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Sample records for single rna molecule

  1. An RNA toolbox for single-molecule force spectroscopy studies

    NARCIS (Netherlands)

    Vilfan, I.D.; Kamping, W.; Van den Hout, M.; Candelli, A.; Hage, S.; Dekker, N.H.

    2007-01-01

    Precise, controllable single-molecule force spectroscopy studies of RNA and RNA-dependent processes have recently shed new light on the dynamics and pathways of RNA folding and RNAenzyme interactions. A crucial component of this research is the design and assembly of an appropriate RNA construct.

  2. Single-molecule correlated chemical probing of RNA.

    Science.gov (United States)

    Homan, Philip J; Favorov, Oleg V; Lavender, Christopher A; Kursun, Olcay; Ge, Xiyuan; Busan, Steven; Dokholyan, Nikolay V; Weeks, Kevin M

    2014-09-23

    Complex higher-order RNA structures play critical roles in all facets of gene expression; however, the through-space interaction networks that define tertiary structures and govern sampling of multiple conformations are poorly understood. Here we describe single-molecule RNA structure analysis in which multiple sites of chemical modification are identified in single RNA strands by massively parallel sequencing and then analyzed for correlated and clustered interactions. The strategy thus identifies RNA interaction groups by mutational profiling (RING-MaP) and makes possible two expansive applications. First, we identify through-space interactions, create 3D models for RNAs spanning 80-265 nucleotides, and characterize broad classes of intramolecular interactions that stabilize RNA. Second, we distinguish distinct conformations in solution ensembles and reveal previously undetected hidden states and large-scale structural reconfigurations that occur in unfolded RNAs relative to native states. RING-MaP single-molecule nucleic acid structure interrogation enables concise and facile analysis of the global architectures and multiple conformations that govern function in RNA.

  3. Single-molecule observations of RNA-RNA kissing interactions in a DNA nanostructure.

    Science.gov (United States)

    Takeuchi, Yosuke; Endo, Masayuki; Suzuki, Yuki; Hidaka, Kumi; Durand, Guillaume; Dausse, Eric; Toulmé, Jean-Jacques; Sugiyama, Hiroshi

    2016-01-01

    RNA molecules uniquely form a complex through specific hairpin loops, called a kissing complex. The kissing complex is widely investigated and used for the construction of RNA nanostructures. Molecular switches have also been created by combining a kissing loop and a ligand-binding aptamer to control the interactions of RNA molecules. In this study, we incorporated two kinds of RNA molecules into a DNA origami structure and used atomic force microscopy to observe their ligand-responsive interactions at the single-molecule level. We used a designed RNA aptamer called GTPswitch, which has a guanosine triphosphate (GTP) responsive domain and can bind to the target RNA hairpin named Aptakiss in the presence of GTP. We observed shape changes of the DNA/RNA strands in the DNA origami, which are induced by the GTPswitch, into two different shapes in the absence and presence of GTP, respectively. We also found that the switching function in the nanospace could be improved by using a cover strand over the kissing loop of the GTPswitch or by deleting one base from this kissing loop. These newly designed ligand-responsive aptamers can be used for the controlled assembly of the various DNA and RNA nanostructures.

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

    Science.gov (United States)

    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.

  5. Simultaneous detection of mRNA and protein in single cells using immunofluorescence-combined single-molecule RNA FISH.

    Science.gov (United States)

    Kochan, Jakub; Wawro, Mateusz; Kasza, Aneta

    2015-10-01

    Although the concept of combining immunofluorescence (IF) with single-molecule RNA fluorescence in situ hybridization (smRNA FISH) seems obvious, the specific materials used during IF and smRNA FISH make it difficult to perform these procedures simultaneously on the same specimen. Even though there are reports where IF and smRNA FISH were combined with success, these were insufficient in terms of signal intensities, staining patterns, and GFP-compatibility, and a detailed exploration of the various factors that influence IF and smRNA FISH outcome has not been published yet. Here, we report a detailed study of conditions and reagents used in classic IF and smRNA FISH that allowed us to establish an easy, robust, and GFP-compatible procedure. Our protocol enables simultaneous detection of mRNA and protein quantity as well as the subcellular distribution of these molecules in single cells by combining an RNase-free modification of the IF technique and the more recent smRNA FISH method. Using this procedure, we have shown the direct interaction of RNase MCPIP1 with IL-6 mRNA. We also demonstrate the use of our protocol in heterogeneous cell population analysis, revealing cell-to-cell differences in mRNA and protein content.

  6. 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.

  7. Molecular-crowding effects on single-molecule RNA folding/unfolding thermodynamics and kinetics

    Science.gov (United States)

    Dupuis, Nicholas F.; Holmstrom, Erik D.; Nesbitt, David J.

    2014-01-01

    The effects of “molecular crowding” on elementary biochemical processes due to high solute concentrations are poorly understood and yet clearly essential to the folding of nucleic acids and proteins into correct, native structures. The present work presents, to our knowledge, first results on the single-molecule kinetics of solute molecular crowding, specifically focusing on GAAA tetraloop–receptor folding to isolate a single RNA tertiary interaction using time-correlated single-photon counting and confocal single-molecule FRET microscopy. The impact of crowding by high–molecular-weight polyethylene glycol on the RNA folding thermodynamics is dramatic, with up to ΔΔG° ∼ −2.5 kcal/mol changes in free energy and thus >60-fold increase in the folding equilibrium constant (Keq) for excluded volume fractions of 15%. Most importantly, time-correlated single-molecule methods permit crowding effects on the kinetics of RNA folding/unfolding to be explored for the first time (to our knowledge), which reveal that this large jump in Keq is dominated by a 35-fold increase in tetraloop–receptor folding rate, with only a modest decrease in the corresponding unfolding rate. This is further explored with temperature-dependent single-molecule RNA folding measurements, which identify that crowding effects are dominated by entropic rather than enthalpic contributions to the overall free energy change. Finally, a simple “hard-sphere” treatment of the solute excluded volume is invoked to model the observed kinetic trends, and which predict ΔΔG° ∼ −5 kcal/mol free-energy stabilization at excluded volume fractions of 30%. PMID:24850865

  8. 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.

  9. 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.

  10. Live-Cell Visualization of Pre-mRNA Splicing with Single-Molecule Sensitivity

    Directory of Open Access Journals (Sweden)

    Robert M. Martin

    2013-09-01

    Full Text Available Removal of introns from pre-messenger RNAs (pre-mRNAs via splicing provides a versatile means of genetic regulation that is often disrupted in human diseases. To decipher how splicing occurs in real time, we directly examined with single-molecule sensitivity the kinetics of intron excision from pre-mRNA in the nucleus of living human cells. By using two different RNA labeling methods, MS2 and λN, we show that β-globin introns are transcribed and excised in 20–30 s. Furthermore, we show that replacing the weak polypyrimidine (Py tract in mouse immunoglobulin μ (IgM pre-mRNA by a U-rich Py decreases the intron lifetime, thus providing direct evidence that splice-site strength influences splicing kinetics. We also found that RNA polymerase II transcribes at elongation rates ranging between 3 and 6 kb min−1 and that transcription can be rate limiting for splicing. These results have important implications for a mechanistic understanding of cotranscriptional splicing regulation in the live-cell context.

  11. Genome-wide Single-Molecule Footprinting Reveals High RNA Polymerase II Turnover at Paused Promoters.

    Science.gov (United States)

    Krebs, Arnaud R; Imanci, Dilek; Hoerner, Leslie; Gaidatzis, Dimos; Burger, Lukas; Schübeler, Dirk

    2017-08-03

    Transcription initiation entails chromatin opening followed by pre-initiation complex formation and RNA polymerase II recruitment. Subsequent polymerase elongation requires additional signals, resulting in increased residence time downstream of the start site, a phenomenon referred to as pausing. Here, we harnessed single-molecule footprinting to quantify distinct steps of initiation in vivo throughout the Drosophila genome. This identifies the impact of promoter structure on initiation dynamics in relation to nucleosomal occupancy. Additionally, perturbation of transcriptional initiation reveals an unexpectedly high turnover of polymerases at paused promoters-an observation confirmed at the level of nascent RNAs. These observations argue that absence of elongation is largely caused by premature termination rather than by stable polymerase stalling. In support of this non-processive model, we observe that induction of the paused heat shock promoter depends on continuous initiation. Our study provides a framework to quantify protein binding at single-molecule resolution and refines concepts of transcriptional pausing. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  12. 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

  13. Single-Molecule Spectroscopy

    Indian Academy of Sciences (India)

    IAS Admin

    RESONANCE. February 2015. GENERAL ARTICLE. Single-Molecule Spectroscopy. Every Molecule is Different! Kankan Bhattacharyya. Keywords. Single-molecule ..... Resonance Energy. Transfer (FRET) is an elegant technique to measure the distance between a donor and an acceptor molecule. FRET refers to the.

  14. Single-Molecule Spectroscopy

    Indian Academy of Sciences (India)

    IAS Admin

    GENERAL ARTICLE. Single-Molecule Spectroscopy. Every Molecule is Different! Kankan Bhattacharyya. Keywords. Single-molecule spectroscopy. (SMS), confocal microscopy,. FCS, sm-FRET, FLIM. 1 High-resolution spectrum re- fers to a spectrum consisting of very sharp lines. The sharp lines clearly display transitions to ...

  15. 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.

  16. How to switch the motor on: RNA polymerase initiation steps at the single-molecule level

    NARCIS (Netherlands)

    Marchetti, M.; Malinowska, A.; Heller, I.; Wuite, G. J. L.

    RNA polymerase (RNAP) is the central motor of gene expression since it governs the process of transcription. In prokaryotes, this holoenzyme is formed by the RNAP core and a sigma factor. After approaching and binding the specific promoter site on the DNA, the holoenzyme-promoter complex undergoes

  17. Molecular dynamics simulations of RNA: An in silico single molecule approach

    Czech Academy of Sciences Publication Activity Database

    McDowell, S.E.; Špačková, Naďa; Šponer, Jiří; Walter, N.G.

    2006-01-01

    Roč. 85, č. 2 (2006), s. 169-184 ISSN 0006-3525 R&D Projects: GA ČR(CZ) GA203/05/0009; GA ČR(CZ) GA203/05/0388; GA AV ČR(CZ) 1QS500040581; GA MŠk(CZ) LC06030; GA MŠk(CZ) LC512 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z40550506 Keywords : hydration and cation binding * molecular dynamics * RNA Subject RIV: BO - Biophysics Impact factor: 2.480, year: 2006

  18. 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.

  19. Widespread Polycistronic Transcripts in Fungi Revealed by Single-Molecule mRNA Sequencing.

    Directory of Open Access Journals (Sweden)

    Sean P Gordon

    Full Text Available Genes in prokaryotic genomes are often arranged into clusters and co-transcribed into polycistronic RNAs. Isolated examples of polycistronic RNAs were also reported in some higher eukaryotes but their presence was generally considered rare. Here we developed a long-read sequencing strategy to identify polycistronic transcripts in several mushroom forming fungal species including Plicaturopsis crispa, Phanerochaete chrysosporium, Trametes versicolor, and Gloeophyllum trabeum. We found genome-wide prevalence of polycistronic transcription in these Agaricomycetes, involving up to 8% of the transcribed genes. Unlike polycistronic mRNAs in prokaryotes, these co-transcribed genes are also independently transcribed. We show that polycistronic transcription may interfere with expression of the downstream tandem gene. Further comparative genomic analysis indicates that polycistronic transcription is conserved among a wide range of mushroom forming fungi. In summary, our study revealed, for the first time, the genome prevalence of polycistronic transcription in a phylogenetic range of higher fungi. Furthermore, we systematically show that our long-read sequencing approach and combined bioinformatics pipeline is a generic powerful tool for precise characterization of complex transcriptomes that enables identification of mRNA isoforms not recovered via short-read assembly.

  20. Single-Molecule Spectroscopy

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 20; Issue 2. Single-Molecule Spectroscopy: Every Molecule is Different! Kankan Bhattacharyya. General Article Volume 20 Issue 2 February 2015 pp 151-164. Fulltext. Click here to view fulltext PDF. Permanent link:

  1. Single-Molecule Spectroscopy

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 20; Issue 2. Single-Molecule Spectroscopy: Every Molecule is Different! ... Author Affiliations. Kankan Bhattacharyya1. Department of Physical Chemistry, Indian Association for the Cultivation of Science Jadavpur, Kolkata 700 032 India.

  2. Construction of a laser combiner for dual fluorescent single molecule imaging of pRNA of phi29 DNA packaging motor.

    Science.gov (United States)

    Zhang, Hui; Shu, Dan; Browne, Mark; Guo, Peixuan

    2010-02-01

    A customized laser combiner was designed and constructed for dual channel single molecule imaging. The feasibility of a combiner-incorporated imaging system was demonstrated in studies of single molecule FRET. Distance rulers made of dual-labeled dsDNA were used to evaluate the system by determining the distance between one FRET pair. The results showed that the system is sensitive enough to distinguish between distances differing by two base pair and the distances calculated from FRET efficiencies are close to those documented in the literature. The single molecule FRET with the dual-color imaging system was also applied to reconstructed phi29 motor pRNA monomers. Finally, techniques for dual laser alignment and tuning of laser power for dual-color excitation are discussed.

  3. Watching single molecules dance

    Science.gov (United States)

    Mehta, Amit Dinesh

    Molecular motors convert chemical energy, from ATP hydrolysis or ion flow, into mechanical motion. A variety of increasingly precise mechanical probes have been developed to monitor and perturb these motors at the single molecule level. Several outstanding questions can be best approached at the single molecule level. These include: how far does a motor progress per energy quanta consumed? how does its reaction cycle respond to load? how many productive catalytic cycles can it undergo per diffusional encounter with its track? and what is the mechanical stiffness of a single molecule connection? A dual beam optical trap, in conjunction with in vitro ensemble motility assays, has been used to characterize two members of the myosin superfamily: muscle myosin II and chick brain myosin V. Both move the helical polymer actin, but myosin II acts in large ensembles to drive muscle contraction or cytokinesis, while myosin V acts in small numbers to transport vesicles. An optical trapping apparatus was rendered sufficiently precise to identify a myosin working stroke with 1nm or so, barring systematic errors such as those perhaps due to random protein orientations. This and other light microscopic motility assays were used to characterize myosin V: unlike myosin II this vesicle transport protein moves through many increments of travel while remaining strongly bound to a single actin filament. The step size, stall force, and travel distance of myosin V reveal a remarkably efficient motor capable of moving along a helical track for over a micrometer without significantly spiraling around it. Such properties are fully consistent with the putative role of an organelle transport motor, present in small numbers to maintain movement over long ranges relative to cellular size scales. The contrast between myosin II and myosin V resembles that between a human running on the moon and one walking on earth, where the former allows for faster motion when in larger ensembles but for less

  4. Single-Molecule Nanomagnets

    Science.gov (United States)

    Friedman, Jonathan R.; Sarachik, Myriam P.

    2010-04-01

    Single-molecule magnets straddle the classical and quantum mechanical worlds, displaying many fascinating phenomena. They may have important technological applications in information storage and quantum computation. We review the physical properties of two prototypical molecular nanomagnets, Mn12-acetate and Fe8: Each behaves as a rigid, spin-10 object and exhibits tunneling between up and down directions. As temperature is lowered, the spin-reversal process evolves from thermal activation to pure quantum tunneling. At low temperatures, magnetic avalanches occur in which the magnetization of an entire sample rapidly reverses. We discuss the important role that symmetry-breaking fields play in driving tunneling and in producing Berry-phase interference. Recent experimental advances indicate that quantum coherence can be maintained on timescales sufficient to allow a meaningful number of quantum computing operations to be performed. Efforts are under way to create monolayers and to address and manipulate individual molecules.

  5. Lanthanide single molecule magnets

    CERN Document Server

    Tang, Jinkui

    2015-01-01

    This book begins by providing basic information on single-molecule magnets (SMMs), covering the magnetism of lanthanide, the characterization and relaxation dynamics of SMMs, and advanced means of studying lanthanide SMMs. It then systematically introduces lanthanide SMMs ranging from mononuclear and dinuclear to polynuclear complexes, classifying them and highlighting those SMMs with high barrier and blocking temperatures – an approach that provides some very valuable indicators for the structural features needed to optimize the contribution of an Ising type spin to a molecular magnet. The final chapter presents some of the newest developments in the lanthanide SMM field, such as the design of multifunctional and stimuli-responsive magnetic materials as well as the anchoring and organization of the SMMs on surfaces. In addition, the crystal structure and magnetic data are clearly presented with a wealth of illustrations in each chapter, helping newcomers and experts alike to better grasp ongoing trends and...

  6. Lanthanide single molecule magnets

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Jinkui; Zhang, Peng [Chinese Academy of Sciences, Changchun (China). Changchun Inst. of Applied Chemistry

    2015-10-01

    This book begins by providing basic information on single-molecule magnets (SMMs), covering the magnetism of lanthanide, the characterization and relaxation dynamics of SMMs and advanced means of studying lanthanide SMMs. It then systematically introduces lanthanide SMMs ranging from mononuclear and dinuclear to polynuclear complexes, classifying them and highlighting those SMMs with high barrier and blocking temperatures - an approach that provides some very valuable indicators for the structural features needed to optimize the contribution of an Ising type spin to a molecular magnet. The final chapter presents some of the newest developments in the lanthanide SMM field, such as the design of multifunctional and stimuli-responsive magnetic materials as well as the anchoring and organization of the SMMs on surfaces. In addition, the crystal structure and magnetic data are clearly presented with a wealth of illustrations in each chapter, helping newcomers and experts alike to better grasp ongoing trends and explore new directions.

  7. Quantum transport through single molecules

    NARCIS (Netherlands)

    Osorio Oliveros, E.A.

    2009-01-01

    This thesis describes three-terminal transport measurements through single molecules. The interest in this field stems from the dream that single molecules will form the building blocks for future nanoscale electronic devices. The advantages are their small size -nanometers-, and their synthetic

  8. Single-Molecule Imaging of PSD-95 mRNA Translation in Dendrites and Its Dysregulation in a Mouse Model of Fragile X Syndrome.

    Science.gov (United States)

    Ifrim, Marius F; Williams, Kathryn R; Bassell, Gary J

    2015-05-06

    Fragile X syndrome (FXS) is caused by the loss of the fragile X mental retardation protein (FMRP), an RNA binding protein that regulates translation of numerous target mRNAs, some of which are dendritically localized. Our previous biochemical studies using synaptoneurosomes demonstrate a role for FMRP and miR-125a in regulating the translation of PSD-95 mRNA. However, the local translation of PSD-95 mRNA within dendrites and spines, as well as the roles of FMRP or miR-125a, have not been directly studied. Herein, local synthesis of a Venus-PSD-95 fusion protein was directly visualized in dendrites and spines using single-molecule imaging of a diffusion-restricted Venus-PSD-95 reporter under control of the PSD-95 3'UTR. The basal translation rates of Venus-PSD-95 mRNA was increased in cultured hippocampal neurons from Fmr1 KO mice compared with WT neurons, which correlated with a transient elevation of endogenous PSD-95 within dendrites. Following mGluR stimulation with (S)-3,5-dihydroxyphenylglycine, the rate of Venus-PSD-95 mRNA translation increased rapidly in dendrites of WT hippocampal neurons, but not in those of Fmr1 KO neurons or when the binding site of miR125a, previously shown to bind PSD-95 3'UTR, was mutated. This study provides direct support for the hypothesis that local translation within dendrites and spines is dysregulated in FXS. Impairments in the regulated local synthesis of PSD-95, a critical regulator of synaptic structure and function, may affect the spatiotemporal control of PSD-95 levels and affect dendritic spine development and synaptic plasticity in FXS. Copyright © 2015 the authors 0270-6474/15/357116-15$15.00/0.

  9. Theoretical Investigations Regarding Single Molecules

    DEFF Research Database (Denmark)

    Pedersen, Kim Georg Lind

    Neoclassical Valence Bond Theory, Quantum Transport, Quantum Interference, Kondo Effect, and Electron Pumping. Trap a single organic molecule between two electrodes and apply a bias voltage across this "molecular junction". When electrons pass through the molecule, the different electron paths can...... interfere destructively or constructively. Destructive interference effects in electron transport could potentially improve thermo-electrics, organic logic circuits and energy harvesting. We have investigated destructive interference in off-resonant transport through organic molecules, and have found a set...

