemical biological approaches had played a major role in driving these exciting discoveries. The discovery and use of small molecule probes in cell death research has not only revealed significant insights into the regulatory mechanism of cell death but also provided new drug targets and lead drug candidates for developing therapeutics of human diseases with huge unmet need. Here, we provide an overview of small molecule modulators for necroptosis and ferroptosis, two non-apoptotic cell death mechanisms, and discuss the molecular pathways and relevant pathophysiological mechanisms revealed by the judicial applications of such small molecule probes. We suggest that the development and applications of small molecule probes for non-apoptotic cell death mechanisms provide an outstanding example showcasing the power of chemical biology in exploring novel biological mechanisms. Copyright © 2017 Elsevier Ltd. All rights reserved.

MIRNA-DISTILLER: a stand-alone application to compile microRNA data from databases

Directory of Open Access Journals (Sweden)

Jessica K. Rieger

2011-07-01

Full Text Available MicroRNAs (miRNA are small non-coding RNA molecules of ~22 nucleotides which regulate large numbers of genes by binding to seed sequences at the 3’-UTR of target gene transcripts. The target mRNA is then usually degraded or translation is inhibited, although thus resulting in posttranscriptional down regulation of gene expression at the mRNA and/or protein level. Due to the bioinformatic difficulties in predicting functional miRNA binding sites, several publically available databases have been developed that predict miRNA binding sites based on different algorithms. The parallel use of different databases is currently indispensable, but highly uncomfortable and time consuming, especially when working with numerous genes of interest. We have therefore developed a new stand-alone program, termed MIRNA-DISTILLER, which allows to compile miRNA data for given target genes from public databases. Currently implemented are TargetScan, microCosm, and miRDB, which may be queried independently, pairwise, or together to calculate the respective intersections. Data are stored locally for application of further analysis tools including freely definable biological parameter filters, customized output-lists for both miRNAs and target genes, and various graphical facilities. The software, a data example file and a tutorial are freely available at http://www.ikp-stuttgart.de/content/language1/html/10415.asp

MIRNA-DISTILLER: A Stand-Alone Application to Compile microRNA Data from Databases.

Science.gov (United States)

Rieger, Jessica K; Bodan, Denis A; Zanger, Ulrich M

2011-01-01

MicroRNAs (miRNA) are small non-coding RNA molecules of ∼22 nucleotides which regulate large numbers of genes by binding to seed sequences at the 3'-untranslated region of target gene transcripts. The target mRNA is then usually degraded or translation is inhibited, although thus resulting in posttranscriptional down regulation of gene expression at the mRNA and/or protein level. Due to the bioinformatic difficulties in predicting functional miRNA binding sites, several publically available databases have been developed that predict miRNA binding sites based on different algorithms. The parallel use of different databases is currently indispensable, but highly uncomfortable and time consuming, especially when working with numerous genes of interest. We have therefore developed a new stand-alone program, termed MIRNA-DISTILLER, which allows to compile miRNA data for given target genes from public databases. Currently implemented are TargetScan, microCosm, and miRDB, which may be queried independently, pairwise, or together to calculate the respective intersections. Data are stored locally for application of further analysis tools including freely definable biological parameter filters, customized output-lists for both miRNAs and target genes, and various graphical facilities. The software, a data example file and a tutorial are freely available at http://www.ikp-stuttgart.de/content/language1/html/10415.asp.

MicroRNA Expression Changes during Interferon-Beta Treatment in the Peripheral Blood of Multiple Sclerosis Patients

Directory of Open Access Journals (Sweden)

Georg Füllen

2013-08-01

Full Text Available MicroRNAs (miRNAs are small non-coding RNA molecules acting as post-transcriptional regulators of gene expression. They are involved in many biological processes, and their dysregulation is implicated in various diseases, including multiple sclerosis (MS. Interferon-beta (IFN-beta is widely used as a first-line immunomodulatory treatment of MS patients. Here, we present the first longitudinal study on the miRNA expression changes in response to IFN-beta therapy. Peripheral blood mononuclear cells (PBMC were obtained before treatment initiation as well as after two days, four days, and one month, from patients with clinically isolated syndrome (CIS and patients with relapsing-remitting MS (RRMS. We measured the expression of 651 mature miRNAs and about 19,000 mRNAs in parallel using real-time PCR arrays and Affymetrix microarrays. We observed that the up-regulation of IFN-beta-responsive genes is accompanied by a down-regulation of several miRNAs, including members of the mir-29 family. These differentially expressed miRNAs were found to be associated with apoptotic processes and IFN feedback loops. A network of miRNA-mRNA target interactions was constructed by integrating the information from different databases. Our results suggest that miRNA-mediated regulation plays an important role in the mechanisms of action of IFN-beta, not only in the treatment of MS but also in normal immune responses. miRNA expression levels in the blood may serve as a biomarker of the biological effects of IFN-beta therapy that may predict individual disease activity and progression.

High-throughput SHAPE analysis reveals structures in HIV-1 genomic RNA strongly conserved across distinct biological states.

Directory of Open Access Journals (Sweden)

Kevin A Wilkinson

2008-04-01

Full Text Available Replication and pathogenesis of the human immunodeficiency virus (HIV is tightly linked to the structure of its RNA genome, but genome structure in infectious virions is poorly understood. We invent high-throughput SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension technology, which uses many of the same tools as DNA sequencing, to quantify RNA backbone flexibility at single-nucleotide resolution and from which robust structural information can be immediately derived. We analyze the structure of HIV-1 genomic RNA in four biologically instructive states, including the authentic viral genome inside native particles. Remarkably, given the large number of plausible local structures, the first 10% of the HIV-1 genome exists in a single, predominant conformation in all four states. We also discover that noncoding regions functioning in a regulatory role have significantly lower (p-value < 0.0001 SHAPE reactivities, and hence more structure, than do viral coding regions that function as the template for protein synthesis. By directly monitoring protein binding inside virions, we identify the RNA recognition motif for the viral nucleocapsid protein. Seven structurally homologous binding sites occur in a well-defined domain in the genome, consistent with a role in directing specific packaging of genomic RNA into nascent virions. In addition, we identify two distinct motifs that are targets for the duplex destabilizing activity of this same protein. The nucleocapsid protein destabilizes local HIV-1 RNA structure in ways likely to facilitate initial movement both of the retroviral reverse transcriptase from its tRNA primer and of the ribosome in coding regions. Each of the three nucleocapsid interaction motifs falls in a specific genome domain, indicating that local protein interactions can be organized by the long-range architecture of an RNA. High-throughput SHAPE reveals a comprehensive view of HIV-1 RNA genome structure, and further

Synthesis and biological incorporation of icons into macromolecules for NMR study. Final report, June 1, 1977--May 31, 1978

International Nuclear Information System (INIS)

Grant, D.M.; Horton, W.J.

1978-01-01

Carbon-13 enrichment synthesis and incorporation into three important biological systems have been carried out to provide materials for carbon-13 magnetic resonance studies. These systems include antibody-labeled haptens, labeled t-RNA and 5S-RNA molecules, and pyridoxal-5'-phosphate-labeled substrate mixtures. The synthesis phase of the work has been completed in all three cases, and the NMR studies completed on all but the antibody-hapten system which is still in process having been absorbed into other supported projects. Publications are now in preparation for the RNA and pyridoxal work. Preliminary results on the antibody-haptens work are encouraging as signals of antibody absorbed haptens have been observed but the results are still not yet conclusive

Re-criticizing RNA-mediated cell evolution: a radical perspective

Science.gov (United States)

Kotakis, Christos

2016-01-01

Genetic inter-communication of the nucleic-organellar dual in eukaryotes is dominated by DNA-directed phenomena. RNA regulatory circuits have also been observed in artificial laboratory prototypes where gene transfer events are reconstructed, but they are excluded from the primary norm due to their rarity. Recent technical advances in organellar biotechnology, genome engineering and single-molecule tracking give novel experimental insights on RNA metabolism not only at cellular level, but also on organismal survival. Here, I put forward a hypothesis for RNA's involvement in gene piece transfer, taken together the current knowledge on the primitive RNA character as a biochemical modulator with model organisms from peculiar natural habitats. It is proposed that RNA molecules of special structural signature and functional identity can drive evolution, integrating the ecological pressure of environmental oscillations into genome imprinting by buffering-out epigenetic aberrancies.

Rational Design and Tuning of Functional RNA Switch to Control an Allosteric Intermolecular Interaction.

Science.gov (United States)

Endoh, Tamaki; Sugimoto, Naoki

2015-08-04

Conformational transitions of biomolecules in response to specific stimuli control many biological processes. In natural functional RNA switches, often called riboswitches, a particular RNA structure that has a suppressive or facilitative effect on gene expression transitions to an alternative structure with the opposite effect upon binding of a specific metabolite to the aptamer region. Stability of RNA secondary structure (-ΔG°) can be predicted based on thermodynamic parameters and is easily tuned by changes in nucleobases. We envisioned that tuning of a functional RNA switch that causes an allosteric interaction between an RNA and a peptide would be possible based on a predicted switching energy (ΔΔG°) that corresponds to the energy difference between the RNA secondary structure before (-ΔG°before) and after (-ΔG°after) the RNA conformational transition. We first selected functional RNA switches responsive to neomycin with predicted ΔΔG° values ranging from 5.6 to 12.2 kcal mol(-1). We then demonstrated a simple strategy to rationally convert the functional RNA switch to switches responsive to natural metabolites thiamine pyrophosphate, S-adenosyl methionine, and adenine based on the predicted ΔΔG° values. The ΔΔG° values of the designed RNA switches proportionally correlated with interaction energy (ΔG°interaction) between the RNA and peptide, and we were able to tune the sensitivity of the RNA switches for the trigger molecule. The strategy demonstrated here will be generally applicable for construction of functional RNA switches and biosensors in which mechanisms are based on conformational transition of nucleic acids.