  10. Single Molecule Electronics and Devices

    Science.gov (United States)

    Tsutsui, Makusu; Taniguchi, Masateru

    2012-01-01

    The manufacture of integrated circuits with single-molecule building blocks is a goal of molecular electronics. While research in the past has been limited to bulk experiments on self-assembled monolayers, advances in technology have now enabled us to fabricate single-molecule junctions. This has led to significant progress in understanding electron transport in molecular systems at the single-molecule level and the concomitant emergence of new device concepts. Here, we review recent developments in this field. We summarize the methods currently used to form metal-molecule-metal structures and some single-molecule techniques essential for characterizing molecular junctions such as inelastic electron tunnelling spectroscopy. We then highlight several important achievements, including demonstration of single-molecule diodes, transistors, and switches that make use of electrical, photo, and mechanical stimulation to control the electron transport. We also discuss intriguing issues to be addressed further in the future such as heat and thermoelectric transport in an individual molecule. PMID:22969345

  11. Single molecule tracking

    Science.gov (United States)

    Shera, E. Brooks

    1988-01-01

    A detection system is provided for identifying individual particles or molecules having characteristic emission in a flow train of the particles in a flow cell. A position sensitive sensor is located adjacent the flow cell in a position effective to detect the emissions from the particles within the flow cell and to assign spatial and temporal coordinates for the detected emissions. A computer is then enabled to predict spatial and temporal coordinates for the particle in the flow train as a function of a first detected emission. Comparison hardware or software then compares subsequent detected spatial and temporal coordinates with the predicted spatial and temporal coordinates to determine whether subsequently detected emissions originate from a particle in the train of particles. In one embodiment, the particles include fluorescent dyes which are excited to fluoresce a spectrum characteristic of the particular particle. Photones are emitted adjacent at least one microchannel plate sensor to enable spatial and temporal coordinates to be assigned. The effect of comparing detected coordinates with predicted coordinates is to define a moving sample volume which effectively precludes the effects of background emissions.

  12. Cartography of neurexin alternative splicing mapped by single-molecule long-read mRNA sequencing.

    Science.gov (United States)

    Treutlein, Barbara; Gokce, Ozgun; Quake, Stephen R; Südhof, Thomas C

    2014-04-01

    Neurexins are evolutionarily conserved presynaptic cell-adhesion molecules that are essential for normal synapse formation and synaptic transmission. Indirect evidence has indicated that extensive alternative splicing of neurexin mRNAs may produce hundreds if not thousands of neurexin isoforms, but no direct evidence for such diversity has been available. Here we use unbiased long-read sequencing of full-length neurexin (Nrxn)1α, Nrxn1β, Nrxn2β, Nrxn3α, and Nrxn3β mRNAs to systematically assess how many sites of alternative splicing are used in neurexins with a significant frequency, and whether alternative splicing events at these sites are independent of each other. In sequencing more than 25,000 full-length mRNAs, we identified a novel, abundantly used alternatively spliced exon of Nrxn1α and Nrxn3α (referred to as alternatively spliced sequence 6) that encodes a 9-residue insertion in the flexible hinge region between the fifth LNS (laminin-α, neurexin, sex hormone-binding globulin) domain and the third EGF-like sequence. In addition, we observed several larger-scale events of alternative splicing that deleted multiple domains and were much less frequent than the canonical six sites of alternative splicing in neurexins. All of the six canonical events of alternative splicing appear to be independent of each other, suggesting that neurexins may exhibit an even larger isoform diversity than previously envisioned and comprise thousands of variants. Our data are consistent with the notion that α-neurexins represent extracellular protein-interaction scaffolds in which different LNS and EGF domains mediate distinct interactions that affect diverse functions and are independently regulated by independent events of alternative splicing.

  13. RNA as a small molecule druggable target.

    Science.gov (United States)

    Rizvi, Noreen F; Smith, Graham F

    2017-12-01

    Small molecule drugs have readily been developed against many proteins in the human proteome, but RNA has remained an elusive target for drug discovery. Increasingly, we see that RNA, and to a lesser extent DNA elements, show a persistent tertiary structure responsible for many diverse and complex cellular functions. In this digest, we have summarized recent advances in screening approaches for RNA targets and outlined the discovery of novel, drug-like small molecules against RNA targets from various classes and therapeutic areas. The link of structure, function, and small-molecule Druggability validates now for the first time that RNA can be the targets of therapeutic agents. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Single-molecule nanopore enzymology

    Science.gov (United States)

    Wloka, Carsten; Maglia, Giovanni

    2017-01-01

    Biological nanopores are a class of membrane proteins that open nanoscale water-conduits in biological membranes. When they are reconstituted in artificial membranes and a bias voltage is applied across the membrane, the ionic current passing through individual nanopores can be used to monitor chemical reactions, to recognize individual molecules and, of most interest, to sequence DNA. More recently, proteins and enzymes have started being analysed with nanopores. Monitoring enzymatic reactions with nanopores, i.e. nanopore enzymology, has the unique advantage that it allows long-timescale observations of native proteins at the single-molecule level. Here we describe the approaches and challenges in nanopore enzymology. PMID:28630164

  15. Automated imaging system for single molecules

    Science.gov (United States)

    Schwartz, David Charles; Runnheim, Rodney; Forrest, Daniel

    2012-09-18

    There is provided a high throughput automated single molecule image collection and processing system that requires minimal initial user input. The unique features embodied in the present disclosure allow automated collection and initial processing of optical images of single molecules and their assemblies. Correct focus may be automatically maintained while images are collected. Uneven illumination in fluorescence microscopy is accounted for, and an overall robust imaging operation is provided yielding individual images prepared for further processing in external systems. Embodiments described herein are useful in studies of any macromolecules such as DNA, RNA, peptides and proteins. The automated image collection and processing system and method of same may be implemented and deployed over a computer network, and may be ergonomically optimized to facilitate user interaction.

  16. Isolation and detection of small RNA molecules

    Czech Academy of Sciences Publication Activity Database

    Fulneček, Jaroslav

    2007-01-01

    Roč. 53, - (2007), s. 451-455 ISSN 1214-1178 R&D Projects: GA ČR(CZ) GA204/06/1432 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : small RNA molecules * electrophoresis Subject RIV: BO - Biophysics

  17. Single-molecule magnet engineering

    DEFF Research Database (Denmark)

    Pedersen, Kasper Steen; Bendix, Jesper; Clérac, Rodolphe

    2014-01-01

    Tailoring the specific magnetic properties of any material relies on the topological control of the constituent metal ion building blocks. Although this general approach does not seem to be easily applied to traditional inorganic bulk magnets, coordination chemistry offers a unique tool...... to delicately tune, for instance, the properties of molecules that behave as "magnets", the so-called single-molecule magnets (SMMs). Although many interesting SMMs have been prepared by a more or less serendipitous approach, the assembly of predesigned, isolatable molecular entities into higher nuclearity...... complexes constitutes an elegant and fascinating strategy. This Feature article focuses on the use of building blocks or modules (both terms being used indiscriminately) to direct the structure, and therefore also the magnetic properties, of metal ion complexes exhibiting SMM behaviour. This journal is...

  18. Single-Molecule Stochastic Resonance

    Directory of Open Access Journals (Sweden)

    K. Hayashi

    2012-08-01

    Full Text Available Stochastic resonance (SR is a well-known phenomenon in dynamical systems. It consists of the amplification and optimization of the response of a system assisted by stochastic (random or probabilistic noise. Here we carry out the first experimental study of SR in single DNA hairpins which exhibit cooperatively transitions from folded to unfolded configurations under the action of an oscillating mechanical force applied with optical tweezers. By varying the frequency of the force oscillation, we investigate the folding and unfolding kinetics of DNA hairpins in a periodically driven bistable free-energy potential. We measure several SR quantifiers under varied conditions of the experimental setup such as trap stiffness and length of the molecular handles used for single-molecule manipulation. We find that a good quantifier of the SR is the signal-to-noise ratio (SNR of the spectral density of measured fluctuations in molecular extension of the DNA hairpins. The frequency dependence of the SNR exhibits a peak at a frequency value given by the resonance-matching condition. Finally, we carry out experiments on short hairpins that show how SR might be useful for enhancing the detection of conformational molecular transitions of low SNR.

  19. Recent advances in developing small molecules targeting RNA.

    Science.gov (United States)

    Guan, Lirui; Disney, Matthew D

    2012-01-20

    RNAs are underexploited targets for small molecule drugs or chemical probes of function. This may be due, in part, to a fundamental lack of understanding of the types of small molecules that bind RNA specifically and the types of RNA motifs that specifically bind small molecules. In this review, we describe recent advances in the development and design of small molecules that bind to RNA and modulate function that aim to fill this void.

  20. Transcriptome Profiling Using Single-Molecule Direct RNA Sequencing Approach for In-depth Understanding of Genes in Secondary Metabolism Pathways of Camellia sinensis

    Directory of Open Access Journals (Sweden)

    Qingshan Xu

    2017-07-01

    Full Text Available Characteristic secondary metabolites, including flavonoids, theanine and caffeine, are important components of Camellia sinensis, and their biosynthesis has attracted widespread interest. Previous studies on the biosynthesis of these major secondary metabolites using next-generation sequencing technologies limited the accurately prediction of full-length (FL splice isoforms. Herein, we applied single-molecule sequencing to pooled tea plant tissues, to provide a more complete transcriptome of C. sinensis. Moreover, we identified 94 FL transcripts and four alternative splicing events for enzyme-coding genes involved in the biosynthesis of flavonoids, theanine and caffeine. According to the comparison between long-read isoforms and assemble transcripts, we improved the quality and accuracy of genes sequenced by short-read next-generation sequencing technology. The resulting FL transcripts, together with the improved assembled transcripts and identified alternative splicing events, enhance our understanding of genes involved in the biosynthesis of characteristic secondary metabolites in C. sinensis.

  1. Sub-millimetre wave absorption spectra of artificial RNA molecules

    CERN Document Server

    Globus, T; Woolard, D; Gelmont, B

    2003-01-01

    We demonstrate submillimetre-wave Fourier transform spectroscopy as a novel technique for biological molecule characterization. Transmission measurements are reported at frequencies 10-25 cm sup - sup 1 for single- and double-stranded RNA molecules of known base-pair sequences: homopolymers poly[A], poly[U], poly[C] and poly[G], and double-stranded homopolymers poly[A]-poly[U] and poly[C]-poly[G]. Multiple resonances are observed (i.e. in the microwave through terahertz frequency regime). We also present a computational method to predict the low-frequency absorption spectra of short artificial DNA and RNA. Theoretical conformational analysis of molecules was utilized to derive the low-frequency vibrational modes. Oscillator strengths were calculated for all the vibrational modes in order to evaluate their weight in the absorption spectrum of a molecule. Normal modes and absorption spectra of the double-stranded RNA chain poly[C]-poly[G] were calculated. The absorption spectra extracted from the experiment wer...

  2. Site-Specific Chemical Labeling of Long RNA Molecules

    DEFF Research Database (Denmark)

    Jahn, Kasper; Olsen, Eva Maria; Nielsen, Morten Muhlig

    2011-01-01

    Site-specific labeling of RNA molecules is a valuable tool for studying their structure and function. Here, we describe a new site-specific RNA labeling method, which utilizes a DNA-templated chemical reaction to attach a label at a specific internal nucleotide in an RNA molecule. The method...

  3. Nano-manipulation of single DNA molecules

    International Nuclear Information System (INIS)

    Hu Jun; Shanghai Jiaotong Univ., Shanghai; Lv Junhong; Wang Guohua; Wang Ying; Li Minqian; Zhang Yi; Li Bin; Li Haikuo; An Hongjie

    2004-01-01

    Nano-manipulation of single atoms and molecules is a critical technique in nanoscience and nanotechnology. This review paper will focus on the recent development of the manipulation of single DNA molecules based on atomic force microscopy (AFM). Precise manipulation has been realized including varied manipulating modes such as 'cutting', 'pushing', 'folding', 'kneading', 'picking up', 'dipping', etc. The cutting accuracy is dominated by the size of the AFM tip, which is usually 10 nm or less. Single DNA fragments can be cut and picked up and then amplified by single molecule PCR. Thus positioning isolation and sequencing can be performed. (authors)

  4. Single Molecule Applications of Quantum Dots

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Elmelund; Jauffred, Liselotte; Brewer, Jonathan R.

    2013-01-01

    Fluorescent nanocrystals composed of semiconductor materials were first introduced for biological applications in the late 1990s. The focus of this review is to give a brief survey of biological applications of quantum dots (QDs) at the single QD sensitivity level. These are described as follows:...... experiments held together with the prospects in localization microscopy and single molecule manipulation experiments gave QDs a promising future in single molecule research....

  5. Watching single protein molecules in action

    DEFF Research Database (Denmark)

    Heiðarsson, Pétur Orri

    (NCS1). The NMR solution structure of NCS1, in combination with fluorescence spectroscopy and mutational analysis, suggested a novel role for the C-terminal tail in regulating conformational stability. On the single-molecule level, the C-domain folded through a partially folded intermediate state....... This knowledge-gap is partly due to our inability to unveil the details of folding mechanisms that can be buried in the ensemble-averaged output of traditional bulk methods. Single-molecule techniques have provided a perspective beyond the ensemble average and enable studying the folding trajectories of protein...... molecules in unprecedented detail. These methods can, in principle, detect rare folding or misfolding events, and ultimately lead to a reconstruction of the free energy landscape. In this thesis, the folding mechanism of both single- and double-domain proteins is unraveled using single-molecule optical...

  6. Single Molecule Spectroscopy of Electron Transfer

    International Nuclear Information System (INIS)

    Holman, Michael; Zang, Ling; Liu, Ruchuan; Adams, David M.

    2009-01-01

    The objectives of this research are threefold: (1) to develop methods for the study electron transfer processes at the single molecule level, (2) to develop a series of modifiable and structurally well defined molecular and nanoparticle systems suitable for detailed single molecule/particle and bulk spectroscopic investigation, (3) to relate experiment to theory in order to elucidate the dependence of electron transfer processes on molecular and electronic structure, coupling and reorganization energies. We have begun the systematic development of single molecule spectroscopy (SMS) of electron transfer and summaries of recent studies are shown. There is a tremendous need for experiments designed to probe the discrete electronic and molecular dynamic fluctuations of single molecules near electrodes and at nanoparticle surfaces. Single molecule spectroscopy (SMS) has emerged as a powerful method to measure properties of individual molecules which would normally be obscured in ensemble-averaged measurement. Fluctuations in the fluorescence time trajectories contain detailed molecular level statistical and dynamical information of the system. The full distribution of a molecular property is revealed in the stochastic fluctuations, giving information about the range of possible behaviors that lead to the ensemble average. In the case of electron transfer, this level of understanding is particularly important to the field of molecular and nanoscale electronics: from a device-design standpoint, understanding and controlling this picture of the overall range of possible behaviors will likely prove to be as important as designing ia the ideal behavior of any given molecule.

  7. Relaxation dynamics of a single DNA molecule

    Science.gov (United States)

    Goshen, E.; Zhao, W. Z.; Carmon, G.; Rosen, S.; Granek, R.; Feingold, M.

    2005-06-01

    The relaxation of a single DNA molecule is studied. The experimental system consists of optical tweezers and a micron-sized bead that is tethered to the bottom of the sample by a single double-stranded DNA molecule. The bead slows down the DNA relaxation from a strongly stretched configuration such that it is passing through stretched equilibrium states. This allows for a theoretical description of the relaxation trajectory, which is in good agreement with experiment.

  8. DNA heterogeneity and phosphorylation unveiled by single-molecule electrophoresis

    Science.gov (United States)

    Wang, Hui; Dunning, James E.; Huang, Albert P.-H.; Nyamwanda, Jacqueline A.; Branton, Daniel

    2004-09-01

    Broad-spectrum analysis of DNA and RNA samples is of increasing importance in the growing field of biotechnology. We show that nanopore measurements may be used to assess the purity, phosphorylation state, and chemical integrity of nucleic acid preparations. In contrast with gel electrophoresis and mass spectrometry, an unprecedented dynamic range of DNA sizes and concentrations can be evaluated in a single data acquisition process that spans minutes. Because the molecule information is quantized and digitally recorded with single-molecule resolution, the sensitivity of the system can be adjusted in real time to detect trace amounts of a particular DNA species.

  9. Single Molecule Biophysics Experiments and Theory

    CERN Document Server

    Komatsuzaki, Tamiki; Takahashi, Satoshi; Yang, Haw; Silbey, Robert J; Rice, Stuart A; Dinner, Aaron R

    2011-01-01

    Discover the experimental and theoretical developments in optical single-molecule spectroscopy that are changing the ways we think about molecules and atoms The Advances in Chemical Physics series provides the chemical physics field with a forum for critical, authoritative evaluations of advances in every area of the discipline. This latest volume explores the advent of optical single-molecule spectroscopy, and how atomic force microscopy has empowered novel experiments on individual biomolecules, opening up new frontiers in molecular and cell biology and leading to new theoretical approaches

  10. 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 area of remarkable current interest. For this purpose, a basic understanding of the molecular aspects of the interaction of small molecules with various RNA structures is essential. Alkaloids are a group of ...

  11. Watching single protein molecules in action

    DEFF Research Database (Denmark)

    Heiðarsson, Pétur Orri

    . This knowledge-gap is partly due to our inability to unveil the details of folding mechanisms that can be buried in the ensemble-averaged output of traditional bulk methods. Single-molecule techniques have provided a perspective beyond the ensemble average and enable studying the folding trajectories of protein...... molecules in unprecedented detail. These methods can, in principle, detect rare folding or misfolding events, and ultimately lead to a reconstruction of the free energy landscape. In this thesis, the folding mechanism of both single- and double-domain proteins is unraveled using single-molecule optical......, with transition states located almost halfway between the native and unfolded states. When pulled from the N- and C-termini, both experiments and simulations suggested that the molecule populates a transition state that resembles that observed during chemical denaturation, with respect to structure and position...

  12. 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.

  13. Trapping and manipulating single molecules of DNA

    Science.gov (United States)

    Shon, Min Ju

    This thesis presents the development and application of nanoscale techniques to trap and manipulate biomolecules, with a focus on DNA. These methods combine single-molecule microscopy and nano- and micro-fabrication to study biophysical properties of DNA and proteins. The Dimple Machine is a lab-on-a-chip device that can isolate and confine a small number of molecules from a bulk solution. It traps molecules in nanofabricated chambers, or "dimples", and the trapped molecules are then studied on a fluorescence microscope at the single-molecule level. The sampling of bulk solution by dimples is representative, reproducible, and automated, enabling highthroughput single-molecule experiments. The device was applied to study hybridization of oligonucleotides, particularly in the context of reaction thermodynamics and kinetics in nanoconfinement. The DNA Pulley is a system to study protein binding and the local mechanical properties of DNA. A molecule of DNA is tethered to a surface on one end, and a superparamagnetic bead is attached to the other. A magnet pulls the DNA taut, and a silicon nitride knife with a nanoscale blade scans the DNA along its contour. Information on the local properties of the DNA is extracted by tracking the bead with nanometer precision in a white-light microscope. The system can detect proteins bound to DNA and localize their recognition sites, as shown with a model protein, EcoRI restriction enzyme. Progress on the measurements of nano-mechanical properties of DNA is included.

  14. Single-molecule manipulation and detection.

    Science.gov (United States)

    Zhao, Deyu; Liu, Siyun; Gao, Ying

    2018-01-25

    Compared to conventional ensemble methods, studying macromolecules at single-molecule level can reveal extraordinary clear and even surprising views for a biological reaction. In the past 20 years, single-molecule techniques have been undergoing a very rapid development, and these cutting edge technologies have revolutionized the biological research by facilitating single-molecule manipulation and detection. Here we give a brief review about these advanced techniques, including optical tweezers, magnetic tweezers, atomic force microscopy (AFM), hydrodynamic flow-stretching assay, and single-molecule fluorescence resonance energy transfer (smFRET). We are trying to describe their basic principles and provide a few examples of applications for each technique. This review aims to give a rather introductory survey of single-molecule techniques for audiences with biological or biophysical background. © The Author(s) 2018. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  15. Single-Molecule Interfacial Electron Transfer

    Energy Technology Data Exchange (ETDEWEB)

    Lu, H. Peter [Bowling Green State Univ., Bowling Green, OH (United States). Dept. of Chemistry and Center for Photochemical Sciences

    2017-11-28

    This project is focused on the use of single-molecule high spatial and temporal resolved techniques to study molecular dynamics in condensed phase and at interfaces, especially, the complex reaction dynamics associated with electron and energy transfer rate processes. The complexity and inhomogeneity of the interfacial ET dynamics often present a major challenge for a molecular level comprehension of the intrinsically complex systems, which calls for both higher spatial and temporal resolutions at ultimate single-molecule and single-particle sensitivities. Combined single-molecule spectroscopy and electrochemical atomic force microscopy approaches are unique for heterogeneous and complex interfacial electron transfer systems because the static and dynamic inhomogeneities can be identified and characterized by studying one molecule at a specific nanoscale surface site at a time. The goal of our project is to integrate and apply these spectroscopic imaging and topographic scanning techniques to measure the energy flow and electron flow between molecules and substrate surfaces as a function of surface site geometry and molecular structure. We have been primarily focusing on studying interfacial electron transfer under ambient condition and electrolyte solution involving both single crystal and colloidal TiO2 and related substrates. The resulting molecular level understanding of the fundamental interfacial electron transfer processes will be important for developing efficient light harvesting systems and broadly applicable to problems in fundamental chemistry and physics. We have made significant advancement on deciphering the underlying mechanism of the complex and inhomogeneous interfacial electron transfer dynamics in dyesensitized TiO2 nanoparticle systems that strongly involves with and regulated by molecule-surface interactions. We have studied interfacial electron transfer on TiO2 nanoparticle surfaces by using ultrafast single-molecule

  16. Quantum transport of the single metallocene molecule

    Science.gov (United States)

    Yu, Jing-Xin; Chang, Jing; Wei, Rong-Kai; Liu, Xiu-Ying; Li, Xiao-Dong

    2016-10-01

    The Quantum transport of three single metallocene molecule is investigated by performing theoretical calculations using the non-equilibrium Green's function method combined with density functional theory. We find that the three metallocen molecules structure become stretched along the transport direction, the distance between two Cp rings longer than the other theory and experiment results. The lager conductance is found in nickelocene molecule, the main transmission channel is the electron coupling between molecule and the electrodes is through the Ni dxz and dyz orbitals and the s, dxz, dyz of gold. This is also confirmed by the highest occupied molecular orbital resonance at Fermi level. In addition, negative differential resistance effect is found in the ferrocene, cobaltocene molecules, this is also closely related with the evolution of the transmission spectrum under applied bias.

  17. Single molecule microscopy and spectroscopy: concluding remarks.

    Science.gov (United States)

    van Hulst, Niek F

    2015-01-01

    Chemistry is all about molecules: control, synthesis, interaction and reaction of molecules. All too easily on a blackboard, one draws molecules, their structures and dynamics, to create an insightful picture. The dream is to see these molecules in reality. This is exactly what "Single Molecule Detection" provides: a look at molecules in action at ambient conditions; a breakthrough technology in chemistry, physics and biology. Within the realms of the Royal Society of Chemistry, the Faraday Discussion on "Single Molecule Microscopy and Spectroscopy" was a very appropriate topic for presentation, deliberation and debate. Undoubtedly, the Faraday Discussions have a splendid reputation in stimulating scientific debates along the traditions set by Michael Faraday. Interestingly, back in the 1830's, Faraday himself pursued an experiment that led to the idea that atoms in a compound were joined by an electrical component. He placed two opposite electrodes in a solution of water containing a dissolved compound, and observed that one of the elements of the compound accumulated on one electrode, while the other was deposited on the opposite electrode. Although Faraday was deeply opposed to atomism, he had to recognize that electrical forces were responsible for the joining of atoms. Probably a direct view on the atoms or molecules in his experiment would have convinced him. As such, Michael Faraday might have liked the gathering at Burlington House in September 2015 (). Surely, with the questioning eyes of his bust on the 1st floor corridor, the non-believer Michael Faraday has incited each passer-by to enter into discussion and search for deeper answers at the level of single molecules. In these concluding remarks, highlights of the presented papers and discussions are summarized, complemented by a conclusion on future perspectives.

  18. Nanopore analytics: sensing of single molecules.

    Science.gov (United States)

    Howorka, Stefan; Siwy, Zuzanna

    2009-08-01

    In nanopore analytics, individual molecules pass through a single nanopore giving rise to detectable temporary blockades in ionic pore current. Reflecting its simplicity, nanopore analytics has gained popularity and can be conducted with natural protein as well as man-made polymeric and inorganic pores. The spectrum of detectable analytes ranges from nucleic acids, peptides, proteins, and biomolecular complexes to organic polymers and small molecules. Apart from being an analytical tool, nanopores have developed into a general platform technology to investigate the biophysics, physicochemistry, and chemistry of individual molecules (critical review, 310 references).

  19. Single molecule transcription profiling with AFM

    Science.gov (United States)

    Reed, Jason; Mishra, Bud; Pittenger, Bede; Magonov, Sergei; Troke, Joshua; Teitell, Michael A.; Gimzewski, James K.

    2007-01-01

    Established techniques for global gene expression profiling, such as microarrays, face fundamental sensitivity constraints. Due to greatly increasing interest in examining minute samples from micro-dissected tissues, including single cells, unorthodox approaches, including molecular nanotechnologies, are being explored in this application. Here, we examine the use of single molecule, ordered restriction mapping, combined with AFM, to measure gene transcription levels from very low abundance samples. We frame the problem mathematically, using coding theory, and present an analysis of the critical error sources that may serve as a guide to designing future studies. We follow with experiments detailing the construction of high density, single molecule, ordered restriction maps from plasmids and from cDNA molecules, using two different enzymes, a result not previously reported. We discuss these results in the context of our calculations. Based on invited talk at the International Conference on Nanoscience and Technology 2006.

  20. Single molecule transcription profiling with AFM

    International Nuclear Information System (INIS)

    Reed, Jason; Mishra, Bud; Pittenger, Bede; Magonov, Sergei; Troke, Joshua; Teitell, Michael A; Gimzewski, James K

    2007-01-01

    Established techniques for global gene expression profiling, such as microarrays, face fundamental sensitivity constraints. Due to greatly increasing interest in examining minute samples from micro-dissected tissues, including single cells, unorthodox approaches, including molecular nanotechnologies, are being explored in this application. Here, we examine the use of single molecule, ordered restriction mapping, combined with AFM, to measure gene transcription levels from very low abundance samples. We frame the problem mathematically, using coding theory, and present an analysis of the critical error sources that may serve as a guide to designing future studies. We follow with experiments detailing the construction of high density, single molecule, ordered restriction maps from plasmids and from cDNA molecules, using two different enzymes, a result not previously reported. We discuss these results in the context of our calculations

  1. Telomeric noncoding RNA TERRA is induced by telomere shortening to nucleate telomerase molecules at short telomeres.

    Science.gov (United States)

    Cusanelli, Emilio; Romero, Carmina Angelica Perez; Chartrand, Pascal

    2013-09-26

    Elongation of a short telomere depends on the action of multiple telomerase molecules, which are visible as telomerase RNA foci or clusters associated with telomeres in yeast and mammalian cells. How several telomerase molecules act on a single short telomere is unknown. Herein, we report that the telomeric noncoding RNA TERRA is involved in the nucleation of telomerase molecules into clusters prior to their recruitment at a short telomere. We find that telomere shortening induces TERRA expression, leading to the accumulation of TERRA molecules into a nuclear focus. Simultaneous time-lapse imaging of telomerase RNA and TERRA reveals spontaneous events of telomerase nucleation on TERRA foci in early S phase, generating TERRA-telomerase clusters. This cluster is subsequently recruited to the short telomere from which TERRA transcripts originate during S phase. We propose that telomere shortening induces noncoding RNA expression to coordinate the recruitment and activity of telomerase molecules at short telomeres. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Imaging of tautomerism in a single molecule.

    Science.gov (United States)

    Piwoński, Hubert; Stupperich, Clemens; Hartschuh, Achim; Sepioł, Jerzy; Meixner, Alfred; Waluk, Jacek

    2005-04-20

    Fluorescence imaging is used to visualize directly the transfer of two inner hydrogen atoms in single porphycene molecules. This reaction leads to a chemically equivalent but differently oriented structure and hence results in a rotation of the transition dipole moments. By probing single immobilized molecules with an azimuthally polarized laser beam in the focal spot of a confocal microscope we observe ring-like emission patterns, possible only for a chromophore with two nearly orthogonal transition dipole moments. Numerical simulations of the observed emission patterns yield a value of 72 degrees for the angle between the S0-S1 transition moments in the two tautomeric forms.

  3. Small Molecule Modifiers of the microRNA and RNA Interference Pathway

    OpenAIRE

    Deiters, Alexander

    2009-01-01

    Recently, the RNA interference (RNAi) pathway has become the target of small molecule inhibitors and activators. RNAi has been well established as a research tool in the sequence-specific silencing of genes in eukaryotic cells and organisms by using exogenous, small, double-stranded RNA molecules of approximately 20 nucleotides. Moreover, a recently discovered post-transcriptional gene regulatory mechanism employs microRNAs (miRNAs), a class of endogenously expressed small RNA molecules, whic...

  4. Single-molecule studies using magnetic traps.

    Science.gov (United States)

    Lionnet, Timothée; Allemand, Jean-François; Revyakin, Andrey; Strick, Terence R; Saleh, Omar A; Bensimon, David; Croquette, Vincent

    2012-01-01

    In recent years, techniques have been developed to study and manipulate single molecules of DNA and other biopolymers. In one such technique, the magnetic trap, a single DNA molecule is bound at one end to a glass surface and at the other to a magnetic microbead. Small magnets, whose position and rotation can be controlled, pull on and rotate the microbead. This provides a simple method to stretch and twist the molecule. The system allows one to apply and measure forces ranging from 10(-3) to >100 pN. In contrast to other techniques, the force measurement is absolute and does not require calibration of the sensor. In this article, we describe the principle of the magnetic trap, as well as its use in the measurement of the elastic properties of DNA and the study of DNA-protein interactions.

  5. Small molecule alteration of RNA sequence in cells and animals.

    Science.gov (United States)

    Guan, Lirui; Luo, Yiling; Ja, William W; Disney, Matthew D

    2017-10-18

    RNA regulation and maintenance are critical for proper cell function. Small molecules that specifically alter RNA sequence would be exceptionally useful as probes of RNA structure and function or as potential therapeutics. Here, we demonstrate a photochemical approach for altering the trinucleotide expanded repeat causative of myotonic muscular dystrophy type 1 (DM1), r(CUG) exp . The small molecule, 2H-4-Ru, binds to r(CUG) exp and converts guanosine residues to 8-oxo-7,8-dihydroguanosine upon photochemical irradiation. We demonstrate targeted modification upon irradiation in cell culture and in Drosophila larvae provided a diet containing 2H-4-Ru. Our results highlight a general chemical biology approach for altering RNA sequence in vivo by using small molecules and photochemistry. Furthermore, these studies show that addition of 8-oxo-G lesions into RNA 3' untranslated regions does not affect its steady state levels. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Single Molecule Nanoelectrochemistry in Electrical Junctions.

    Science.gov (United States)

    Nichols, Richard J; Higgins, Simon J

    2016-11-15

    It is now possible to reliably measure single molecule conductance in a wide variety of environments including organic liquids, ultrahigh vacuum, water, ionic liquids, and electrolytes. The most commonly used methods deploy scanning probe microscopes, mechanically formed break junctions, or lithographically formed nanogap contacts. Molecules are generally captured between a pair of facing electrodes, and the junction current response is measured as a function of bias voltage. Gating electrodes can also be added so that the electrostatic potential at the molecular bridge can be independently controlled by this third noncontacting electrode. This can also be achieved in an electrolytic environment using a four-electrode bipotentiostatic configuration, which allows independent electrode potential control of the two contacting electrodes. This is commonly realized using an electrochemical STM and enables single molecule electrical characterization as a function of electrode potential and redox state of the molecular bridge. This has emerged as a powerful tool in modern interfacial electrochemistry and nanoelectrochemistry for studying charge transport across single molecules as a function of electrode potential and the electrolytic environments. Such measurements are possible in electrolytes ranging from aqueous buffers to nonaqueous ionic liquids. In this Account, we illustrate a number of examples of single molecule electrical measurements under electrode potential control use a scanning tunneling microscope (STM) and demonstrate how these can help in the understanding of charge transport in single molecule junctions. Examples showing charge transport following phase coherent tunneling to incoherent charge hopping across redox active molecular bridges are shown. In the case of bipyridinium (or viologen) molecular wires, it is shown how electrochemical reduction leads to an increase of the single molecule conductance, which is controlled by the liquid electrochemical

  7. Single molecule Studies of DNA Mismatch Repair

    Science.gov (United States)

    Erie, Dorothy A.; Weninger, Keith R.

    2015-01-01

    DNA mismatch repair involves is a widely conserved set of proteins that is essential to limit genetic drift in all organisms. The same system of proteins plays key roles in many cancer related cellular transactions in humans. Although the basic process has been reconstituted in vitro using purified components, many fundamental aspects of DNA mismatch repair remain hidden due in part to the complexity and transient nature of the interactions between the mismatch repair proteins and DNA substrates. Single molecule methods offer the capability to uncover these transient but complex interactions and allow novel insights into mechanisms that underlie DNA mismatch repair. In this review, we discuss applications of single molecule methodology including electron microscopy, atomic force microscopy, particle tracking, FRET, and optical trapping to studies of DNA mismatch repair. These studies have led to formulation of mechanistic models of how proteins identify single base mismatches in the vast background of matched DNA and signal for their repair. PMID:24746644

  8. Handbook of Single-Molecule Biophysics

    CERN Document Server

    Hinterdorfer, Peter

    2009-01-01

    The last decade has seen the development of a number of novel biophysical methods that allow the manipulation and study of individual biomolecules. The ability to monitor biological processes at this fundamental level of sensitivity has given rise to an improved understanding of the underlying molecular mechanisms. Through the removal of ensemble averaging, distributions and fluctuations of molecular properties can be characterized, transient intermediates identified, and catalytic mechanisms elucidated. By applying forces on biomolecules while monitoring their activity, important information can be obtained on how proteins couple function to structure. The Handbook of Single-Molecule Biophysics provides an introduction to these techniques and presents an extensive discussion of the new biological insights obtained from them. Coverage includes: Experimental techniques to monitor and manipulate individual biomolecules The use of single-molecule techniques in super-resolution and functional imaging Single-molec...

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

    Indian Academy of Sciences (India)

    For this purpose, a basic understanding of the molecular aspects of the interaction of small molecules with various RNA structures is essential. Alkaloids are a group of natural products with potential therapeutic utility, and very recently, their interaction with many RNA structures have been reported. Especially noteworthy are ...

  10. Preface: Special Topic on Single-Molecule Biophysics.

    Science.gov (United States)

    Makarov, Dmitrii E; Schuler, Benjamin

    2018-03-28

    Single-molecule measurements are now almost routinely used to study biological systems and processes. The scope of this special topic emphasizes the physics side of single-molecule observations, with the goal of highlighting new developments in physical techniques as well as conceptual insights that single-molecule measurements bring to biophysics. This issue also comprises recent advances in theoretical physical models of single-molecule phenomena, interpretation of single-molecule signals, and fundamental areas of statistical mechanics that are related to single-molecule observations. A particular goal is to illustrate the increasing synergy between theory, simulation, and experiment in single-molecule biophysics.

  11. Defining RNA-Small Molecule Affinity Landscapes Enables Design of a Small Molecule Inhibitor of an Oncogenic Noncoding RNA.

    Science.gov (United States)

    Velagapudi, Sai Pradeep; Luo, Yiling; Tran, Tuan; Haniff, Hafeez S; Nakai, Yoshio; Fallahi, Mohammad; Martinez, Gustavo J; Childs-Disney, Jessica L; Disney, Matthew D

    2017-03-22

    RNA drug targets are pervasive in cells, but methods to design small molecules that target them are sparse. Herein, we report a general approach to score the affinity and selectivity of RNA motif-small molecule interactions identified via selection. Named High Throughput Structure-Activity Relationships Through Sequencing (HiT-StARTS), HiT-StARTS is statistical in nature and compares input nucleic acid sequences to selected library members that bind a ligand via high throughput sequencing. The approach allowed facile definition of the fitness landscape of hundreds of thousands of RNA motif-small molecule binding partners. These results were mined against folded RNAs in the human transcriptome and identified an avid interaction between a small molecule and the Dicer nuclease-processing site in the oncogenic microRNA (miR)-18a hairpin precursor, which is a member of the miR-17-92 cluster. Application of the small molecule, Targapremir-18a, to prostate cancer cells inhibited production of miR-18a from the cluster, de-repressed serine/threonine protein kinase 4 protein (STK4), and triggered apoptosis. Profiling the cellular targets of Targapremir-18a via Chemical Cross-Linking and Isolation by Pull Down (Chem-CLIP), a covalent small molecule-RNA cellular profiling approach, and other studies showed specific binding of the compound to the miR-18a precursor, revealing broadly applicable factors that govern small molecule drugging of noncoding RNAs.

  12. Single molecule SERS: Perspectives of analytical applications

    Czech Academy of Sciences Publication Activity Database

    Vlčková, B.; Pavel, I.; Sládková, M.; Šišková, K.; Šlouf, Miroslav

    834-836, - (2007), s. 42-47 ISSN 0022-2860. [European Congress on Molecular Spectroscopy /28./. Istanbul, 03.09.2006-08.09.2006] R&D Projects: GA ČR GA203/04/0688 Institutional research plan: CEZ:AV0Z40500505 Keywords : surface-enhanced Raman scattering (SERS) * surface-enhanced resonance Raman (SERRS) * single molecule SERS Subject RIV: CD - Macromolecular Chemistry Impact factor: 1.486, year: 2007

  13. Single-Molecule Imaging of GPCR Interactions.

    Science.gov (United States)

    Calebiro, Davide; Sungkaworn, Titiwat

    2018-02-01

    G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are of great interest as pharmacological targets. Although the occurrence of GPCR signaling nanodomains has long been hypothesized based on indirect evidence, this and other fundamental aspects of GPCR signaling have been difficult to prove. The advent of single-molecule microscopy methods, which allow direct visualization of individual membrane proteins with unprecedented spatiotemporal resolution, provides unique opportunities to address several of these open questions. Indeed, recent single-molecule studies have revealed that GPCRs and G proteins transiently interact with each other as well as with structural components of the plasma membrane, leading to the formation of dynamic complexes and 'hot spots' for GPCR signaling. Whereas we are only beginning to understand the implications of this unexpected level of complexity, single-molecule approaches are likely to play a crucial role to further dissect the protein-protein interactions that are at the heart of GPCR signaling. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Graphical models for inferring single molecule dynamics

    Directory of Open Access Journals (Sweden)

    Gonzalez Ruben L

    2010-10-01

    Full Text Available Abstract Background The recent explosion of experimental techniques in single molecule biophysics has generated a variety of novel time series data requiring equally novel computational tools for analysis and inference. This article describes in general terms how graphical modeling may be used to learn from biophysical time series data using the variational Bayesian expectation maximization algorithm (VBEM. The discussion is illustrated by the example of single-molecule fluorescence resonance energy transfer (smFRET versus time data, where the smFRET time series is modeled as a hidden Markov model (HMM with Gaussian observables. A detailed description of smFRET is provided as well. Results The VBEM algorithm returns the model’s evidence and an approximating posterior parameter distribution given the data. The former provides a metric for model selection via maximum evidence (ME, and the latter a description of the model’s parameters learned from the data. ME/VBEM provide several advantages over the more commonly used approach of maximum likelihood (ML optimized by the expectation maximization (EM algorithm, the most important being a natural form of model selection and a well-posed (non-divergent optimization problem. Conclusions The results demonstrate the utility of graphical modeling for inference of dynamic processes in single molecule biophysics.

  15. Origins and Early Evolution of the tRNA Molecule

    Directory of Open Access Journals (Sweden)

    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.

  16. Origins and Early Evolution of the tRNA Molecule.

    Science.gov (United States)

    Tamura, Koji

    2015-12-03

    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.