Comparison of protocols and RNA carriers for plasma miRNA isolation. Unraveling RNA carrier influence on miRNA isolation

Science.gov (United States)

Martos, Laura; Fernández-Pardo, Álvaro; Oto, Julia; Medina, Pilar; España, Francisco; Navarro, Silvia

2017-01-01

microRNAs are promising biomarkers in biological fluids in several diseases. Different plasma RNA isolation protocols and carriers are available, but their efficiencies have been scarcely compared. Plasma microRNAs were isolated using a phenol and column-based procedure and a column-based procedure, in the presence or absence of two RNA carriers (yeast RNA and MS2 RNA). We evaluated the presence of PCR inhibitors and the relative abundance of certain microRNAs by qRT-PCR. Furthermore, we analyzed the association between different isolation protocols, the relative abundance of the miRNAs in the sample, the GC content and the free energy of microRNAs. In all microRNAs analyzed, the addition of yeast RNA as a carrier in the different isolation protocols used gave lower raw Cq values, indicating higher microRNA recovery. Moreover, this increase in microRNAs recovery was dependent on their own relative abundance in the sample, their GC content and the free-energy of their own most stable secondary structure. Furthermore, the normalization of microRNA levels by an endogenous microRNA is more reliable than the normalization by plasma volume, as it reduced the difference in microRNA fold abundance between the different isolation protocols evaluated. Our thorough study indicates that a standardization of pre- and analytical conditions is necessary to obtain reproducible inter-laboratory results in plasma microRNA studies. PMID:29077772

2'-O-methylation in mRNA disrupts tRNA decoding during translation elongation.

Science.gov (United States)

Choi, Junhong; Indrisiunaite, Gabriele; DeMirci, Hasan; Ieong, Ka-Weng; Wang, Jinfan; Petrov, Alexey; Prabhakar, Arjun; Rechavi, Gideon; Dominissini, Dan; He, Chuan; Ehrenberg, Måns; Puglisi, Joseph D

2018-03-01

Chemical modifications of mRNA may regulate many aspects of mRNA processing and protein synthesis. Recently, 2'-O-methylation of nucleotides was identified as a frequent modification in translated regions of human mRNA, showing enrichment in codons for certain amino acids. Here, using single-molecule, bulk kinetics and structural methods, we show that 2'-O-methylation within coding regions of mRNA disrupts key steps in codon reading during cognate tRNA selection. Our results suggest that 2'-O-methylation sterically perturbs interactions of ribosomal-monitoring bases (G530, A1492 and A1493) with cognate codon-anticodon helices, thereby inhibiting downstream GTP hydrolysis by elongation factor Tu (EF-Tu) and A-site tRNA accommodation, leading to excessive rejection of cognate aminoacylated tRNAs in initial selection and proofreading. Our current and prior findings highlight how chemical modifications of mRNA tune the dynamics of protein synthesis at different steps of translation elongation.

Defining the RNA Internal Loops Preferred by Benzimidazole Derivatives via Two-Dimensional Combinatorial Screening and Computational Analysis

Science.gov (United States)

Velagapudi, Sai Pradeep; Seedhouse, Steven J.; French, Jonathan

2011-01-01

RNA is an important therapeutic target, however, RNA targets are generally underexploited due to a lack of understanding of the small molecules that bind RNA and the RNA motifs that bind small molecules. Herein, we describe the identification of the RNA internal loops derived from a 4096-member 3×3 nucleotide loop library that are the most specific and highest affinity binders to a series of four designer, drug-like benzimidazoles. These studies establish a potentially general protocol to define the highest affinity and most specific RNA motif targets for heterocyclic small molecules. Such information could be used to target functionally important RNAs in genomic sequence. PMID:21604752

Single-cell mRNA transfection studies: delivery, kinetics and statistics by numbers.

Science.gov (United States)

Leonhardt, Carolin; Schwake, Gerlinde; Stögbauer, Tobias R; Rappl, Susanne; Kuhr, Jan-Timm; Ligon, Thomas S; Rädler, Joachim O