  17. Single-molecule theory of enzymatic inhibition.

    Science.gov (United States)

    Robin, Tal; Reuveni, Shlomi; Urbakh, Michael

    2018-02-22

    The classical theory of enzymatic inhibition takes a deterministic, bulk based approach to quantitatively describe how inhibitors affect the progression of enzymatic reactions. Catalysis at the single-enzyme level is, however, inherently stochastic which could lead to strong deviations from classical predictions. To explore this, we take the single-enzyme perspective and rebuild the theory of enzymatic inhibition from the bottom up. We find that accounting for multi-conformational enzyme structure and intrinsic randomness should strongly change our view on the uncompetitive and mixed modes of inhibition. There, stochastic fluctuations at the single-enzyme level could make inhibitors act as activators; and we state-in terms of experimentally measurable quantities-a mathematical condition for the emergence of this surprising phenomenon. Our findings could explain why certain molecules that inhibit enzymatic activity when substrate concentrations are high, elicit a non-monotonic dose response when substrate concentrations are low.

  18. 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.

  19. Single-Molecule Interfacial Electron Transfer

    Energy Technology Data Exchange (ETDEWEB)

    Ho, Wilson [Univ. of California, Irvine, CA (United States)

    2018-02-03

    Interfacial electron transfer (ET) plays an important role in many chemical and biological processes. Specifically, interfacial ET in TiO2-based systems is important to solar energy technology, catalysis, and environmental remediation technology. However, the microscopic mechanism of interfacial ET is not well understood with regard to atomic surface structure, molecular structure, bonding, orientation, and motion. In this project, we used two complementary methodologies; single-molecule fluorescence spectroscopy, and scanning-tunneling microscopy and spectroscopy (STM and STS) to address this scientific need. The goal of this project was to integrate these techniques and measure the molecular dependence of ET between adsorbed molecules and TiO2 semiconductor surfaces and the ET induced reactions such as the splitting of water. The scanning probe techniques, STM and STS, are capable of providing the highest spatial resolution but not easily time-resolved data. Single-molecule fluorescence spectroscopy is capable of good time resolution but requires further development to match the spatial resolution of the STM. The integrated approach involving Peter Lu at Bowling Green State University (BGSU) and Wilson Ho at the University of California, Irvine (UC Irvine) produced methods for time and spatially resolved chemical imaging of interfacial electron transfer dynamics and photocatalytic reactions. An integral aspect of the joint research was a significant exchange of graduate students to work at the two institutions. This project bridged complementary approaches to investigate a set of common problems by working with the same molecules on a variety of solid surfaces, but using appropriate techniques to probe under ambient (BGSU) and ultrahigh vacuum (UCI) conditions. The molecular level understanding of the fundamental interfacial electron transfer processes obtained in this joint project will be important for developing efficient light harvesting

  20. Deep learning for single-molecule science

    Science.gov (United States)

    Albrecht, Tim; Slabaugh, Gregory; Alonso, Eduardo; Al-Arif, SM Masudur R.

    2017-10-01

    Exploring and making predictions based on single-molecule data can be challenging, not only due to the sheer size of the datasets, but also because a priori knowledge about the signal characteristics is typically limited and poor signal-to-noise ratio. For example, hypothesis-driven data exploration, informed by an expectation of the signal characteristics, can lead to interpretation bias or loss of information. Equally, even when the different data categories are known, e.g., the four bases in DNA sequencing, it is often difficult to know how to make best use of the available information content. The latest developments in machine learning (ML), so-called deep learning (DL) offer interesting, new avenues to address such challenges. In some applications, such as speech and image recognition, DL has been able to outperform conventional ML strategies and even human performance. However, to date DL has not been applied much in single-molecule science, presumably in part because relatively little is known about the ‘internal workings’ of such DL tools within single-molecule science as a field. In this Tutorial, we make an attempt to illustrate in a step-by-step guide how one of those, a convolutional neural network (CNN), may be used for base calling in DNA sequencing applications. We compare it with a SVM as a more conventional ML method, and discuss some of the strengths and weaknesses of the approach. In particular, a ‘deep’ neural network has many features of a ‘black box’, which has important implications on how we look at and interpret data.

  1. Biological Nanopores: Confined Spaces for Electrochemical Single-Molecule Analysis.

    Science.gov (United States)

    Cao, Chan; Long, Yi-Tao

    2018-02-20

    Nanopore sensing is developing into a powerful single-molecule approach to investigate the features of biomolecules that are not accessible by studying ensemble systems. When a target molecule is transported through a nanopore, the ions occupying the pore are excluded, resulting in an electrical signal from the intermittent ionic blockade event. By statistical analysis of the amplitudes, duration, frequencies, and shapes of the blockade events, many properties of the target molecule can be obtained in real time at the single-molecule level, including its size, conformation, structure, charge, geometry, and interactions with other molecules. With the development of the use of α-hemolysin to characterize individual polynucleotides, nanopore technology has attracted a wide range of research interest in the fields of biology, physics, chemistry, and nanoscience. As a powerful single-molecule analytical method, nanopore technology has been applied for the detection of various biomolecules, including oligonucleotides, peptides, oligosaccharides, organic molecules, and disease-related proteins. In this Account, we highlight recent developments of biological nanopores in DNA-based sensing and in studying the conformational structures of DNA and RNA. Furthermore, we introduce the application of biological nanopores to investigate the conformations of peptides affected by charge, length, and dipole moment and to study disease-related proteins' structures and aggregation transitions influenced by an inhibitor, a promoter, or an applied voltage. To improve the sensing ability of biological nanopores and further extend their application to a wider range of molecular sensing, we focus on exploring novel biological nanopores, such as aerolysin and Stable Protein 1. Aerolysin exhibits an especially high sensitivity for the detection of single oligonucleotides both in current separation and duration. Finally, to facilitate the use of nanopore measurements and statistical analysis

  2. Mechanoenzymatics and Nanoassembly of Single Molecules

    Science.gov (United States)

    Puchner, Elias M.; Gaub, Hermann E.

    We investigated the muscle enzyme, titin kinase, by means of single-molecule force spectroscopy. Our results show that the binding of ATP, which is the first step of its signaling cascade controlling the muscle gene expression and protein turnover, is mechanically induced. The detailed determination of barrier positions in the mechanical activation pathway and the corresponding functional states allow structural insight, by comparing the experiment with molecular dynamics simulations. From our results, we conclude that titin kinase acts as a natural force sensor controlling the muscle build-up. To study the interplay of functional units, we developed the single-molecule cut-and-paste technique, which combines the precision of AFM with the selectivity of DNA hybridization. Functional units can be assembled one-by-one in an arbitrarily predefined pattern, with an accuracy that is better than 11 nm. The cyclic assembly process is optically monitored and mechanically recorded by force-extension traces. Using biotin as a functional unit attached to the transported DNA, patterns of binding sites may be created, to which streptavidin-modified nanoobjects like fluorescent nanoparticles can specifically self-assemble in a second step.

  3. Single particle tracking and single molecule energy transfer

    CERN Document Server

    Bräuchle, Christoph; Michaelis, Jens

    2009-01-01

    Closing a gap in the literature, this handbook gathers all the information on single particle tracking and single molecule energy transfer. It covers all aspects of this hot and modern topic, from detecting virus entry to membrane diffusion, and from protein folding using spFRET to coupled dye systems, as well recent achievements in the field. Throughout, the first-class editors and top international authors present content of the highest quality, making this a must-have for physical chemists, spectroscopists, molecular physicists and biochemists.

  4. Spectrally resolved single-molecule electrometry

    Science.gov (United States)

    Ruggeri, F.; Krishnan, M.

    2018-03-01

    Escape-time electrometry is a recently developed experimental technique that offers the ability to measure the effective electrical charge of a single biomolecule in solution with sub-elementary charge precision. The approach relies on measuring the average escape-time of a single charged macromolecule or molecular species transiently confined in an electrostatic fluidic trap. Comparing the experiments with the predictions of a mean-field model of molecular electrostatics, we have found that the measured effective charge even reports on molecular conformation, e.g., folded or disordered state, and non-uniform charge distribution in disordered proteins or polyelectrolytes. Here we demonstrate the ability to use the spectral dimension to distinguish minute differences in electrical charge between individual molecules or molecular species in a single simultaneous measurement, under identical experimental conditions. Using one spectral channel for referenced measurement, this kind of photophysical distinguishability essentially eliminates the need for accurate knowledge of key experimental parameters, otherwise obtained through intensive characterization of the experimental setup. As examples, we demonstrate the ability to detect small differences (˜5%) in the length of double-stranded DNA fragments as well as single amino acid exchange in an intrinsically disordered protein, prothymosin α.

  5. Molecular electronics: the single molecule switch and transistor

    NARCIS (Netherlands)

    Sotthewes, Kai; Geskin, Victor; Heimbuch, Rene; Kumar, Avijit; Zandvliet, Henricus J.W.

    2014-01-01

    In order to design and realize single-molecule devices it is essential to have a good understanding of the properties of an individual molecule. For electronic applications, the most important property of a molecule is its conductance. Here we show how a single octanethiol molecule can be connected

  6. 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...

  7. 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

  8. 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

  9. Magnetization reversal in single molecule magnets

    Science.gov (United States)

    Bokacheva, Louisa

    2002-09-01

    I have studied the magnetization reversal in single molecule magnets (SMMs). SMMs are Van der Waals crystals, consisting of identical molecules containing transition metal ions, with high spin and large uniaxial magnetic anisotropy. They can be considered as ensembles of identical, iso-oriented nanomagnets. At high temperature, these materials behave as superparamagnets and their magnetization reversal occurs by thermal activation. At low temperature they become blocked, and their magnetic relaxation occurs via thermally assisted tunneling or pure quantum tunneling through the anisotropy barrier. We have conducted detailed experimental studies of the magnetization reversal in SMM material Mn12-acetate (Mn12) with S = 10. Low temperature measurements were conducted using micro-Hall effect magnetometry. We performed hysteresis and relaxation studies as a function of temperature, transverse field, and magnetization state of the sample. We identified magnetic sublevels that dominate the tunneling at a given field, temperature and magnetization. We observed a crossover between thermally assisted and pure quantum tunneling. The form of this crossover depends on the magnitude and direction of the applied field. This crossover is abrupt (first-order) and occurs in a narrow temperature interval (tunneling mechanisms in Mn12.

  10. Grafting single molecule magnets on gold nanoparticles.

    Science.gov (United States)

    Perfetti, Mauro; Pineider, Francesco; Poggini, Lorenzo; Otero, Edwige; Mannini, Matteo; Sorace, Lorenzo; Sangregorio, Claudio; Cornia, Andrea; Sessoli, Roberta

    2014-01-29

    The chemical synthesis and characterization of the first hybrid material composed by gold nanoparticles and single molecule magnets (SMMs) are described. Gold nanoparticles are functionalized via ligand exchange using a tetrairon(III) SMM containing two 1,2-dithiolane end groups. The grafting is evidenced by the shift of the plasmon resonance peak recorded with a UV-vis spectrometer, by the suppression of nuclear magnetic resonance signals, by X-ray photoemission spectroscopy peaks, and by transmission electron microscopy images. The latter evidence the formation of aggregates of nanoparticles as a consequence of the cross-linking ability of Fe4 through the two 1,2-dithiolane rings located on opposite sides of the metal core. The presence of intact Fe4 molecules is directly proven by synchrotron-based X-ray absorption spectroscopy and X-ray magnetic circular dichroism spectroscopy, while a detailed magnetic characterization, obtained using electron paramagnetic resonance and alternating-current susceptibility, confirms the persistence of SMM behavior in this new hybrid nanostructure. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Small Interfering RNA Efficiently Suppresses Adhesion Molecule Expression on Pulmonary Microvascular Endothelium

    Directory of Open Access Journals (Sweden)

    Tobias Walker

    2011-01-01

    Full Text Available Background. Adhesion molecules are known to influence postoperative organ function, they are hardly involved in the inflammatory response following the ischemia-reperfusion injury. We sought to investigate the potency of small interfering RNAs to suppress adhesion molecule expression in human pulmonary microvascular endothelial cells. Methods. Human lung microvascular endothelial cells were transfected with specific siRNA followed by a stimulation of the cells with an inflammatory cytokine. Adhesion molecule expression was determined by FACS-analysis, and reduction of intracellular mRNA was determined by qRT-PCR. Furthermore, the attachment of isolated neutrophils on the endothelial layer was determined after siRNA transfection. Results. In summary, siRNA transfection significantly decreased the percentage positive cells in a single cocktail transfection of each adhesion molecule investigated. Adhering neutrophils were diminished as well. Conclusion. siRNA might be a promising tool for the effective suppression of adhesion molecule expression on pulmonary microvascular cells, potentially minimizing leukocyte-endothelial depending interactions of a pulmonary allograft.

  12. Single Molecule Screening of Disease DNA Without Amplification

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Ji-Young [Iowa State Univ., Ames, IA (United States)

    2006-01-01

    The potential of single molecule detection as an analysis tool in biological and medical fields is well recognized today. This fast evolving technique will provide fundamental sensitivity to pick up individual pathogen molecules, and therefore contribute to a more accurate diagnosis and a better chance for a complete cure. Many studies are being carried out to successfully apply this technique in real screening fields. In this dissertation, several attempts are shown that have been made to test and refine the application of the single molecule technique as a clinical screening method. A basic applicability was tested with a 100% target content sample, using electrophoretic mobility and multiple colors as identification tools. Both electrophoretic and spectral information of individual molecule were collected within a second, while the molecule travels along the flow in a capillary. Insertion of a transmission grating made the recording of the whole spectrum of a dye-stained molecule possible without adding complicated instrumental components. Collecting two kinds of information simultaneously and combining them allowed more thorough identification, up to 98.8% accuracy. Probing mRNA molecules with fluorescently labeled cDNA via hybridization was also carried out. The spectral differences among target, probe, and hybrid were interpreted in terms of dispersion distances after transmission grating, and used for the identification of each molecule. The probes were designed to have the least background when they are free, but have strong fluorescence after hybridization via fluorescence resonance energy transfer. The mRNA-cDNA hybrids were further imaged in whole blood, plasma, and saliva, to test how far a crude preparation can be tolerated. Imaging was possible with up to 50% of clear bio-matrix contents, suggesting a simple lysis and dilution would be sufficient for imaging for some cells. Real pathogen DNA of human papillomavirus (HPV) type-I6 in human genomic DNA

  13. Single Molecule Analysis of Resection Tracks.

    Science.gov (United States)

    Huertas, Pablo; Cruz-García, Andrés

    2018-01-01

    Homologous recombination is initiated by the so-called DNA end resection, the 5'-3' nucleolytic degradation of a single strand of the DNA at each side of the break. The presence of resected DNA is an obligatory step for homologous recombination. Moreover, the amount of resected DNA modulates the prevalence of different recombination pathways. In different model organisms, there are several published ways to visualize and measure with more or less detail the amount of DNA resected. In human cells, however, technical constraints hampered the study of resection at high resolution. Some information might be gathered from the study of endonuclease-created DSBs, in which the resection of breaks at known sites can be followed by PCR or ChIP. In this chapter, we describe in detail a novel assay to study DNA end resection in breaks located on unknown positions. Here, we use ionizing radiation to induce double-strand breaks, but the same approach can be used to monitor resection induced by different DNA damaging agents. By modifying the DNA-combing technique, used for high-resolution replication analyses, we can measure resection progression at the level of individual DNA fibers. Thus, we named the method Single Molecule Analysis of Resection Tracks (SMART). We use human cells in culture as a model system, but in principle the same approach would be feasible to any model organism adjusting accordingly the DNA isolation part of the protocol.

  14. Nucleosomal arrangement affects single-molecule transcription dynamics.

    Science.gov (United States)

    Fitz, Veronika; Shin, Jaeoh; Ehrlich, Christoph; Farnung, Lucas; Cramer, Patrick; Zaburdaev, Vasily; Grill, Stephan W

    2016-10-24

    In eukaryotes, gene expression depends on chromatin organization. However, how chromatin affects the transcription dynamics of individual RNA polymerases has remained elusive. Here, we use dual trap optical tweezers to study single yeast RNA polymerase II (Pol II) molecules transcribing along a DNA template with two nucleosomes. The slowdown and the changes in pausing behavior within the nucleosomal region allow us to determine a drift coefficient, χ, which characterizes the ability of the enzyme to recover from a nucleosomal backtrack. Notably, χ can be used to predict the probability to pass the first nucleosome. Importantly, the presence of a second nucleosome changes χ in a manner that depends on the spacing between the two nucleosomes, as well as on their rotational arrangement on the helical DNA molecule. Our results indicate that the ability of Pol II to pass the first nucleosome is increased when the next nucleosome is turned away from the first one to face the opposite side of the DNA template. These findings help to rationalize how chromatin arrangement affects Pol II transcription dynamics.

  15. Examining small molecule: HIV RNA interactions using arrayed imaging reflectometry

    Science.gov (United States)

    Chaimayo, Wanaruk; Miller, Benjamin L.

    2014-03-01

    Human Immunodeficiency Virus (HIV) has been the subject of intense research for more than three decades as it causes an uncurable disease: Acquired Immunodeficiency Syndrome, AIDS. In the pursuit of a medical treatment, RNAtargeted small molecules are emerging as promising targets. In order to understand the binding kinetics of small molecules and HIV RNA, association (ka) and dissociation (kd) kinetic constants must be obtained, ideally for a large number of sequences to assess selectivity. We have developed Aqueous Array Imaged Reflectometry (Aq-AIR) to address this challenge. Using a simple light interference phenomenon, Aq-AIR provides real-time high-throughput multiplex capabilities to detect binding of targets to surface-immobilized probes in a label-free microarray format. The second generation of Aq-AIR consisting of high-sensitivity CCD camera and 12-μL flow cell was fabricated. The system performance was assessed by real-time detection of MBNL1-(CUG)10 and neomycin B - HIV RNA bindings. The results establish this second-generation Aq-AIR to be able to examine small molecules binding to RNA sequences specific to HIV.

  16. Single-molecule magnets ``without'' intermolecular interactions

    Science.gov (United States)

    Wernsdorfer, W.; Vergnani, L.; Rodriguez-Douton, M. J.; Cornia, A.; Neugebauer, P.; Barra, A. L.; Sorace, L.; Sessoli, R.

    2012-02-01

    Intermolecular magnetic interactions (dipole-dipole and exchange) affect strongly the magnetic relaxation of crystals of single-molecule magnets (SMMs), especially at low temperature, where quantum tunneling of the magnetization (QTM) dominates. This leads to complex many-body problems [l]. Measurements on magnetically diluted samples are desirable to clearly sort out the behaviour of magnetically-isolated SMMs and to reveal, by comparison, the effect of intermolecular interactions. Here, we diluted a Fe4 SMM into a diamagnetic crystal lattice, affording arrays of independent and iso-oriented magnetic units. We found that the resonant tunnel transitions are much sharper, the tunneling efficiency changes significantly, and two-body QTM transitions disappear. These changes have been rationalized on the basis of a dipolar shuffling mechanism and of transverse dipolar fields, whose effect has been analyzed using a multispin model. Our findings directly prove the impact of intermolecular magnetic couplings on the SMM behaviour and disclose the magnetic response of truly-isolated giant spins in a diamagnetic crystalline environment.[4pt] [1] W. Wernsdorfer, at al, PRL 82, 3903 (1999); PRL 89, 197201 (2002); Nature 416, 406 (2002); IS Tupitsyn, PCE Stamp, NV Prokof'ev, PRB 69, 132406 (2004).

  17. Giant magnetoresistance through a single molecule.

    Science.gov (United States)

    Schmaus, Stefan; Bagrets, Alexei; Nahas, Yasmine; Yamada, Toyo K; Bork, Annika; Bowen, Martin; Beaurepaire, Eric; Evers, Ferdinand; Wulfhekel, Wulf

    2011-03-01

    Magnetoresistance is a change in the resistance of a material system caused by an applied magnetic field. Giant magnetoresistance occurs in structures containing ferromagnetic contacts separated by a metallic non-magnetic spacer, and is now the basis of read heads for hard drives and for new forms of random access memory. Using an insulator (for example, a molecular thin film) rather than a metal as the spacer gives rise to tunnelling magnetoresistance, which typically produces a larger change in resistance for a given magnetic field strength, but also yields higher resistances, which are a disadvantage for real device operation. Here, we demonstrate giant magnetoresistance across a single, non-magnetic hydrogen phthalocyanine molecule contacted by the ferromagnetic tip of a scanning tunnelling microscope. We measure the magnetoresistance to be 60% and the conductance to be 0.26G(0), where G(0) is the quantum of conductance. Theoretical analysis identifies spin-dependent hybridization of molecular and electrode orbitals as the cause of the large magnetoresistance.

  18. Single-Molecule Plasmon Sensing: Current Status and Future Prospects.

    Science.gov (United States)

    Taylor, Adam B; Zijlstra, Peter

    2017-08-25

    Single-molecule detection has long relied on fluorescent labeling with high quantum-yield fluorophores. Plasmon-enhanced detection circumvents the need for labeling by allowing direct optical detection of weakly emitting and completely nonfluorescent species. This review focuses on recent advances in single molecule detection using plasmonic metal nanostructures as a sensing platform, particularly using a single particle-single molecule approach. In the past decade two mechanisms for plasmon-enhanced single-molecule detection have been demonstrated: (1) by plasmonically enhancing the emission of weakly fluorescent biomolecules, or (2) by monitoring shifts of the plasmon resonance induced by single-molecule interactions. We begin with a motivation regarding the importance of single molecule detection, and advantages plasmonic detection offers. We describe both detection mechanisms and discuss challenges and potential solutions. We finalize by highlighting the exciting possibilities in analytical chemistry and medical diagnostics.

  19. OPE3 : A model system for single-molecule transport

    NARCIS (Netherlands)

    Frisenda, R.

    2016-01-01

    In this dissertation, charge-transport through individual organic molecules is investigated. The single molecules are contacted with two-terminal mechanically controllable break junction gold electrodes and their electrical and mechanical behavior studied at room and low temperature.

  20. 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)

  1. Transport through a Single Octanethiol Molecule

    NARCIS (Netherlands)

    Kockmann, D.; Poelsema, Bene; Zandvliet, Henricus J.W.

    2009-01-01

    Octanethiol molecules adsorbed on Pt chains are studied with scanning tunneling microscopy and spectroscopy at 77 K. The head of the octanethiol binds to a Pt atom and the tail is lying flat down on the chain. Open-loop current time traces reveal that the molecule wags its tail and attaches to the

  2. 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.

  3. Experimental techniques for single cell and single molecule biomechanics

    International Nuclear Information System (INIS)

    Lim, C.T.; Zhou, E.H.; Li, A.; Vedula, S.R.K.; Fu, H.X.

    2006-01-01

    Stresses and strains that act on the human body can arise either from external physical forces or internal physiological environmental conditions. These biophysical interactions can occur not only at the musculoskeletal but also cellular and molecular levels and can determine the health and function of the human body. Here, we seek to investigate the structure-property-function relationship of cells and biomolecules so as to understand their important physiological functions as well as establish possible connections to human diseases. With the recent advancements in cell and molecular biology, biophysics and nanotechnology, several innovative and state-of-the-art experimental techniques and equipment have been developed to probe the structural and mechanical properties of biostructures from the micro- down to picoscale. Some of these experimental techniques include the optical or laser trap method, micropipette aspiration, step-pressure technique, atomic force microscopy and molecular force spectroscopy. In this article, we will review the basic principles and usage of these techniques to conduct single cell and single molecule biomechanics research

  4. Importance and key events of prokaryotic RNA decay: the ultimate fate of an RNA molecule.

    Science.gov (United States)

    Silva, Inês Jesus; Saramago, Margarida; Dressaire, Clémentine; Domingues, Susana; Viegas, Sandra Cristina; Arraiano, Cecília Maria

    2011-01-01

    RNAs are important effectors in the process of gene expression. In bacteria, constant adaptation to environmental demands is accompanied by a continual adjustment of transcripts' levels. The cellular concentration of a given RNA is the result of the balance between its synthesis and degradation. RNA degradation is a complex process encompassing multiple pathways. Ribonucleases (RNases) are the enzymes that directly process and degrade the transcripts, regulating their amounts. They are also important in quality control of RNAs by detecting and destroying defective molecules. The rate at which RNA decay occurs depends on the availability of ribonucleases and their specificities according to the sequence and/or the structural elements of the RNA molecule. Ribosome loading and the 5'-phosphorylation status can also modulate the stability of transcripts. The wide diversity of RNases present in different microorganisms is another factor that conditions the pathways and mechanisms of RNA degradation. RNases are themselves carefully regulated by distinct mechanisms. Several other factors modulate RNA degradation, namely polyadenylation, which plays a multifunctional role in RNA metabolism. Additionally, small non-coding RNAs are crucial regulators of gene expression, and can directly modulate the stability of their mRNA targets. In many cases this regulation is dependent on Hfq, an RNA binding protein which can act in concert with polyadenylation enzymes and is often necessary for the activity of sRNAs. All of the above-mentioned aspects are discussed in the present review, which also highlights the principal differences between the RNA degradation pathways for the two main Gram-negative and Gram-positive bacterial models. Copyright © 2011 John Wiley & Sons, Ltd.

  5. RNA structure probing : biochemical structure analysis of autoimmune-related RNA molecules

    NARCIS (Netherlands)

    Teunissen, A.W.M.

    1999-01-01

    Next to the well known messenger, ribosomal and transfer RNAs, a large number of small structural RNA molecules exist. These RNAs are bound to proteins, forming ribonucleoprotein particles (RNPs). RNPs are often targets for autoantibodies occurring in an autoimmune disease.Chapter 1 introduces

  6. 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

  7. 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.

  8. Electrical and mechanical effects in single-molecule junctions

    NARCIS (Netherlands)

    Seldenthuis, J.S.

    2011-01-01

    In single-molecule junctions, the behavior of a device is determined by the properties of an individual molecule. In this thesis we develop several models to describe both electrical and mechanical effects in such devices, which can be used to design molecules with a specific functionality. We show

  9. Manipulation of organic polyradicals in a single-molecule transistor

    NARCIS (Netherlands)

    Fock, J.; Leijnse, M.; Jennum, K.; Zyazin, A.S.; Paaske, J.; Hedegard, P.; Brondsted Nielsen, M.; Van der Zant, H.S.J.

    2012-01-01

    Inspired by cotunneling spectroscopy of spin-states in a single OPE5-based molecule, we investigate the prospects for electric control of magnetism in purely organic molecules contacted in a three-terminal geometry. Using the gate electrode, the molecule is reversibly switched between three

  10. Atomic-Scale Control of Electron Transport through Single Molecules

    DEFF Research Database (Denmark)

    Wang, Y. F.; Kroger, J.; Berndt, R.

    2010-01-01

    Tin-phthalocyanine molecules adsorbed on Ag(111) were contacted with the tip of a cryogenic scanning tunneling microscope. Orders-of-magnitude variations of the single-molecule junction conductance were achieved by controllably dehydrogenating the molecule and by modifying the atomic structure...

  11. Single Molecule 3D Orientation in Time and Space

    NARCIS (Netherlands)

    Börner, Richard; Ehrlich, Nicky; Hohlbein, Johannes; Hübner, Christian G.

    2016-01-01

    Interactions between single molecules profoundly depend on their mutual three-dimensional orientation. Recently, we demonstrated a technique that allows for orientation determination of single dipole emitters using a polarization-resolved distribution of fluorescence into several detection

  12. Phenotypic effect of mutations in evolving populations of RNA molecules

    Directory of Open Access Journals (Sweden)

    Manrubia Susanna C

    2010-02-01

    Full Text Available Abstract Background The secondary structure of folded RNA sequences is a good model to map phenotype onto genotype, as represented by the RNA sequence. Computational studies of the evolution of ensembles of RNA molecules towards target secondary structures yield valuable clues to the mechanisms behind adaptation of complex populations. The relationship between the space of sequences and structures, the organization of RNA ensembles at mutation-selection equilibrium, the time of adaptation as a function of the population parameters, the presence of collective effects in quasispecies, or the optimal mutation rates to promote adaptation all are issues that can be explored within this framework. Results We investigate the effect of microscopic mutations on the phenotype of RNA molecules during their in silico evolution and adaptation. We calculate the distribution of the effects of mutations on fitness, the relative fractions of beneficial and deleterious mutations and the corresponding selection coefficients for populations evolving under different mutation rates. Three different situations are explored: the mutation-selection equilibrium (optimized population in three different fitness landscapes, the dynamics during adaptation towards a goal structure (adapting population, and the behavior under periodic population bottlenecks (perturbed population. Conclusions The ratio between the number of beneficial and deleterious mutations experienced by a population of RNA sequences increases with the value of the mutation rate μ at which evolution proceeds. In contrast, the selective value of mutations remains almost constant, independent of μ, indicating that adaptation occurs through an increase in the amount of beneficial mutations, with little variations in the average effect they have on fitness. Statistical analyses of the distribution of fitness effects reveal that small effects, either beneficial or deleterious, are well described by a Pareto

  13. Novel approaches for single molecule activation and detection

    CERN Document Server

    Benfenati, Fabio; Torre, Vincent

    2014-01-01

    How can we obtain tools able to process and exchange information at the molecular scale In order to do this, it is necessary to activate and detect single molecules under controlled conditions. This book focuses on the generation of biologically-inspired molecular devices. These devices are based on the developments of new photonic tools able to activate and stimulate single molecule machines. Additionally, new light sensitive molecules can be selectively activated by photonic tools. These technological innovations will provide a way to control activation of single light-sensitive molecules, a

  14. Single Molecule Studies on Dynamics in Liquid Crystals

    Directory of Open Access Journals (Sweden)

    Daniela Täuber

    2013-09-01

    Full Text Available Single molecule (SM methods are able to resolve structure related dynamics of guest molecules in liquid crystals (LC. Highly diluted small dye molecules on the one hand explore structure formation and LC dynamics, on the other hand they report about a distortion caused by the guest molecules. The anisotropic structure of LC materials is used to retrieve specific conformation related properties of larger guest molecules like conjugated polymers. This in particular sheds light on organization mechanisms within biological cells, where large molecules are found in nematic LC surroundings. This review gives a short overview related to the application of highly sensitive SM detection schemes in LC.

  15. Single molecule studies on dynamics in liquid crystals.

    Science.gov (United States)

    Täuber, Daniela; von Borczyskowski, Christian

    2013-09-26

    Single molecule (SM) methods are able to resolve structure related dynamics of guest molecules in liquid crystals (LC). Highly diluted small dye molecules on the one hand explore structure formation and LC dynamics, on the other hand they report about a distortion caused by the guest molecules. The anisotropic structure of LC materials is used to retrieve specific conformation related properties of larger guest molecules like conjugated polymers. This in particular sheds light on organization mechanisms within biological cells, where large molecules are found in nematic LC surroundings. This review gives a short overview related to the application of highly sensitive SM detection schemes in LC.

  16. Rotation of a single molecule within a supramolecular bearing

    DEFF Research Database (Denmark)

    Gimzewski, J.K.; Joachim, C.; Schlittler, R.R.

    1998-01-01

    Experimental visualization and verification of a single-molecule rotor operating within a supramolecular bearing is reported. Using a scanning tunneling microscope, single molecules were observed to exist in one of two spatially defined states Laterally separated by 0.26 nanometers. One...

  17. Large negative differential conductance in single-molecule break junctions

    NARCIS (Netherlands)

    Perrin, Mickael L.; Frisenda, Riccardo; Koole, Max; Seldenthuis, Johannes S.; Gil, Jose A. Celis; Valkenier, Hennie; Hummelen, Jan C.; Renaud, Nicolas; Grozema, Ferdinand C.; Thijssen, Joseph M.; Dulic, Diana; van der Zant, Herre S. J.

    2014-01-01

    Molecular electronics aims at exploiting the internal structure and electronic orbitals of molecules to construct functional building blocks(1). To date, however, the overwhelming majority of experimentally realized single-molecule junctions can be described as single quantum dots, where transport

  18. Single-molecule probes in organic field-effect transistors

    NARCIS (Netherlands)

    Nicolet, Aurélien Armel Louis

    2007-01-01

    The goal of this thesis is to study charge transport phenomena in organic materials. This is done optically by means of single-molecule spectroscopy in a field-effect transistor based on a molecular crystal. We present (in Chapter 2) a fundamental requirement for single-molecule spectroscopy

  19. A single molecule DNA flow stretching microscope for undergraduates

    NARCIS (Netherlands)

    Williams, Kelly; Grafe, Brendan; Burke, Kathryn M.; Tanner, Nathan; van Oijen, Antoine M.; Loparo, Joseph; Price, Allen C.

    2011-01-01

    The design of a simple, safe, and inexpensive single molecule flow stretching instrument is presented. The instrument uses a low cost upright microscope coupled to a webcam for imaging single DNA molecules that are tethered in an easy to construct microfluidic flow cell. The system requires no

  20. Single molecule insights on conformational selection and induced fit mechanism

    DEFF Research Database (Denmark)

    Hatzakis, Nikos

    2014-01-01

    of unsynchronized molecules, often masking intrinsic dynamic behavior of proteins and biologically significant transient intermediates. Single molecule measurements are emerging as a powerful tool for characterizing protein function. They offer the direct observation and quantification of the activity, abundance...... and lifetime of multiple states and transient intermediates in the energy landscape, that are typically averaged out in non-synchronized ensemble measurements. Here we survey new insights from single molecule studies that advance our understanding of the molecular mechanisms underlying biomolecular recognition....

  1. DNA analysis by single molecule stretching in nanofluidic biochips

    DEFF Research Database (Denmark)

    Abad, E.; Juarros, A.; Retolaza, A.

    2011-01-01

    Stretching single DNA molecules by confinement in nanofluidic channels has attracted a great interest during the last few years as a DNA analysis tool. We have designed and fabricated a sealed micro/nanofluidic device for DNA stretching applications, based on the use of the high throughput Nano......Imprint Lithography (NIL) technology combined with a conventional anodic bonding of the silicon base and Pyrex cover. Using this chip, we have performed single molecule imaging on a bench-top fluorescent microscope system. Lambda phage DNA was used as a model sample to characterize the chip. Single molecules of λ...... a method to determining DNA size. The results of this work prove that the developed fabrication process is a good alternative for the fabrication of single molecule DNA biochips and it allows developing a variety of innovative bio/chemical sensors based on single-molecule DNA sequencing devices....

  2. Computer systems for annotation of single molecule fragments

    Science.gov (United States)

    Schwartz, David Charles; Severin, Jessica

    2016-07-19

    There are provided computer systems for visualizing and annotating single molecule images. Annotation systems in accordance with this disclosure allow a user to mark and annotate single molecules of interest and their restriction enzyme cut sites thereby determining the restriction fragments of single nucleic acid molecules. The markings and annotations may be automatically generated by the system in certain embodiments and they may be overlaid translucently onto the single molecule images. An image caching system may be implemented in the computer annotation systems to reduce image processing time. The annotation systems include one or more connectors connecting to one or more databases capable of storing single molecule data as well as other biomedical data. Such diverse array of data can be retrieved and used to validate the markings and annotations. The annotation systems may be implemented and deployed over a computer network. They may be ergonomically optimized to facilitate user interactions.

  3. Controlled transport through a single molecule

    NARCIS (Netherlands)

    Kumar, Avijit; Heimbuch, Rene; Poelsema, Bene; Zandvliet, Henricus J.W.

    2012-01-01

    We demonstrate how an electrode–molecule–electrode junction can be controllably opened and closed by careful tuning of the contacts' interspace and voltage. The molecule, an octanethiol, flips to bridge a ~1 nm interspace between substrate and scanning tunnelling microscope tip when an electric

  4. Designing Efficient Double RNA trans-Splicing Molecules for Targeted RNA Repair

    Science.gov (United States)

    Hüttner, Clemens; Murauer, Eva M.; Hainzl, Stefan; Kocher, Thomas; Neumayer, Anna; Reichelt, Julia; Bauer, Johann W.; Koller, Ulrich

    2016-01-01

    RNA trans-splicing is a promising tool for mRNA modification in a diversity of genetic disorders. In particular, the substitution of internal exons of a gene by combining 3′ and 5′ RNA trans-splicing seems to be an elegant way to modify especially large pre-mRNAs. Here we discuss a robust method for designing double RNA trans-splicing molecules (dRTM). We demonstrate how the technique can be implemented in an endogenous setting, using COL7A1, the gene encoding type VII collagen, as a target. An RTM screening system was developed with the aim of testing the replacement of two internal COL7A1 exons, harbouring a homozygous mutation, with the wild-type version. The most efficient RTMs from a pool of randomly generated variants were selected via our fluorescence-based screening system and adapted for use in an in vitro disease model system. Transduction of type VII collagen-deficient keratinocytes with the selected dRTM led to accurate replacement of two internal COL7A1 exons resulting in a restored wild-type RNA sequence. This is the first study demonstrating specific exon replacement by double RNA trans-splicing within an endogenous transcript in cultured cells, corroborating the utility of this technology for mRNA repair in a variety of genetic disorders. PMID:27669223

  5. Zero-mode waveguide nanophotonic structures for single molecule characterization

    Science.gov (United States)

    Crouch, Garrison M.; Han, Donghoon; Bohn, Paul W.

    2018-05-01

    Single-molecule characterization has become a crucial research tool in the chemical and life sciences, but limitations, such as limited concentration range, inability to control molecular distributions in space, and intrinsic phenomena, such as photobleaching, present significant challenges. Recent developments in non-classical optics and nanophotonics offer promising routes to mitigating these restrictions, such that even low affinity (K D ~ mM) biomolecular interactions can be studied. Here we introduce and review specific nanophotonic devices used to support single molecule studies. Optical nanostructures, such as zero-mode waveguides (ZMWs), are usually fabricated in thin gold or aluminum films and serve to confine the observation volume of optical microspectroscopy to attoliter to zeptoliter volumes. These simple nanostructures allow individual molecules to be isolated for optical and electrochemical analysis, even when the molecules of interest are present at high concentration (µM–mM) in bulk solution. Arrays of ZMWs may be combined with optical probes such as single molecule fluorescence, single molecule fluorescence resonance energy transfer, and fluorescence correlation spectroscopy for distributed analysis of large numbers of single-molecule reactions or binding events in parallel. Furthermore, ZMWs may be used as multifunctional devices, for example by combining optical and electrochemical functions in a single discrete architecture to achieve electrochemical ZMWs. In this review, we will describe the optical properties, fabrication, and applications of ZMWs for single-molecule studies, as well as the integration of ZMWs into systems for chemical and biochemical analysis.

  6. Did the Pre-RNA World Rest Upon DNA Molecules?

    Science.gov (United States)

    Lazcano, Antonio; Dworkin, Jason P.; Miller, Stanley L.

    2004-01-01

    The isolation of a DNA sequence that catalyzes the ligation of oligodeoxynucleotides via the formation of 3' - 5' phosphodiester linkage significance in selection experiments has been reported. Ball recently used this to discuss the possibility that natural DNA molecules may have formed in the primitive Earth leading to the origin of life. As noted by Ferris and Usher, if metabolic pathways evolved backwards, it could be argued that the biosynthesis of 2-deoxyribose from ribose suggests that RNA came from DNA. As summarized elsewhere, there are several properties of deoxyribose which could be interpreted to support the possibility that DNA-like molecules arose prior to the RNA world. For example, 2-deoxyribose is slightly more soluble than ribose (which may have been an advantage in a drying pool scenario), may have been more reactive under possible prebiotic conditions (it forms a nucleoside approx. 150 times faster than ribose with the alternative base urazole at 25 C), while it decomposes in solution (approximately 2.6 times more slowly than ribose at 100 C). Other advantages of DNA over RNA are that it has one fewer chiral center, has a greater stability at the 8.2 pH value of the current oceans, and does not has the 2'5' and 3'5' ambiguity in polymerizations. Yet, there is strong molecular biological and biochemical evidence that RNA was featured in the biology well before the last common ancestor. The presence of sugar acids, including both ribo- and deoxysugar acids, in the 4.6 Ga old Murchison meteorite suggest that both may have been available in the primitive Earth, derived from the accretion of extraterrestrial sources and/or from endogenous processes involving formaldehyde and its derivatives. However, the abiotic synthesis of deoxyribose, ribose, and other sugars from glyceraldehyde and acetaldehyde under alkaline conditions is inefficient and unespecific. Although sugars are labile compounds, the role of cyanamide or borate minerals in the

  7. Controlling single-molecule junction conductance by molecular interactions

    Science.gov (United States)

    Kitaguchi, Y.; Habuka, S.; Okuyama, H.; Hatta, S.; Aruga, T.; Frederiksen, T.; Paulsson, M.; Ueba, H.