2014-05-01

In artificial gene delivery, messenger RNA (mRNA) is an attractive alternative to plasmid DNA (pDNA) since it does not require transfer into the cell nucleus. Here we show that, unlike for pDNA transfection, the delivery statistics and dynamics of mRNA-mediated expression are generic and predictable in terms of mathematical modeling. We measured the single-cell expression time-courses and levels of enhanced green fluorescent protein (eGFP) using time-lapse microscopy and flow cytometry (FC). The single-cell analysis provides direct access to the distribution of onset times, life times and expression rates of mRNA and eGFP. We introduce a two-step stochastic delivery model that reproduces the number distribution of successfully delivered and translated mRNA molecules and thereby the dose-response relation. Our results establish a statistical framework for mRNA transfection and as such should advance the development of RNA carriers and small interfering/micro RNA-based drugs. This team of authors established a statistical framework for mRNA transfection by using a two-step stochastic delivery model that reproduces the number distribution of successfully delivered and translated mRNA molecules and thereby their dose-response relation. This study establishes a nice connection between theory and experimental planning and will aid the cellular delivery of mRNA molecules. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Challenges of biological sample preparation for SIMS imaging of elements and molecules at subcellular resolution

International Nuclear Information System (INIS)

Chandra, Subhash

2008-01-01

Secondary ion mass spectrometry (SIMS) based imaging techniques capable of subcellular resolution characterization of elements and molecules are becoming valuable tools in many areas of biology and medicine. Due to high vacuum requirements of SIMS, the live cells cannot be analyzed directly in the instrument. The sample preparation, therefore, plays a critical role in preserving the native chemical composition for SIMS analysis. This work focuses on the evaluation of frozen-hydrated and frozen freeze-dried sample preparations for SIMS studies of cultured cells with a CAMECA IMS-3f dynamic SIMS ion microscope instrument capable of producing SIMS images with a spatial resolution of 500 nm. The sandwich freeze-fracture method was used for fracturing the cells. The complimentary fracture planes in the plasma membrane were characterized by field-emission secondary electron microscopy (FESEM) in the frozen-hydrated state. The cells fractured at the dorsal surface were used for SIMS analysis. The frozen-hydrated SIMS analysis of individual cells under dynamic primary ion beam (O 2 + ) revealed local secondary ion signal enhancements correlated with the water image signals of 19 (H 3 O) + . A preferential removal of water from the frozen cell matrix in the Z-axis was also observed. These complications render the frozen-hydrated sample type less desirable for subcellular dynamic SIMS studies. The freeze-drying of frozen-hydrated cells, either inside the instrument or externally in a freeze-drier, allowed SIMS imaging of subcellular chemical composition. Morphological evaluations of fractured freeze-dried cells with SEM and confocal laser scanning microscopy (CLSM) revealed well-preserved mitochondria, Golgi apparatus, and stress fibers. SIMS analysis of fractured freeze-dried cells revealed well-preserved chemical composition of even the most highly diffusible ions like K + and Na + in physiologically relevant concentrations. The high K-low Na signature in individual cells

Challenges of biological sample preparation for SIMS imaging of elements and molecules at subcellular resolution

Science.gov (United States)

Chandra, Subhash

2008-12-01

Secondary ion mass spectrometry (SIMS) based imaging techniques capable of subcellular resolution characterization of elements and molecules are becoming valuable tools in many areas of biology and medicine. Due to high vacuum requirements of SIMS, the live cells cannot be analyzed directly in the instrument. The sample preparation, therefore, plays a critical role in preserving the native chemical composition for SIMS analysis. This work focuses on the evaluation of frozen-hydrated and frozen freeze-dried sample preparations for SIMS studies of cultured cells with a CAMECA IMS-3f dynamic SIMS ion microscope instrument capable of producing SIMS images with a spatial resolution of 500 nm. The sandwich freeze-fracture method was used for fracturing the cells. The complimentary fracture planes in the plasma membrane were characterized by field-emission secondary electron microscopy (FESEM) in the frozen-hydrated state. The cells fractured at the dorsal surface were used for SIMS analysis. The frozen-hydrated SIMS analysis of individual cells under dynamic primary ion beam (O 2+) revealed local secondary ion signal enhancements correlated with the water image signals of 19(H 3O) +. A preferential removal of water from the frozen cell matrix in the Z-axis was also observed. These complications render the frozen-hydrated sample type less desirable for subcellular dynamic SIMS studies. The freeze-drying of frozen-hydrated cells, either inside the instrument or externally in a freeze-drier, allowed SIMS imaging of subcellular chemical composition. Morphological evaluations of fractured freeze-dried cells with SEM and confocal laser scanning microscopy (CLSM) revealed well-preserved mitochondria, Golgi apparatus, and stress fibers. SIMS analysis of fractured freeze-dried cells revealed well-preserved chemical composition of even the most highly diffusible ions like K + and Na + in physiologically relevant concentrations. The high K-low Na signature in individual cells

On RNA-RNA interaction structures of fixed topological genus.