    2015-01-01

    For the rational design of single-molecular electronic devices, it is essential to understand environmental effects on the electronic properties of a working molecule. Here we investigate the impact of molecular interactions on the single-molecule conductance by accurately positioning individual molecules on the electrode. To achieve reproducible and precise conductivity measurements, we utilize relatively weak π-bonding between a phenoxy molecule and a STM-tip to form and cleave one contact to the molecule. The anchoring to the other electrode is kept stable using a chalcogen atom with strong bonding to a Cu(110) substrate. These non-destructive measurements permit us to investigate the variation in single-molecule conductance under different but controlled environmental conditions. Combined with density functional theory calculations, we clarify the role of the electrostatic field in the environmental effect that influences the molecular level alignment. PMID:26135251

  8. Precision control of single-molecule electrical junctions.

    Science.gov (United States)

    Haiss, Wolfgang; Wang, Changsheng; Grace, Iain; Batsanov, Andrei S; Schiffrin, David J; Higgins, Simon J; Bryce, Martin R; Lambert, Colin J; Nichols, Richard J

    2006-12-01

    There is much discussion of molecules as components for future electronic devices. However, the contacts, the local environment and the temperature can all affect their electrical properties. This sensitivity, particularly at the single-molecule level, may limit the use of molecules as active electrical components, and therefore it is important to design and evaluate molecular junctions with a robust and stable electrical response over a wide range of junction configurations and temperatures. Here we report an approach to monitor the electrical properties of single-molecule junctions, which involves precise control of the contact spacing and tilt angle of the molecule. Comparison with ab initio transport calculations shows that the tilt-angle dependence of the electrical conductance is a sensitive spectroscopic probe, providing information about the position of the Fermi energy. It is also shown that the electrical properties of flexible molecules are dependent on temperature, whereas those of molecules designed for their rigidity are not.

  9. 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.

  10. Direct single-molecule dynamic detection of chemical reactions.

    Science.gov (United States)

    Guan, Jianxin; Jia, Chuancheng; Li, Yanwei; Liu, Zitong; Wang, Jinying; Yang, Zhongyue; Gu, Chunhui; Su, Dingkai; Houk, Kendall N; Zhang, Deqing; Guo, Xuefeng

    2018-02-01

    Single-molecule detection can reveal time trajectories and reaction pathways of individual intermediates/transition states in chemical reactions and biological processes, which is of fundamental importance to elucidate their intrinsic mechanisms. We present a reliable, label-free single-molecule approach that allows us to directly explore the dynamic process of basic chemical reactions at the single-event level by using stable graphene-molecule single-molecule junctions. These junctions are constructed by covalently connecting a single molecule with a 9-fluorenone center to nanogapped graphene electrodes. For the first time, real-time single-molecule electrical measurements unambiguously show reproducible large-amplitude two-level fluctuations that are highly dependent on solvent environments in a nucleophilic addition reaction of hydroxylamine to a carbonyl group. Both theoretical simulations and ensemble experiments prove that this observation originates from the reversible transition between the reactant and a new intermediate state within a time scale of a few microseconds. These investigations open up a new route that is able to be immediately applied to probe fast single-molecule physics or biophysics with high time resolution, making an important contribution to broad fields beyond reaction chemistry.

  11. RNA secondary structure diagrams for very large molecules: RNAfdl.

    Science.gov (United States)

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

    2013-11-15

    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 and produces output in common formats. Source code is available under the GNU General Public License v3.0 at http://sourceforge.net/projects/rnafdl for Linux and similar systems or Windows using MinGW. RNAfdl is implemented in C, uses the Cairo 2D graphics library and offers both command line and graphical user interfaces. hecker@rth.dk

  12. Stochastic single-molecule dynamics of synaptic membrane protein domains

    Science.gov (United States)

    Kahraman, Osman; Li, Yiwei; Haselwandter, Christoph A.

    2016-09-01

    Motivated by single-molecule experiments on synaptic membrane protein domains, we use a stochastic lattice model to study protein reaction and diffusion processes in crowded membranes. We find that the stochastic reaction-diffusion dynamics of synaptic proteins provide a simple physical mechanism for collective fluctuations in synaptic domains, the molecular turnover observed at synaptic domains, key features of the single-molecule trajectories observed for synaptic proteins, and spatially inhomogeneous protein lifetimes at the cell membrane. Our results suggest that central aspects of the single-molecule and collective dynamics observed for membrane protein domains can be understood in terms of stochastic reaction-diffusion processes at the cell membrane.

  13. Single-molecule tracking in living cells using single quantum dot applications.

    Science.gov (United States)

    Baba, Koichi; Nishida, Kohji

    2012-01-01

    Revealing the behavior of single molecules in living cells is very useful for understanding cellular events. Quantum dot probes are particularly promising tools for revealing how biological events occur at the single molecule level both in vitro and in vivo. In this review, we will introduce how single quantum dot applications are used for single molecule tracking. We will discuss how single quantum dot tracking has been used in several examples of complex biological processes, including membrane dynamics, neuronal function, selective transport mechanisms of the nuclear pore complex, and in vivo real-time observation. We also briefly discuss the prospects for single molecule tracking using advanced probes.

  14. Single-Molecule Electronics: Chemical and Analytical Perspectives.

    Science.gov (United States)

    Nichols, Richard J; Higgins, Simon J

    2015-01-01

    It is now possible to measure the electrical properties of single molecules using a variety of techniques including scanning probe microcopies and mechanically controlled break junctions. Such measurements can be made across a wide range of environments including ambient conditions, organic liquids, ionic liquids, aqueous solutions, electrolytes, and ultra high vacuum. This has given new insights into charge transport across molecule electrical junctions, and these experimental methods have been complemented with increasingly sophisticated theory. This article reviews progress in single-molecule electronics from a chemical perspective and discusses topics such as the molecule-surface coupling in electrical junctions, chemical control, and supramolecular interactions in junctions and gating charge transport. The article concludes with an outlook regarding chemical analysis based on single-molecule conductance.

  15. In situ temperature monitoring in single-molecule FRET experiments

    Science.gov (United States)

    Hartmann, Andreas; Berndt, Frederic; Ollmann, Simon; Krainer, Georg; Schlierf, Michael

    2018-03-01

    Thermodynamic properties of single molecules including enthalpic and entropic contributions are often determined from experiments by a direct control and precise measurement of the local temperature. However, common temperature monitoring techniques using, for example, ultrafine temperature probes can lead to uncertainties as the probe cannot be placed in the vicinity of the molecule of interest. Here, we devised an approach to measure the local temperature in freely diffusing confocal single-molecule Förster Resonance Energy Transfer (smFRET) experiments in situ by directly adding the temperature-sensitive fluorescent dye Rhodamine B, whose fluorescence lifetime serves as a probe of the local temperature in the confocal volume. We demonstrate that the temperature and FRET efficiencies of static and dynamic molecules can be extracted within one measurement simultaneously, without the need of a reference chamber. We anticipate this technique to be particularly useful in the physicochemical analyses of temperature-dependent biomolecular processes from single-molecule measurements.

  16. Single Molecule Raman Detection of Enkephalin on Silver Colloidal Particles

    DEFF Research Database (Denmark)

    Kneipp, Katrin; Kneipp, Holger; Abdali, Salim

    2004-01-01

    the Raman signal the enkephalin molecules have been attached to silver colloidal cluster structures. The experiments demonstrate that the SERS signal of the strongly enhanced ring breathing vibration of phenylalanine at 1000 cm-1 can be used as “intrinsic marker” for detecting a single enkephalin molecule...... and for monitoring its diffusion on the surface of the silver colloidal cluster without using a specific label molecule....

  17. Experimental Confirmation of a Whole Set of tRNA Molecules in Two Archaeal Species

    Directory of Open Access Journals (Sweden)

    Yoh-ichi Watanabe

    2015-01-01

    Full Text Available Based on the genomic sequences for most archaeal species, only one tRNA gene (isodecoder is predicted for each triplet codon. This observation promotes analysis of a whole set of tRNA molecules and actual splicing patterns of interrupted tRNA in one organism. The entire genomic sequences of two Creanarchaeota, Aeropyrum pernix and Sulfolobus tokodaii, were determined approximately 15 years ago. In these genome datasets, 47 and 46 tRNA genes were detected, respectively. Among them, 14 and 24 genes, respectively, were predicted to be interrupted tRNA genes. To confirm the actual transcription of these predicted tRNA genes and identify the actual splicing patterns of the predicted interrupted tRNA molecules, RNA samples were prepared from each archaeal species and used to synthesize cDNA molecules with tRNA sequence-specific primers. Comparison of the nucleotide sequences of cDNA clones representing unspliced and spliced forms of interrupted tRNA molecules indicated that some introns were located at positions other than one base 3' from anticodon region and that bulge-helix-bulge structures were detected around the actual splicing sites in each interrupted tRNA molecule. Whole-set analyses of tRNA molecules revealed that the archaeal tRNA splicing mechanism may be essential for efficient splicing of all tRNAs produced from interrupted tRNA genes in these archaea.

  18. Investigating single molecule adhesion by atomic force spectroscopy.

    Science.gov (United States)

    Stetter, Frank W S; Kienle, Sandra; Krysiak, Stefanie; Hugel, Thorsten

    2015-02-27

    Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. At the same time the AFM tip is passivated to prevent unspecific interactions between the tip and the substrate, which is a prerequisite to study single molecules attached to the AFM tip. Analyses to determine the adhesion force, the adhesion length, and the free energy of these molecules on solid surfaces and bio-interfaces are shortly presented and external references for further reading are provided. Example molecules are the poly(amino acid) polytyrosine, the graft polymer PI-g-PS and the phospholipid POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine). These molecules are desorbed from different surfaces like CH3-SAMs, hydrogen terminated diamond and supported lipid bilayers under various solvent conditions. Finally, the advantages of force spectroscopic single molecule experiments are discussed including means to decide if truly a single molecule has been studied in the experiment.

  19. Investigating Single Molecule Adhesion by Atomic Force Spectroscopy

    Science.gov (United States)

    Stetter, Frank W. S.; Kienle, Sandra; Krysiak, Stefanie; Hugel, Thorsten

    2015-01-01

    Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. At the same time the AFM tip is passivated to prevent unspecific interactions between the tip and the substrate, which is a prerequisite to study single molecules attached to the AFM tip. Analyses to determine the adhesion force, the adhesion length, and the free energy of these molecules on solid surfaces and bio-interfaces are shortly presented and external references for further reading are provided. Example molecules are the poly(amino acid) polytyrosine, the graft polymer PI-g-PS and the phospholipid POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine). These molecules are desorbed from different surfaces like CH3-SAMs, hydrogen terminated diamond and supported lipid bilayers under various solvent conditions. Finally, the advantages of force spectroscopic single molecule experiments are discussed including means to decide if truly a single molecule has been studied in the experiment. PMID:25867282

  20. Extending Single-Molecule Microscopy Using Optical Fourier Processing

    Science.gov (United States)

    2015-01-01

    This article surveys the recent application of optical Fourier processing to the long-established but still expanding field of single-molecule imaging and microscopy. A variety of single-molecule studies can benefit from the additional image information that can be obtained by modulating the Fourier, or pupil, plane of a widefield microscope. After briefly reviewing several current applications, we present a comprehensive and computationally efficient theoretical model for simulating single-molecule fluorescence as it propagates through an imaging system. Furthermore, we describe how phase/amplitude-modulating optics inserted in the imaging pathway may be modeled, especially at the Fourier plane. Finally, we discuss selected recent applications of Fourier processing methods to measure the orientation, depth, and rotational mobility of single fluorescent molecules. PMID:24745862

  1. Single Molecule Spectroscopy in Chemistry, Physics and Biology Nobel Symposium

    CERN Document Server

    Gräslund, Astrid; Widengren, Jerker

    2010-01-01

    Written by the leading experts in the field, this book describes the development and current state-of-the-art in single molecule spectroscopy. The application of this technique, which started 1989, in physics, chemistry and biosciences is displayed.

  2. Single Molecule Scanning of DNA Radiation Oxidative Damage Project

    Data.gov (United States)

    National Aeronautics and Space Administration — This proposal will develop an assay to map genomic DNA, at the single molecule level and in a nanodevice, for oxidative DNA damage arising from radiation exposure;...

  3. Single-molecule fluorescence microscopy in living Caenorhabditis elegans

    NARCIS (Netherlands)

    van Krugten, Jaap; Peterman, Erwin J.G.

    2018-01-01

    Transportation of organelles and biomolecules is vital for many cellular processes. Single-molecule (SM) fluorescence microscopy can expose molecular aspects of the dynamics that remain unresolved in ensemble experiments. For example, trajectories of individual, moving biomolecules can reveal

  4. Single Molecule Scanning of DNA Radiation Oxidative Damage, Phase I

    Data.gov (United States)

    National Aeronautics and Space Administration — This proposal will develop an assay to map genomic DNA, at the single molecule level and in a nanodevice, for oxidative DNA damage arising from radiation exposure;...

  5. DNA replication at the single-molecule level

    NARCIS (Netherlands)

    Stratmann, S.A.; Oijen, A.M. van

    2014-01-01

    A cell can be thought of as a highly sophisticated micro factory: in a pool of billions of molecules – metabolites, structural proteins, enzymes, oligonucleotides – multi-subunit complexes assemble to perform a large number of basic cellular tasks, such as DNA replication, RNA/protein synthesis or

  6. Electrochemically-gated single-molecule electrical devices

    International Nuclear Information System (INIS)

    Guo, Shaoyin; Artés, Juan Manuel; Díez-Pérez, Ismael

    2013-01-01

    In the last decade, single-molecule electrical contacts have emerged as a new experimental platform that allows exploring charge transport phenomena in individual molecular blocks. This novel tool has evolved into an essential element within the Molecular Electronics field to understand charge transport processes in hybrid (bio)molecule/electrode interfaces at the nanoscale, and prospect the implementation of active molecular components into functional nanoscale optoelectronic devices. Within this area, three-terminal single-molecule devices have been sought, provided that they are highly desired to achieve full functionality in logic electronic circuits. Despite the latest experimental developments offer consistent methods to bridge a molecule between two electrodes (source and drain in a transistor notation), placing a third electrode (gate) close to the single-molecule electrical contact is still technically challenging. In this vein, electrochemically-gated single-molecule devices have emerged as an experimentally affordable alternative to overcome these technical limitations. In this review, the operating principle of an electrochemically-gated single-molecule device is presented together with the latest experimental methodologies to built them and characterize their charge transport characteristics. Then, an up-to-date comprehensive overview of the most prominent examples will be given, emphasizing on the relationship between the molecular structure and the final device electrical behaviour

  7. Fluorescent Biosensors Based on Single-Molecule Counting.

    Science.gov (United States)

    Ma, Fei; Li, Ying; Tang, Bo; Zhang, Chun-Yang

    2016-09-20

    Biosensors for highly sensitive, selective, and rapid quantification of specific biomolecules make great contributions to biomedical research, especially molecular diagnostics. However, conventional methods for biomolecular assays often suffer from insufficient sensitivity and poor specificity. In some case (e.g., early disease diagnostics), the concentration of target biomolecules is too low to be detected by these routine approaches, and cumbersome procedures are needed to improve the detection sensitivity. Therefore, there is an urgent need for rapid and ultrasensitive analytical tools. In this respect, single-molecule fluorescence approaches may well satisfy the requirement and hold promising potential for the development of ultrasensitive biosensors. Encouragingly, owing to the advances in single-molecule microscopy and spectroscopy over past decades, the detection of single fluorescent molecule comes true, greatly boosting the development of highly sensitive biosensors. By in vitro/in vivo labeling of target biomolecules with proper fluorescent tags, the quantification of certain biomolecule at the single-molecule level is achieved. In comparison with conventional ensemble measurements, single-molecule detection-based analytical methods possess the advantages of ultrahigh sensitivity, good selectivity, rapid analysis time, and low sample consumption. Consequently, single-molecule detection may be potentially employed as an ideal analytical approach to quantify low-abundant biomolecules with rapidity and simplicity. In this Account, we will summarize our efforts for developing a series of ultrasensitive biosensors based on single-molecule counting. Single-molecule counting is a member of single-molecule detection technologies and may be used as a very simple and ultrasensitive method to quantify target molecules by simply counting the individual fluorescent bursts. In the fluorescent sensors, the signals of target biomolecules may be translated to the

  8. Single-Molecule Spectroscopic Investigations of Amphipathic Helix Formation

    Science.gov (United States)

    Cunningham, Joy Ann; Okamoto, Kenji; English, Douglas

    2004-03-01

    We are using single molecule spectroscopy to examine surface-induced conformational states occurring through interaction of a polypeptide with an interface. Specifically, we investigate the folding of amphipathic helices by using single-molecule fluorescence resonance energy transfer to construct peptide conformational distributions in solution and at interfaces. Analysis of the conformational distributions and kinetics of peptides in different environments reveals properties of the free energy surface for helix formation at an interface relative to formation in solution.

  9. 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

  10. Novel Fluorescent Dyes for Single DNA Molecule Techniques

    Directory of Open Access Journals (Sweden)

    Alexander Zarkov

    2013-03-01

    Full Text Available To answer the demands of scientific and medical imaging issues, the family of nucleic acid fluorescent dyes is constantly enlarging. Most of the developed dyes reveal high qualities in bulk solution assays but are inefficient to produce a strong and sufficiently stable signal to enable the application of single-molecule techniques. Therefore, we tested 12 novel monomeric and homodimeric cyanine dyes for potential single DNA molecule imaging. Although their qualities in bulk solutions have already been described, nothing was known about their behavior on a single-molecule level. All 12 dyes demonstrated strong emission when intercalated into single DNA molecules and stretched on a silanized surface, which makes them the perfect choice for fluorescent microscopy imaging. A comparison of their fluorescence intensity and photostability with the most applicable dyes in single-molecule techniques, fluorescent dyes YOYO-1 and POPO-3, was carried out. They all exhibited a strong signal, comparable to that of YOYO-1. However, in contrast to YOYO-1, which is visualized under a green filter only, their emission permits red filter visualization. As their photostability highly exceeds that of similar spectrum POPO-3 dye, the studied dyes stand out as the best choice for a broad range of solid surface single-molecule applications when yellow to red DNA backbone fluorescence is needed.

  11. A theoretical justification for single molecule peptide sequencing.

    Directory of Open Access Journals (Sweden)

    Jagannath Swaminathan

    2015-02-01

    Full Text Available The proteomes of cells, tissues, and organisms reflect active cellular processes and change continuously in response to intracellular and extracellular cues. Deep, quantitative profiling of the proteome, especially if combined with mRNA and metabolite measurements, should provide an unprecedented view of cell state, better revealing functions and interactions of cell components. Molecular diagnostics and biomarker discovery should benefit particularly from the accurate quantification of proteomes, since complex diseases like cancer change protein abundances and modifications. Currently, shotgun mass spectrometry is the primary technology for high-throughput protein identification and quantification; while powerful, it lacks high sensitivity and coverage. We draw parallels with next-generation DNA sequencing and propose a strategy, termed fluorosequencing, for sequencing peptides in a complex protein sample at the level of single molecules. In the proposed approach, millions of individual fluorescently labeled peptides are visualized in parallel, monitoring changing patterns of fluorescence intensity as N-terminal amino acids are sequentially removed, and using the resulting fluorescence signatures (fluorosequences to uniquely identify individual peptides. We introduce a theoretical foundation for fluorosequencing and, by using Monte Carlo computer simulations, we explore its feasibility, anticipate the most likely experimental errors, quantify their potential impact, and discuss the broad potential utility offered by a high-throughput peptide sequencing technology.

  12. Site-specific labeling of Saccharomyces cerevisiae ribosomes for single-molecule manipulations

    Science.gov (United States)

    Petrov, Alexey; Puglisi, Joseph D.

    2010-01-01

    Site-specific labeling of Escherichia coli ribosomes has allowed application of single-molecule fluorescence spectroscopy and force methods to probe the mechanism of translation. To apply these approaches to eukaryotic translation, eukaryotic ribosomes must be specifically labeled with fluorescent labels and molecular handles. Here, we describe preparation and labeling of the small and large yeast ribosomal subunits. Phylogenetically variable hairpin loops in ribosomal RNA are mutated to allow hybridization of oligonucleotides to mutant ribosomes. We demonstrate specific labeling of the ribosomal subunits, and their use in single-molecule fluorescence and force experiments. PMID:20501598

  13. Electrochemical proton relay at the single-molecule level

    DEFF Research Database (Denmark)

    Kuznetsov, A. M.; Medvedev, I. G.; Ulstrup, Jens

    2009-01-01

    A scheme for the experimental study of single-proton transfer events, based on proton-coupled two-electron transfer between a proton donor and a proton acceptor molecule confined in the tunneling gap between two metal leads in electrolyte solution is suggested. Expressions for the electric current...... are derived and compared with formalism for electron tunneling through redox molecules. The scheme allows studying the kinetics of proton and hydrogen atom transfer as well as kinetic isotope effects at the single-molecule level under electrochemical potential control....