Science.gov (United States)

Fu, Benjamin M M; Han, Hillary S W; Reidys, Christian M

2015-04-01

Interacting RNA complexes are studied via bicellular maps using a filtration via their topological genus. Our main result is a new bijection for RNA-RNA interaction structures and a linear time uniform sampling algorithm for RNA complexes of fixed topological genus. The bijection allows to either reduce the topological genus of a bicellular map directly, or to lose connectivity by decomposing the complex into a pair of single stranded RNA structures. Our main result is proved bijectively. It provides an explicit algorithm of how to rewire the corresponding complexes and an unambiguous decomposition grammar. Using the concept of genus induction, we construct bicellular maps of fixed topological genus g uniformly in linear time. We present various statistics on these topological RNA complexes and compare our findings with biological complexes. Furthermore we show how to construct loop-energy based complexes using our decomposition grammar. Copyright © 2015 Elsevier Inc. All rights reserved.

Diverging affinity of tospovirus RNA silencing suppressor proteins, NSs, for various RNA duplex molecules

NARCIS (Netherlands)

Schnettler, E.; Hemmes, J.C.; Huisman, R.; Goldbach, R.W.; Prins, M.W.; Kormelink, R.J.M.

2010-01-01

The tospovirus NSs protein was previously shown to suppress the antiviral RNA silencing mechanism in plants. Here the biochemical analysis of NSs proteins from different tospoviruses, using purified NSs or NSs containing cell extracts, is described. The results showed that all tospoviral NSs

The RNAi Universe in Fungi: A Varied Landscape of Small RNAs and Biological Functions.

Science.gov (United States)

Torres-Martínez, Santiago; Ruiz-Vázquez, Rosa M

2017-09-08

RNA interference (RNAi) is a conserved eukaryotic mechanism that uses small RNA molecules to suppress gene expression through sequence-specific messenger RNA degradation, translational repression, or transcriptional inhibition. In filamentous fungi, the protective function of RNAi in the maintenance of genome integrity is well known. However, knowledge of the regulatory role of RNAi in fungi has had to wait until the recent identification of different endogenous small RNA classes, which are generated by distinct RNAi pathways. In addition, RNAi research on new fungal models has uncovered the role of small RNAs and RNAi pathways in the regulation of diverse biological functions. In this review, we give an up-to-date overview of the different classes of small RNAs and RNAi pathways in fungi and their roles in the defense of genome integrity and regulation of fungal physiology and development, as well as in the interaction of fungi with biotic and abiotic environments.

Sharing Structure and Function in Biological Design with SBOL 2.0.

Science.gov (United States)

Roehner, Nicholas; Beal, Jacob; Clancy, Kevin; Bartley, Bryan; Misirli, Goksel; Grünberg, Raik; Oberortner, Ernst; Pocock, Matthew; Bissell, Michael; Madsen, Curtis; Nguyen, Tramy; Zhang, Michael; Zhang, Zhen; Zundel, Zach; Densmore, Douglas; Gennari, John H; Wipat, Anil; Sauro, Herbert M; Myers, Chris J

2016-06-17

The Synthetic Biology Open Language (SBOL) is a standard that enables collaborative engineering of biological systems across different institutions and tools. SBOL is developed through careful consideration of recent synthetic biology trends, real use cases, and consensus among leading researchers in the field and members of commercial biotechnology enterprises. We demonstrate and discuss how a set of SBOL-enabled software tools can form an integrated, cross-organizational workflow to recapitulate the design of one of the largest published genetic circuits to date, a 4-input AND sensor. This design encompasses the structural components of the system, such as its DNA, RNA, small molecules, and proteins, as well as the interactions between these components that determine the system's behavior/function. The demonstrated workflow and resulting circuit design illustrate the utility of SBOL 2.0 in automating the exchange of structural and functional specifications for genetic parts, devices, and the biological systems in which they operate.

Construction of RNA nanocages by re-engineering the packaging RNA of Phi29 bacteriophage

Science.gov (United States)

Hao, Chenhui; Li, Xiang; Tian, Cheng; Jiang, Wen; Wang, Guansong; Mao, Chengde

2014-05-01

RNA nanotechnology promises rational design of RNA nanostructures with wide array of structural diversities and functionalities. Such nanostructures could be used in applications such as small interfering RNA delivery and organization of in vivo chemical reactions. Though having impressive development in recent years, RNA nanotechnology is still quite limited and its programmability and complexity could not rival the degree of its closely related cousin: DNA nanotechnology. Novel strategies are needed for programmed RNA self-assembly. Here, we have assembled RNA nanocages by re-engineering a natural, biological RNA motif: the packaging RNA of phi29 bacteriophage. The resulting RNA nanostructures have been thoroughly characterized by gel electrophoresis, cryogenic electron microscopy imaging and dynamic light scattering.

Fast, clash-free RNA conformational morphing using molecular junctions.