  14. Application of Recognition Tunneling in Single Molecule Identification

    Science.gov (United States)

    Zhao, Yanan

    Single molecule identification is one essential application area of nanotechnology. The application areas including DNA sequencing, peptide sequencing, early disease detection and other industrial applications such as quantitative and quantitative analysis of impurities, etc. The recognition tunneling technique we have developed shows that after functionalization of the probe and substrate of a conventional Scanning Tunneling Microscope with recognition molecules ("tethered molecule-pair" configuration), analyte molecules trapped in the gap that is formed by probe and substrate will bond with the reagent molecules. The stochastic bond formation/breakage fluctuations give insight into the nature of the intermolecular bonding at a single molecule-pair level. The distinct time domain and frequency domain features of tunneling signals were extracted from raw signals of analytes such as amino acids and their enantiomers. The Support Vector Machine (a machine-learning method) was used to do classification and predication based on the signal features generated by analytes, giving over 90% accuracy of separation of up to seven analytes. This opens up a new interface between chemistry and electronics with immediate implications for rapid Peptide/DNA sequencing and molecule identification at single molecule level.

  15. Single Molecule Raman Detection of Enkephalin on Silver Colloidal Particles

    DEFF Research Database (Denmark)

    Kneipp, Katrin; Kneipp, Holger; Abdali, Salim

    2004-01-01

    Enkephalin, an endogeneous substance in the human brain showing morphine-like biological functions, has been detected at the single molecule level based on the surface-enhanced Raman signal of the ring breathing mode of phenylalanine, which is one building block of the molecule. For enhancing...... the Raman signal the enkephalin molecules have been attached to silver colloidal cluster structures. The experiments demonstrate that the SERS signal of the strongly enhanced ring breathing vibration of phenylalanine at 1000 cm-1 can be used as “intrinsic marker” for detecting a single enkephalin molecule...... and for monitoring its diffusion on the surface of the silver colloidal cluster without using a specific label molecule....

  16. Single Molecule Junctions: Probing Contact Chemistry and Fundamental Circuit Laws

    Energy Technology Data Exchange (ETDEWEB)

    Hybertsen M. S.

    2013-04-11

    By exploiting selective link chemistry, formation of single molecule junctions with reproducible conductance has become established. Systematic studies reveal the structure-conductance relationships for diverse molecules. I will draw on experiments from my collaborators at Columbia University, atomic-scale calculations and theory to describe progress in two areas. First, I will describe a novel route to form single molecule junctions, based on SnMe3 terminated molecules, in which gold directly bonds to carbon in the molecule backbone resulting in near ideal contact resistance [1]. Second, comparison of the conductance of junctions formed with molecular species containing either one backbone or two backbones in parallel allows demonstration of the role of quantum interference in the conductance superposition law at the molecular scale [2].

  17. Vesicle Encapsulation Studies Reveal that Single Molecule Ribozyme Heterogeneities Are Intrinsic

    Science.gov (United States)

    Okumus, Burak; Wilson, Timothy J.; Lilley, David M. J.; Ha, Taekjip

    2004-01-01

    Single-molecule measurements have revealed that what were assumed to be identical molecules can differ significantly in their static and dynamic properties. One of the most striking examples is the hairpin ribozyme, which was shown to exhibit two to three orders of magnitude variation in folding kinetics between molecules. Although averaged behavior of single molecules matched the bulk solution data, it was not possible to exclude rigorously the possibility that the variations around the mean values arose from different ways of interacting with the surface environment. To test this, we minimized the molecules' interaction with the surface by encapsulating DNA or RNA molecules inside 100- to 200-nm diameter unilamellar vesicles, following the procedures described by Haran and coworkers. Vesicles were immobilized on a supported lipid bilayer via biotin-streptavidin linkages. We observed no direct binding of DNA or RNA on the supported bilayer even at concentrations exceeding 100 nM, indicating that these molecules do not bind stably on the membrane. Since the vesicle diameter is smaller than the resolution of optical microscopy, the lateral mobility of the molecules is severely constrained, allowing long observation periods. We used fluorescence correlation spectroscopy, nuclease digestion, and external buffer exchange to show that the molecules were indeed encapsulated within the vesicles. When contained within vesicles, the natural form of the hairpin ribozyme exhibited 50-fold variation in both folding and unfolding rates in 0.5 mM Mg2+, which is identical to what was observed from the molecules tethered directly on the surface. This strongly indicates that the observed heterogeneity in dynamic properties does not arise as an artifact of surface attachment, but is intrinsic to the nature of the molecules. PMID:15454471

  18. Lattice diffusion of a single molecule in solution

    Science.gov (United States)

    Ruggeri, Francesca; Krishnan, Madhavi

    2017-12-01

    The ability to trap a single molecule in an electrostatic potential well in solution has opened up new possibilities for the use of molecular electrical charge to study macromolecular conformation and dynamics at the level of the single entity. Here we study the diffusion of a single macromolecule in a two-dimensional lattice of electrostatic traps in solution. We report the ability to measure both the size and effective electrical charge of a macromolecule by observing single-molecule transport trajectories, typically a few seconds in length, using fluorescence microscopy. While, as shown previously, the time spent by the molecule in a trap is a strong function of its effective charge, we demonstrate here that the average travel time between traps in the landscape yields its hydrodynamic radius. Tailoring the pitch of the lattice thus yields two different experimentally measurable time scales that together uniquely determine both the size and charge of the molecule. Since no information is required on the location of the molecule between consecutive departure and arrival events at lattice sites, the technique is ideally suited to measurements on weakly emitting entities such as single molecules.

  19. Massively Parallel Single-Molecule Manipulation Using Centrifugal Force

    Science.gov (United States)

    Wong, Wesley; Halvorsen, Ken

    2011-03-01

    Precise manipulation of single molecules has led to remarkable insights in physics, chemistry, biology, and medicine. However, two issues that have impeded the widespread adoption of these techniques are equipment cost and the laborious nature of making measurements one molecule at a time. To meet these challenges, we have developed an approach that enables massively parallel single- molecule force measurements using centrifugal force. This approach is realized in the centrifuge force microscope, an instrument in which objects in an orbiting sample are subjected to a calibration-free, macroscopically uniform force- field while their micro-to-nanoscopic motions are observed. We demonstrate high- throughput single-molecule force spectroscopy with this technique by performing thousands of rupture experiments in parallel, characterizing force-dependent unbinding kinetics of an antibody-antigen pair in minutes rather than days. Currently, we are taking steps to integrate high-resolution detection, fluorescence, temperature control and a greater dynamic range in force. With significant benefits in efficiency, cost, simplicity, and versatility, single-molecule centrifugation has the potential to expand single-molecule experimentation to a wider range of researchers and experimental systems.

  20. Localization and abundance analysis of human lncRNAs at single-cell and single-molecule resolution.

    Science.gov (United States)

    Cabili, Moran N; Dunagin, Margaret C; McClanahan, Patrick D; Biaesch, Andrew; Padovan-Merhar, Olivia; Regev, Aviv; Rinn, John L; Raj, Arjun

    2015-01-29

    Long non-coding RNAs (lncRNAs) have been implicated in diverse biological processes. In contrast to extensive genomic annotation of lncRNA transcripts, far fewer have been characterized for subcellular localization and cell-to-cell variability. Addressing this requires systematic, direct visualization of lncRNAs in single cells at single-molecule resolution. We use single-molecule RNA-FISH to systematically quantify and categorize the subcellular localization patterns of a representative set of 61 lncRNAs in three different cell types. Our survey yields high-resolution quantification and stringent validation of the number and spatial positions of these lncRNA, with an mRNA set for comparison. Using this highly quantitative image-based dataset, we observe a variety of subcellular localization patterns, ranging from bright sub-nuclear foci to almost exclusively cytoplasmic localization. We also find that the low abundance of lncRNAs observed from cell population measurements cannot be explained by high expression in a small subset of 'jackpot' cells. Additionally, nuclear lncRNA foci dissolve during mitosis and become widely dispersed, suggesting these lncRNAs are not mitotic bookmarking factors. Moreover, we see that divergently transcribed lncRNAs do not always correlate with their cognate mRNA, nor do they have a characteristic localization pattern. Our systematic, high-resolution survey of lncRNA localization reveals aspects of lncRNAs that are similar to mRNAs, such as cell-to-cell variability, but also several distinct properties. These characteristics may correspond to particular functional roles. Our study also provides a quantitative description of lncRNAs at the single-cell level and a universally applicable framework for future study and validation of lncRNAs.

  1. Chum-RNA allows preparation of a high-quality cDNA library from a single-cell quantity of mRNA without PCR amplification.

    Science.gov (United States)

    Tougan, Takahiro; Okuzaki, Daisuke; Nojima, Hiroshi

    2008-09-01

    Linear RNA amplification using T7 RNA polymerase is useful in genome-wide analysis of gene expression using DNA microarrays, but exponential amplification using polymerase chain reaction (PCR) is still required for cDNA library preparation from single-cell quantities of RNA. We have designed a small RNA molecule called chum-RNA that has enabled us to prepare a single-cell cDNA library after four rounds of T7-based linear amplification, without using PCR amplification. Chum-RNA drove cDNA synthesis from only 0.49 femtograms of mRNA (730 mRNA molecules) as a substrate, a quantity that corresponds to a minor population of mRNA molecules in a single mammalian cell. Analysis of the independent cDNA clone of this library (6.6 x 10(5) cfu) suggests that 30-fold RNA amplification occurred in each round of the amplification process. The size distribution and representation of mRNAs in the resulting one-cell cDNA library retained its similarity to that of the million-cell cDNA library. The use of chum-RNA might also facilitate reactions involving other DNA/RNA modifying enzymes whose Michaelis constant (K(m)) values are around 1 mM, allowing them to be activated in the presence of only small quantities of substrate.

  2. Single-Molecule Electronics with Cross- Conjugated Molecules: Quantum Interference, IETS and Non-Equilibrium "Temperatures"

    DEFF Research Database (Denmark)

    Jørgensen, Jacob Lykkebo

    Abstract The idea of using single-molecules as components in electronic devices is fas- cinating. For this idea to come into fruition, a number of technical and theo- retical challenges must be overcome. In this PhD thesis, the electron-phonon interaction is studied for a special class of molecules......, which is characterised by destructive quantum interference. The molecules are cross-conjugated, which means that the two parts of the molecules are conjugated to a third part, but not to each other. This gives rise to an anti-resonance in the trans- mission. In the low bias and low temperature regime......-conjugated molecules. We nd that the vibrational modes that would be expected to dominate, following the propensity, rules are very weak. Instead, other modes are found to be the dominant ones. We study this phenomenon for a number of cross-conjugated molecules, and link these ndings to the anti...

  3. BRCA Testing by Single-Molecule Molecular Inversion Probes

    NARCIS (Netherlands)

    Neveling, K.; Mensenkamp, A.R.; Derks, R; Kwint, M.P.; Ouchene, H.; Steehouwer, M.; Lier, L.A. van; Bosgoed, E.A.J.; Rikken, A.; Tychon, M.W.J.; Zafeiropoulou, D.; Castelein, S.; Hehir-Kwa, J.Y.; Thung, G.W.; Hofste, T.; Lelieveld, S.H.; Bertens, S.M.; Adan, I.B.; Eijkelenboom, A.; Tops, B.B.J.; Yntema, H.G.; Stokowy, T.; Knappskog, P.M.; Hoberg-Vetti, H.; Steen, V.M.; Boyle, E.; Martin, B.; Ligtenberg, M.J.L.; Shendure, J.; Nelen, M.R.; Hoischen, A.

    2017-01-01

    BACKGROUND: Despite advances in next generation DNA sequencing (NGS), NGS-based single gene tests for diagnostic purposes require improvements in terms of completeness, quality, speed, and cost. Single-molecule molecular inversion probes (smMIPs) are a technology with unrealized potential in the

  4. The influence of morphology on excitons in single conjugated molecules

    Science.gov (United States)

    Thiessen, Alexander

    The electronic properties of pi-conjugated molecules are strongly related to their molecular shape and morphology of assembly in three-dimensional space. Understanding the various structure-property relationships is relevant to the applications of these materials in optoelectronic devices such as organic light-emitting diodes, field effect transistors and photovoltaic cells. The fact that conjugated systems interact with visible light opens these materials to a plethora of noninvasive spectroscopic investigation techniques. In this work, electronic properties of different pi-conjugated systems are studied spectroscopically on the ensemble and the single molecule levels. Single molecule spectroscopy is advantageous in that it allows the investigation of the individual nuclear building blocks that contribute to the properties of the ensemble. Additionally, transient photoluminescence spectroscopy methods can provide useful insight into the temporal evolution of the emissive states. In combination with these methods, novel pi-conjugated model molecules are used to probe processes related to exciton dynamics. For the first time, the spatial localization of excited states is probed experimentally in a molecule with a circular chromophoric structure. In addition, a set of model molecules with different geometries is employed to study exciton relaxation in pi-conjugated systems. The molecular morphology is utilized to distinguish between processes such as nuclear reorganization and torsional relaxation. Furthermore, single molecule spectroscopy is used to study the electronic structure of individual polymer chains in the photovoltaic cell material poly-(3-hexylthiophene). Optical spectra of this polymer are known to change with the morphology of the bulk film. Single molecule studies reveal that individual polymer chains exhibit similar behavior and indicate that spectral diversity is an intrinsic property of single P3HT molecules. The main results of this work are the

  5. Fast recognition of single molecules based on single-event photon statistics

    International Nuclear Information System (INIS)

    Dong Shuangli; Huang Tao; Liu Yuan; Wang Jun; Zhang Guofeng; Xiao Liantuan; Jia Suotang

    2007-01-01

    Mandel's Q parameter, which is determined from single-event photon statistics, provides an alternative way to recognize single molecules with fluorescence detection, other than the second-order correlation function. It is shown that the Q parameter of an assumed ideal double-molecule fluorescence with the same average photon number as that of the sample fluorescence can act as the criterion for single-molecule recognition. The influence of signal-to-background ratio and the error estimates for photon statistics are also presented. We have applied this method to ascertain single Cy5 dye molecules within hundreds of milliseconds

  6. Challenges for single molecule electronic devices with nanographene and organic molecules. Do single molecules offer potential as elements of electronic devices in the next generation?

    Science.gov (United States)

    Enoki, Toshiaki; Kiguchi, Manabu

    2018-03-01

    Interest in utilizing organic molecules to fabricate electronic materials has existed ever since organic (molecular) semiconductors were first discovered in the 1950s. Since then, scientists have devoted serious effort to the creation of various molecule-based electronic systems, such as molecular metals and molecular superconductors. Single-molecule electronics and the associated basic science have emerged over the past two decades and provided hope for the development of highly integrated molecule-based electronic devices in the future (after the Si-based technology era has ended). Here, nanographenes (nano-sized graphene) with atomically precise structures are among the most promising molecules that can be utilized for electronic/spintronic devices. To manipulate single small molecules for an electronic device, a single molecular junction has been developed. It is a powerful tool that allows even small molecules to be utilized. External electric, magnetic, chemical, and mechanical perturbations can change the physical and chemical properties of molecules in a way that is different from bulk materials. Therefore, the various functionalities of molecules, along with changes induced by external perturbations, allows us to create electronic devices that we cannot create using current top-down Si-based technology. Future challenges that involve the incorporation of condensed matter physics, quantum chemistry calculations, organic synthetic chemistry, and electronic device engineering are expected to open a new era in single-molecule device electronic technology.

  7. Electrochemical single-molecule conductivity of duplex and quadruplex DNA

    DEFF Research Database (Denmark)

    Zhang, Ling; Zhang, Jingdong; Ulstrup, Jens

    2017-01-01

    Photoinduced and electrochemical charge transport in DNA (oligonucleotides, OGNs) and the notions “hopping”, superexchange, polaron, and vibrationally gated charge transport have been in focus over more than two decades. In recent years mapping of electrochemical charge transport of pure and redox...... marked single- and double-strand OGNs has reached the single-molecule level based i.a. on electrochemical in situ scanning tunnelling microscopy (STM) and break-junction (B-J) STM. There are much fewer such reports on “non-canonical” OGN structures such as G-quadruplexes. We discuss first single......-molecule electrochemical conductivity of pure and redox marked duplex OGNs, and address next electrochemistry and electrochemical conductivity in the few reported monolayer and single-molecule G-quadruplex studies. Facile electrochemical electron transfer of iron protoporphyrin IX stacked onto three-quartet 12-guanine...

  8. Methods to enable the design of bioactive small molecules targeting RNA.

    Science.gov (United States)

    Disney, Matthew D; Yildirim, Ilyas; Childs-Disney, Jessica L

    2014-02-21

    RNA is an immensely important target for small molecule therapeutics or chemical probes of function. However, methods that identify, annotate, and optimize RNA-small molecule interactions that could enable the design of compounds that modulate RNA function are in their infancies. This review describes recent approaches that have been developed to understand and optimize RNA motif-small molecule interactions, including structure-activity relationships through sequencing (StARTS), quantitative structure-activity relationships (QSAR), chemical similarity searching, structure-based design and docking, and molecular dynamics (MD) simulations. Case studies described include the design of small molecules targeting RNA expansions, the bacterial A-site, viral RNAs, and telomerase RNA. These approaches can be combined to afford a synergistic method to exploit the myriad of RNA targets in the transcriptome.

  9. Rationale for single molecule detection by means of Raman spectroscopy

    International Nuclear Information System (INIS)

    Gaponenko, S.V.; Guzatov, D.V.

    2009-01-01

    A consistent quantum electrodynamical description is proposed of Raman scattering of light by a molecule in a medium with a modified photon density of states. Enhanced local density of states near a metal nanobody is shown to increase a scattering rate by several orders of magnitude, thus providing a rationale for experimental detection of single molecules by means of Raman spectroscopy. For an ellipsoidal particle 10 14 -fold enhancement of the Raman scattering cross-section is obtained. (authors)

  10. Electron transfer dynamics of bistable single-molecule junctions

    DEFF Research Database (Denmark)

    Danilov, A.V; Kubatkin, S.; Kafanov, S. G.

    2006-01-01

    We present transport measurements of single-molecule junctions bridged by a molecule with three benzene rings connected by two double bonds and with thiol end-groups that allow chemical binding to gold electrodes. The I-V curves show switching behavior between two distinct states. By statistical ...... analysis of the switching events, we show that a 300 meV mode mediates the transition between the two states. We propose that breaking and reformation of a S-H bond in the contact zone between molecule and electrode explains the observed bistability....

  11. A modified single-cell electroporation method for molecule delivery into a motile protist, Euglena gracilis.

    Science.gov (United States)

    Ohmachi, Masashi; Fujiwara, Yoshie; Muramatsu, Shuki; Yamada, Koji; Iwata, Osamu; Suzuki, Kengo; Wang, Dan Ohtan

    2016-11-01

    Single-cell transfection is a powerful technique for delivering chemicals, drugs, or probes into arbitrary, specific single cells. This technique is especially important when the analysis of molecular function and cellular behavior in individual microscopic organisms such as protists requires the precise identification of the target cell, as fluorescence labeling of bulk populations makes tracking of individual motile protists virtually impossible. Herein, we have modified current single-cell electroporation techniques for delivering fluorescent markers into single Euglena gracilis, a motile photosynthetic microalga. Single-cell electroporation introduced molecules into individual living E. gracilis cells after a negative pressure was applied through a syringe connected to the micropipette to the target cell. The new method achieves high transfection efficiency and viability after electroporation. With the new technique, we successfully introduced a variety of molecules such as GFP, Alexa Fluor 488, and exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) RNA probes into individual motile E. gracilis cells. We demonstrate imaging of endogenous mRNA in living E. gracilis without interfering with their physiological functions, such as swimming or division, over an extended period of time. Thus the modified single-cell electroporation technique is suitable for delivering versatile functional molecules into individual motile protists. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Single Molecule Study of Photoconversion and Spectral Heterogeneities of Fluorophores

    DEFF Research Database (Denmark)

    Liao, Zhiyu

    of conformational changes and dynamics. The photophysical properties of organic dyes directly determine the quality of the experiments. So the better understanding of the photophysical properties of organic dyes, the better we are able to design the experiments and interpret the data, especially in single molecule...... 104 single molecule measurements. A simple and practical method is introduced to study the characteristics of the photoproducts at the ensemble level. Control experiments reveal that the reaction leading to photobleaching is oxygen related, but the composition of the photoproducts remains inconclusive...... stimulate new pathways in engineering and designing photoconvertible fluorophores, based on the reaction with oxygen or other chemicals. Besides, this results show that dyes that convert into other emissive species could give problems when interpreting single molecule FRET systems. The revealed mechanism...