Science.gov (United States)

Héliou, Amélie; Budday, Dominik; Fonseca, Rasmus; van den Bedem, Henry

2017-07-15

Non-coding ribonucleic acids (ncRNA) are functional RNA molecules that are not translated into protein. They are extremely dynamic, adopting diverse conformational substates, which enables them to modulate their interaction with a large number of other molecules. The flexibility of ncRNA provides a challenge for probing their complex 3D conformational landscape, both experimentally and computationally. Despite their conformational diversity, ncRNAs mostly preserve their secondary structure throughout the dynamic ensemble. Here we present a kinematics-based procedure to morph an RNA molecule between conformational substates, while avoiding inter-atomic clashes. We represent an RNA as a kinematic linkage, with fixed groups of atoms as rigid bodies and rotatable bonds as degrees of freedom. Our procedure maintains RNA secondary structure by treating hydrogen bonds between base pairs as constraints. The constraints define a lower-dimensional, secondary-structure constraint manifold in conformation space, where motions are largely governed by molecular junctions of unpaired nucleotides. On a large benchmark set, we show that our morphing procedure compares favorably to peer algorithms, and can approach goal conformations to within a low all-atom RMSD by directing fewer than 1% of its atoms. Our results suggest that molecular junctions can modulate 3D structural rearrangements, while secondary structure elements guide large parts of the molecule along the transition to the correct final conformation. The source code, binaries and data are available at https://simtk.org/home/kgs . amelie.heliou@polytechnique.edu or vdbedem@stanford.edu. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

MicroRNA expression profile in head and neck cancer: HOX-cluster embedded microRNA-196a and microRNA-10b dysregulation implicated in cell proliferation

International Nuclear Information System (INIS)

Severino, Patricia; Mathor, Monica Beatriz; Nunes, Fabio Daumas; Ragoussis, Jiannis; Tajara, Eloiza Helena; Brüggemann, Holger; Andreghetto, Flavia Maziero; Camps, Carme; Klingbeil, Maria de Fatima Garrido; Pereira, Welbert Oliveira de; Soares, Renata Machado; Moyses, Raquel; Wünsch-Filho, Victor

2013-01-01

Current evidence implicates aberrant microRNA expression patterns in human malignancies; measurement of microRNA expression may have diagnostic and prognostic applications. Roles for microRNAs in head and neck squamous cell carcinomas (HNSCC) are largely unknown. HNSCC, a smoking-related cancer, is one of the most common malignancies worldwide but reliable diagnostic and prognostic markers have not been discovered so far. Some studies have evaluated the potential use of microRNA as biomarkers with clinical application in HNSCC. MicroRNA expression profile of oral squamous cell carcinoma samples was determined by means of DNA microarrays. We also performed gain-of-function assays for two differentially expressed microRNA using two squamous cell carcinoma cell lines and normal oral keratinocytes. The effect of the over-expression of these molecules was evaluated by means of global gene expression profiling and cell proliferation assessment. Altered microRNA expression was detected for a total of 72 microRNAs. Among these we found well studied molecules, such as the miR-17-92 cluster, comprising potent oncogenic microRNA, and miR-34, recently found to interact with p53. HOX-cluster embedded miR-196a/b and miR-10b were up- and down-regulated, respectively, in tumor samples. Since validated HOX gene targets for these microRNAs are not consistently deregulated in HNSCC, we performed gain-of-function experiments, in an attempt to outline their possible role. Our results suggest that both molecules interfere in cell proliferation through distinct processes, possibly targeting a small set of genes involved in cell cycle progression. Functional data on miRNAs in HNSCC is still scarce. Our data corroborate current literature and brings new insights into the role of microRNAs in HNSCC. We also show that miR-196a and miR-10b, not previously associated with HNSCC, may play an oncogenic role in this disease through the deregulation of cell proliferation. The study of microRNA

Improved Dye Stability in Single-Molecule Fluorescence Experiments

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EcheverrÍa Aitken, Colin; Marshall, R. Andrew; Pugi, Joseph D.

Complex biological systems challenge existing single-molecule methods. In particular, dye stability limits observation time in singlemolecule fluorescence applications. Current approaches to improving dye performance involve the addition of enzymatic oxygen scavenging systems and small molecule additives. We present an enzymatic oxygen scavenging system that improves dye stability in single-molecule experiments. Compared to the currently-employed glucose-oxidase/catalase system, the protocatechuate-3,4-dioxygenase system achieves lower dissolved oxygen concentration and stabilizes single Cy3, Cy5, and Alexa488 fluorophores. Moreover, this system possesses none of the limitations associated with the glucose oxidase/catalase system. We also tested the effects of small molecule additives in this system. Biological reducing agents significantly destabilize the Cy5 fluorophore as a function of reducing potential. In contrast, anti-oxidants stabilize the Cy3 and Alexa488 fluorophores. We recommend use of the protocatechuate-3,4,-dioxygenase system with antioxidant additives, and in the absence of biological reducing agents. This system should have wide application to single-molecule fluorescence experiments.

MicroRNA-directed siRNA biogenesis in Caenorhabditis elegans.

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Corrêa, Régis L; Steiner, Florian A; Berezikov, Eugene; Ketting, René F

2010-04-08

RNA interference (RNAi) is a post-transcriptional silencing process, triggered by double-stranded RNA (dsRNA), leading to the destabilization of homologous mRNAs. A distinction has been made between endogenous RNAi-related pathways and the exogenous RNAi pathway, the latter being essential for the experimental use of RNAi. Previous studies have shown that, in Caenorhabditis elegans, a complex containing the enzymes Dicer and the Argonaute RDE-1 process dsRNA. Dicer is responsible for cleaving dsRNA into short interfering RNAs (siRNAs) while RDE-1 acts as the siRNA acceptor. RDE-1 then guides a multi-protein complex to homologous targets to trigger mRNA destabilization. However, endogenous role(s) for RDE-1, if any, have remained unexplored. We here show that RDE-1 functions as a scavenger protein, taking up small RNA molecules from many different sources, including the microRNA (miRNA) pathway. This is in striking contrast to Argonaute proteins functioning directly in the miRNA pathway, ALG-1 and ALG-2: these proteins exclusively bind miRNAs. While playing no significant role in the biogenesis of the main pool of miRNAs, RDE-1 binds endogenous miRNAs and triggers RdRP activity on at least one perfectly matching, endogenous miRNA target. The resulting secondary siRNAs are taken up by a set of Argonaute proteins known to act as siRNA acceptors in exogenous RNAi, resulting in strong mRNA destabilization. Our results show that RDE-1 in an endogenous setting is actively screening the transcriptome using many different small RNAs, including miRNAs, as a guide, with implications for the evolution of transcripts with a potential to be recognized by Dicer.

Highly stable aptamers selected from a 2'-fully modified fGmH RNA library for targeting biomaterials.

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Friedman, Adam D; Kim, Dongwook; Liu, Rihe

2015-01-01

When developed as targeting ligands for the in vivo delivery of biomaterials to biological systems, RNA aptamers immediately face numerous obstacles, in particular nuclease degradation and post-selection 2' modification. This study aims to develop a novel class of highly stable, 2'-fully modified RNA aptamers that are ideal for the targeted delivery of biomaterials. We demonstrated the facile transcription of a fGmH (2'-F-dG, 2'-OMe-dA/dC/dU) RNA library with unexpected hydrophobicity, the direct selection of aptamers from a fGmH RNA library that bind Staphylococcus aureus Protein A (SpA) as a model target, and the superior nuclease and serum stability of these aptamers compared to 2'-partially modified RNA variants. Characterizations of fGmH RNA aptamers binding to purified SpA and to endogenous SpA present on the surface of S. aureus cells demonstrate fGmH RNA aptamer selectivity and stability. Significantly, fGmH RNA aptamers were able to functionalize, stabilize, and specifically deliver aggregation-prone silver nanoparticles (AgNPs) to S. aureus with SpA-dependent antimicrobial effects. This study describes a novel aptamer class with considerable potential to improve the in vivo applicability of nucleic acid-based affinity molecules to biomaterials.

Detection of Viral RNA in Tissues following Plasma Clearance from an Ebola Virus Infected Patient.

Directory of Open Access Journals (Sweden)

Mirella Biava

2017-01-01

Full Text Available An unprecedented Ebola virus (EBOV epidemic occurred in 2013-2016 in West Africa. Over this time the epidemic exponentially grew and moved to Europe and North America, with several imported cases and many Health Care Workers (HCW infected. Better understanding of EBOV infection patterns in different body compartments is mandatory to develop new countermeasures, as well as to fully comprehend the pathways of human-to-human transmission. We have longitudinally explored the persistence of EBOV-specific negative sense genomic RNA (neg-RNA and the presence of positive sense RNA (pos-RNA, including both replication intermediate (antigenomic-RNA and messenger RNA (mRNA molecules, in the upper and lower respiratory tract, as compared to plasma, in a HCW infected with EBOV in Sierra Leone, who was hospitalized in the high isolation facility of the National Institute for Infectious Diseases "Lazzaro Spallanzani" (INMI, Rome, Italy. We observed persistence of pos-RNA and neg-RNAs in longitudinally collected specimens of the lower respiratory tract, even after viral clearance from plasma, suggesting possible local replication. The purpose of the present study is to enhance the knowledge on the biological features of EBOV that can contribute to the human-to-human transmissibility and to develop effective intervention strategies. However, further investigation is needed in order to better understand the clinical meaning of viral replication and shedding in the respiratory tract.

BEAM web server: a tool for structural RNA motif discovery.

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Pietrosanto, Marco; Adinolfi, Marta; Casula, Riccardo; Ausiello, Gabriele; Ferrè, Fabrizio; Helmer-Citterich, Manuela

2018-03-15

RNA structural motif finding is a relevant problem that becomes computationally hard when working on high-throughput data (e.g. eCLIP, PAR-CLIP), often represented by thousands of RNA molecules. Currently, the BEAM server is the only web tool capable to handle tens of thousands of RNA in input with a motif discovery procedure that is only limited by the current secondary structure prediction accuracies. The recently developed method BEAM (BEAr Motifs finder) can analyze tens of thousands of RNA molecules and identify RNA secondary structure motifs associated to a measure of their statistical significance. BEAM is extremely fast thanks to the BEAR encoding that transforms each RNA secondary structure in a string of characters. BEAM also exploits the evolutionary knowledge contained in a substitution matrix of secondary structure elements, extracted from the RFAM database of families of homologous RNAs. The BEAM web server has been designed to streamline data pre-processing by automatically handling folding and encoding of RNA sequences, giving users a choice for the preferred folding program. The server provides an intuitive and informative results page with the list of secondary structure motifs identified, the logo of each motif, its significance, graphic representation and information about its position in the RNA molecules sharing it. The web server is freely available at http://beam.uniroma2.it/ and it is implemented in NodeJS and Python with all major browsers supported. marco.pietrosanto@uniroma2.it. Supplementary data are available at Bioinformatics online.

Modification degrees at specific sites on heparan sulphate: an approach to measure chemical modifications on biological molecules with stable isotope labelling

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Wu, Zhengliang L.; Lech, Miroslaw

2005-01-01

Chemical modification of biological molecules is a general mechanism for cellular regulation. A quantitative approach has been developed to measure the extent of modification on HS (heparan sulphates). Sulphation on HS by sulphotransferases leads to variable sulphation levels, which allows cells to tune their affinities to various extracellular proteins, including growth factors. With stable isotope labelling and HPLC-coupled MS, modification degrees at various O-sulphation sites could be determined. A bovine kidney HS sample was first saturated in vitro with 34S by an OST (O-sulphotransferase), then digested with nitrous acid and analysed with HPLC-coupled MS. The 34S-labelled oligosaccharides were identified based on their unique isotope clusters. The modification degrees at the sulphotransferase recognition sites were obtained by calculating the intensities of isotopic peaks in the isotope clusters. The modification degrees at 3-OST-1 and 6-OST-1 sites were examined in detail. This approach can also be used to study other types of chemical modifications on biological molecules. PMID:15743272

A Brief Introduction to Single-Molecule Fluorescence Methods.

Science.gov (United States)

van den Wildenberg, Siet M J L; Prevo, Bram; Peterman, Erwin J G

2018-01-01

One of the more popular single-molecule approaches in biological science is single-molecule fluorescence microscopy, which will be the subject of the following section of this volume. Fluorescence methods provide the sensitivity required to study biology on the single-molecule level, but they also allow access to useful measurable parameters on time and length scales relevant for the biomolecular world. Before several detailed experimental approaches will be addressed, we will first give a general overview of single-molecule fluorescence microscopy. We start with discussing the phenomenon of fluorescence in general and the history of single-molecule fluorescence microscopy. Next, we will review fluorescent probes in more detail and the equipment required to visualize them on the single-molecule level. We will end with a description of parameters measurable with such approaches, ranging from protein counting and tracking, single-molecule localization super-resolution microscopy, to distance measurements with Förster Resonance Energy Transfer and orientation measurements with fluorescence polarization.

Biological response of HeLa cells to gold nanoparticles coated with organic molecules.

Science.gov (United States)

Cardoso Avila, P E; Rangel Mendoza, A; Pichardo Molina, J L; Flores Villavicencio, L L; Castruita Dominguez, J P; Chilakapati, M K; Sabanero Lopez, M

2017-08-01

In this work, gold nanospheres functionalized with low weight organic molecules (4-aminothiphenol and cysteamine) were synthesized in a one-step method for their in vitro cytotoxic evaluation on HeLa cells. To enhance the biocompatibility of the cysteamine-capped GNPs, BSA was used due to its broad PH stability and high binding affinity to gold nanoparticles. Besides, the widely reported silica coated gold nanorods were tested here to contrast their toxic response against our nanoparticles coated with organic molecules. Our results shown, the viability measured at 1.9×10 -5 M did not show significant differences against negative controls for all the samples; however, the metabolic activity of HeLa cells dropped when they were exposed to silica gold nanorods in the range of concentrations from 2.9×10 -7 M to 3.0×10 -4 M, while in the cases of gold nanospheres, we found that only at concentrations below 1.9×10 -5 M metabolic activity was normal. Our preliminary results did not indicate any perceivable harmful toxicity to cell membrane, cytoskeleton or nucleus due to our nanospheres at 1.9×10 -5 M. Additional test should be conducted in order to ensure a safe use of them for biological applications, and to determine the extent of possible damage. Copyright © 2017 Elsevier Ltd. All rights reserved.

MicroRNA Biomarkers in Neurodegenerative Diseases and Emerging Nano-Sensors Technology