  13. Single Molecule Spectroscopy on Photosynthetic Pigment-Protein Complexes

    CERN Document Server

    Jelezko, F; Schuler, S; Thews, E; Tietz, C; Wechsler, A; Wrachtrup, J

    2001-01-01

    Single molecule spectroscopy was applied to unravel the energy transfer pathway in photosynthetic pigment-protein complexes. Detailed analysis of excitation and fluorescence emission spectra has been made for peripheral plant antenna LHC II and Photosystem I from cyanobacterium Synechococcus elongatus. Optical transitions of individual pigments were resolved under nonselective excitation of antenna chlorophylls. High-resolution fluorescence spectroscopy of individual plant antenna LHC II indicates that at low temperatures, the excitation energy is localized on the red-most Chl a pool absorbing at 680 nm. More than one pigment molecule is responsible for the fluorescence emission of the LHC II trimer. The spectral lines of single Chl a molecules absorbing at 675 nm are broadened because of the Foerster energy transfer towards the red-most pigments. Low-temperature spectroscopy on single PS I trimers indicates that two subgroups of pigments, which are present in the red antenna pool, differ by the strength of t...

  14. Coherent interaction of single molecules and plasmonic nanowires

    Science.gov (United States)

    Gerhardt, Ilja; Grotz, Bernhard; Siyushev, Petr; Wrachtrup, Jörg

    2017-09-01

    Quantum plasmonics opens the option to integrate complex quantum optical circuitry onto chip scale devices. In the past, often external light sources were used and nonclassical light was coupled in and out of plasmonic structures, such as hole arrays or waveguide structures. Another option to launch single plasmonic excitations is the coupling of single emitters in the direct proximity of, e.g., a silver or gold nanostructure. Here, we present our attempts to integrate the research of single emitters with wet-chemically grown silver nanowires. The emitters of choice are single organic dye molecules under cryogenic conditions, which are known to act as high-brightness and extremely narrow-band single photon sources. Another advantage is their high optical nonlinearity, such that they might mediate photon-photon interactions on the nanoscale. We report on the coupling of a single molecule fluorescence emission through the wire over the length of several wavelengths. The transmission of coherently emitted photons is proven by an extinction type experiment. As for influencing the spectral properties of a single emitter, we are able to show a remote change of the line-width of a single terrylene molecule, which is in close proximity to the nanowire.

  15. Single-molecule imaging towards precise detection of individual photophysics

    International Nuclear Information System (INIS)

    Tani, Toshiro; Oda, Masaru; Mashimo, Kei; Tachibana, Fumi; Horiuchi, Hiromi

    2006-01-01

    We present our recent study of single fluorescent molecules with specific structure, i.e. tetramethylrhodamine derivative linked with a propyl chain onto silica glass surface. For fluorescent reagent in its synthesis, we used a mixture of two kinds of isomers, which provides a sample with single molecules photophysically different each other even if chemically the same. The isomeric structural difference so introduced in the molecules will provide rather small but probably distinctive photophysical difference, for example, in non-radiative relaxation rates, which we try to detect out with our improved single-molecule microscope imaging technique. To make clear the detectability of such weak inter- or intra-molecular interactions microscopically is significant for versatile applications of single-molecule detections in life science. Our present observation at room temperatures shows so far that such decoupled contributions can be discriminated in the histograms of the intensities of the observed fluorescent spots as broader but separated multi-component structures in the distribution under specific experimental configurations. We will discuss some of the prerequisite for such detections; suitable spatio-temporal resolutions with sufficient S/N ratio, algorithms for data analysis, etc. but also precise sample operations are inevitable

  16. Electrochemical Single-Molecule Transistors with Optimized Gate Coupling

    DEFF Research Database (Denmark)

    Osorio, Henrry M.; Catarelli, Samantha; Cea, Pilar

    2015-01-01

    . These data are rationalized in terms of a two-step electrochemical model for charge transport across the redox bridge. In this model the gate coupling in the ionic liquid is found to be fully effective with a modeled gate coupling parameter, ξ, of unity. This compares to a much lower gate coupling parameter......Electrochemical gating at the single molecule level of viologen molecular bridges in ionic liquids is examined. Contrary to previous data recorded in aqueous electrolytes, a clear and sharp peak in the single molecule conductance versus electrochemical potential data is obtained in ionic liquids...

  17. Towards single molecule biosensors using super-resolution fluorescence microscopy.

    Science.gov (United States)

    Lu, Xun; Nicovich, Philip R; Gaus, Katharina; Gooding, J Justin

    2017-07-15

    Conventional immunosensors require many binding events to give a single transducer output which represents the concentration of the analyte in the sample. Because of the requirements to selectively detect species in complex samples, immunosensing interfaces must allow immobilisation of antibodies while repelling nonspecific adsorption of other species. These requirements lead to quite sophisticated interfacial design, often with molecular level control, but we have no tools to characterise how well these interfaces work at the molecular level. The work reported herein is an initial feasibility study to show that antibody-antigen binding events can be monitored at the single molecule level using single molecule localisation microscopy (SMLM). The steps to achieve this first requires showing that indium tin oxide surfaces can be used for SMLM, then that these surfaces can be modified with self-assembled monolayers using organophosphonic acid derivatives, that the amount of antigens and antibodies on the surface can be controlled and monitored at the single molecule level and finally antibody binding to antigen modified surfaces can be monitored. The results show the amount of antibody that binds to an antigen modified surface is dependent on both the concentration of antigen on the surface and the concentration of antibody in solution. This study demonstrates the potential of SMLM for characterising biosensing interfaces and as the transducer in a massively parallel, wide field, single molecule detection scheme for quantitative analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. A new microcavity design for single molecule detection

    International Nuclear Information System (INIS)

    Steiner, M.; Schleifenbaum, F.; Stupperich, C.; Failla, A.V.; Hartschuh, A.; Meixner, A.J.

    2006-01-01

    We present a new microcavity design which allows for efficient detection of single molecules by measuring the molecular fluorescence emission coupled into a resonant cavity mode. The Fabry-Perot-type microresonator consists of two silver mirrors separated by a thin polymer film doped with dye molecules in ultralow concenctration. By slightly tilting one of the mirrors different cavity lengths can be selected within the same sample. Locally, on a μm scale, the microcavity still acts as a planar Fabry-Perot resonator. Using scanning confocal fluorescence microscopy, single emitters on resonance with a single mode of the microresonator can be spatially addressed. Our microcavity is demonstrated to be well-suited for investigating the coupling mechanism between single quantum emitters and single modes of the electromagnetic field. The microcavity layout could be integrated in a lab-on-a-microchip design for ultrasensitive microfluidic analytics and can be considered as an important improvement for single photon sources based on single molecules operating at room temperature

  19. Expression analysis of multiple myeloma CD138 negative progenitor cells using single molecule microarray readout

    Science.gov (United States)

    Jacak, Jaroslaw; Schnidar, Harald; Muresan, Leila; Regl, Gerhard; Frischauf, Annemarie; Aberger, Fritz; Schütz, Gerhard J.; Hesse, Jan

    2013-01-01

    We present a highly sensitive bioanalytical microarray assay that enables the analysis of small genomic sample material. By combining an optimized cDNA purification step with single molecule cDNA detection on the microarray, the platform has improved sensitivity compared to conventional systems, allowing amplification-free determination of expression profiles with as little as 600 ng total RNA. Total RNA from cells was reverse transcribed into fluorescently labeled cDNA and purified employing a precipitation method that minimizes loss of cDNA material. The microarray was scanned on a fluorescence chip-reader with single molecule sensitivity. Using the newly developed platform we were able to analyze the RNA expression profile of a subpopulation of rare multiple myeloma CD138 negative progenitor (MM CD138neg) cells. The high-sensitivity microarray approach led to the identification of a set of 20 genes differentially expressed in MM CD138neg cells. Our work demonstrates the applicability of a straight-forward single-molecule DNA array technology to current topics of molecular and cellular cancer research, which are otherwise difficult to address due to the limited amount of sample material. PMID:23416329

  20. A Single Molecule Investigation of the Photostability of Quantum Dots

    DEFF Research Database (Denmark)

    Christensen, Eva Arnspang; Kulatunga, Pasad; Lagerholm, B. Christoffer

    2012-01-01

    Quantum dots (QDs) are very attractive probes for multi-color fluorescence applications. We report here however that single QDs that are subject to continuous blue excitation from a 100W mercury arc lamp will undergo a continuous blue-switching of the emission wavelength eventually reaching a per...... is especially detrimental for multi-color single molecule applications, as we regularly observe spectral blue-shifts of 50 nm, or more even after only ten seconds of illumination....

  1. Novel AgoshRNA molecules for silencing of the CCR5 co-receptor for HIV-1 infection.

    Science.gov (United States)

    Herrera-Carrillo, Elena; Berkhout, Ben

    2017-01-01

    Allogeneic transplantation of blood stem cells from a CCR5-Δ32 homozygous donor to an HIV-infected individual, the "Berlin patient", led to a cure. Since then there has been a search for approaches that mimic this intervention in a gene therapy setting. RNA interference (RNAi) has evolved as a powerful tool to regulate gene expression in a sequence-specific manner and can be used to inactivate the CCR5 mRNA. Short hairpin RNA (shRNA) molecules can impair CCR5 expression, but these molecules may cause unintended side effects and they will not be processed in cells that lack Dicer, such as monocytes. Dicer-independent RNAi pathways have opened opportunities for new AgoshRNA designs that rely exclusively on Ago2 for maturation. Furthermore, AgoshRNA processing yields a single active guide RNA, thus reducing off-target effects. In this study, we tested different AgoshRNA designs against CCR5. We selected AgoshRNAs that potently downregulated CCR5 expression on human T cells and peripheral blood mononuclear cells (PBMC) and that had no apparent adverse effect on T cell development as assessed in a competitive cell growth assay. CCR5 knockdown significantly protected T cells from CCR5 tropic HIV-1 infection.

  2. Single molecule DNA detection with an atomic vapor notch filter

    Energy Technology Data Exchange (ETDEWEB)

    Uhland, Denis; Rendler, Torsten; Widmann, Matthias; Lee, Sang-Yun [University of Stuttgart and Stuttgart Research Center of Photonic Engineering (SCoPE) and IQST, 3rd Physics Institute, Stuttgart (Germany); Wrachtrup, Joerg; Gerhardt, Ilja [University of Stuttgart and Stuttgart Research Center of Photonic Engineering (SCoPE) and IQST, 3rd Physics Institute, Stuttgart (Germany); Max Planck Institute for Solid State Research, Stuttgart (Germany)

    2015-12-01

    The detection of single molecules has facilitated many advances in life- and material-science. Commonly the fluorescence of dye molecules is detected, which are attached to a non-fluorescent structure under study. For fluorescence microscopy one desires to maximize the detection efficiency together with an efficient suppression of undesired laser leakage. Here we present the use of the narrow-band filtering properties of hot atomic sodium vapor to selectively filter the excitation light from the red-shifted fluorescence of dye labeled single-stranded DNA molecules. A statistical analysis proves an enhancement in detection efficiency of more than 15% in a confocal and in a wide-field configuration. (orig.)

  3. Force-induced tautomerization in a single molecule.

    Science.gov (United States)

    Ladenthin, Janina N; Frederiksen, Thomas; Persson, Mats; Sharp, John C; Gawinkowski, Sylwester; Waluk, Jacek; Kumagai, Takashi

    2016-10-01

    Heat transfer, electrical potential and light energy are common ways to activate chemical reactions. Applied force is another way, but dedicated studies for such a mechanical activation are limited, and this activation is poorly understood at the single-molecule level. Here, we report force-induced tautomerization in a single porphycene molecule on a Cu(110) surface at 5 K, which is studied by scanning probe microscopy and density functional theory calculations. Force spectroscopy quantifies the force needed to trigger tautomerization with submolecular spatial resolution. The calculations show how the reaction pathway and barrier of tautomerization are modified in the presence of a copper tip and reveal the atomistic origin of the process. Moreover, we demonstrate that a chemically inert tip whose apex is terminated by a xenon atom cannot induce the reaction because of a weak interaction with porphycene and a strong relaxation of xenon on the tip as contact to the molecule is formed.

  4. Single-molecule spectroscopy of amino acids and peptides by recognition tunnelling

    Science.gov (United States)

    Zhao, Yanan; Ashcroft, Brian; Zhang, Peiming; Liu, Hao; Sen, Suman; Song, Weisi; Im, Jongone; Gyarfas, Brett; Manna, Saikat; Biswas, Sovan; Borges, Chad; Lindsay, Stuart

    2014-06-01

    The human proteome has millions of protein variants due to alternative RNA splicing and post-translational modifications, and variants that are related to diseases are frequently present in minute concentrations. For DNA and RNA, low concentrations can be amplified using the polymerase chain reaction, but there is no such reaction for proteins. Therefore, the development of single-molecule protein sequencing is a critical step in the search for protein biomarkers. Here, we show that single amino acids can be identified by trapping the molecules between two electrodes that are coated with a layer of recognition molecules, then measuring the electron tunnelling current across the junction. A given molecule can bind in more than one way in the junction, and we therefore use a machine-learning algorithm to distinguish between the sets of electronic `fingerprints' associated with each binding motif. With this recognition tunnelling technique, we are able to identify D and L enantiomers, a methylated amino acid, isobaric isomers and short peptides. The results suggest that direct electronic sequencing of single proteins could be possible by sequentially measuring the products of processive exopeptidase digestion, or by using a molecular motor to pull proteins through a tunnel junction integrated with a nanopore.

  5. Dynamic protein assemblies in homologous recombination with single DNA molecules

    NARCIS (Netherlands)

    van der Heijden, A.H.

    2007-01-01

    What happens when your DNA breaks? This thesis describes experimental work on the single-molecule level focusing on the interaction between DNA and DNA-repair proteins, in particular bacterial RecA and human Rad51, involved in homologous recombination. Homologous recombination and its central event

  6. Single-molecule experiments in biophysics: Exploring the thermal ...

    Indian Academy of Sciences (India)

    cules contributes to our understanding of the nonequilibrium thermal behavior of small systems. Keywords. Biophysics; single-molecule experiments; fluctuation theorems. PACS Nos 05.70.Ln; 82.37.Rs; 87.10.+e; 87.15.Ya. 1. Biomolecules, molecular demons and statistical physics. Biophysics is a relatively young discipline ...

  7. Computing magnetic anisotropy constants of single molecule magnets

    Indian Academy of Sciences (India)

    Administrator

    Abstract. We present here a theoretical approach to compute the molecular magnetic anisotropy parameters, DM and EM for single molecule magnets in any given spin eigenstate of exchange spin Hami- ltonian. We first describe a hybrid constant MS-valence bond (VB) technique of solving spin Hamilto- nians employing ...

  8. Computing magnetic anisotropy constants of single molecule magnets

    Indian Academy of Sciences (India)

    We present here a theoretical approach to compute the molecular magnetic anisotropy parameters, and for single molecule magnets in any given spin eigenstate of exchange spin Hamiltonian. We first describe a hybrid constant -valence bond (VB) technique of solving spin Hamiltonians employing full spatial ...

  9. Single Molecule Study of Cellulase Hydrolysis of Crystalline Cellulose

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Y.-S.; Luo, Y.; Baker, J. O.; Zeng, Y.; Himmel, M. E.; Smith, S.; Ding, S.-Y.

    2009-12-01

    This report seeks to elucidate the role of cellobiohydrolase-I (CBH I) in the hydrolysis of crystalline cellulose. A single-molecule approach uses various imaging techniques to investigate the surface structure of crystalline cellulose and changes made in the structure by CBH I.

  10. Single-molecule denaturation mapping of DNA in nanofluidic channels

    DEFF Research Database (Denmark)

    Reisner, Walter; Larsen, Niels Bent; Silahtaroglu, Asli

    2010-01-01

    Here we explore the potential power of denaturation mapping as a single-molecule technique. By partially denaturing YOYO (R)-1-labeled DNA in nanofluidic channels with a combination of formamide and local heating, we obtain a sequence-dependent "barcode" corresponding to a series of local dips...

  11. Single molecule microscopy in 3D cell cultures and tissues.

    Science.gov (United States)

    Lauer, Florian M; Kaemmerer, Elke; Meckel, Tobias

    2014-12-15

    From the onset of the first microscopic visualization of single fluorescent molecules in living cells at the beginning of this century, to the present, almost routine application of single molecule microscopy, the method has well-proven its ability to contribute unmatched detailed insight into the heterogeneous and dynamic molecular world life is composed of. Except for investigations on bacteria and yeast, almost the entire story of success is based on studies on adherent mammalian 2D cell cultures. However, despite this continuous progress, the technique was not able to keep pace with the move of the cell biology community to adapt 3D cell culture models for basic research, regenerative medicine, or drug development and screening. In this review, we will summarize the progress, which only recently allowed for the application of single molecule microscopy to 3D cell systems and give an overview of the technical advances that led to it. While initially posing a challenge, we finally conclude that relevant 3D cell models will become an integral part of the on-going success of single molecule microscopy. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. A local view on single and coupled molecules

    NARCIS (Netherlands)

    van Dijk, E.M.H.P.; Hernando Campos, J.; Hoogenboom, Jacob; Garcia Parajo, M.F.

    2005-01-01

    The paper focuses on a novel approach to reveal ultrafast dynamics in single molecules. The main strength of the approach is towards ultrafast processes in extended multi-chromophoric molecular assemblies. Excitonically coupled systems consisting of 2 and 3 rigidly linked perylene-diimide units in a

  13. Isolation of Microarray-Grade Total RNA, MicroRNA, and DNA from a Single PAXgene Blood RNA Tube

    DEFF Research Database (Denmark)

    Kruhøffer, Mogens; Andersen, Lars Dyrskjøt; Voss, Thorsten

    2007-01-01

    We have developed a procedure for isolation of microRNA and genomic DNA in addition to total RNA from whole blood stabilized in PAXgene Blood RNA tubes. The procedure is based on automatic extraction on a BioRobot MDx and includes isolation of DNA from a fraction of the stabilized blood......RNA was tested using spotted locked nucleic acid-based microarrays. We conclude that the yield and quality of total RNA, microRNA, and DNA from a single PAXgene blood RNA tube is sufficient for downstream microarray analysis....

  14. Quantum-Sequencing: Fast electronic single DNA molecule sequencing

    Science.gov (United States)

    Casamada Ribot, Josep; Chatterjee, Anushree; Nagpal, Prashant

    2014-03-01

    A major goal of third-generation sequencing technologies is to develop a fast, reliable, enzyme-free, high-throughput and cost-effective, single-molecule sequencing method. Here, we present the first demonstration of unique ``electronic fingerprint'' of all nucleotides (A, G, T, C), with single-molecule DNA sequencing, using Quantum-tunneling Sequencing (Q-Seq) at room temperature. We show that the electronic state of the nucleobases shift depending on the pH, with most distinct states identified at acidic pH. We also demonstrate identification of single nucleotide modifications (methylation here). Using these unique electronic fingerprints (or tunneling data), we report a partial sequence of beta lactamase (bla) gene, which encodes resistance to beta-lactam antibiotics, with over 95% success rate. These results highlight the potential of Q-Seq as a robust technique for next-generation sequencing.

  15. Modulation of intermolecular interactions in single-molecule magnets

    Science.gov (United States)

    Heroux, Katie Jeanne

    Polynuclear manganese clusters exhibiting interesting magnetic and quantum properties have been an area of intense research since the discovery of the first single-molecule magnet (SMM) in 1993. These molecules, below their blocking temperature, function as single-domain magnetic particles which exhibit classical macroscale magnetic properties as well as quantum mechanical phenomena such as quantum tunnelling of magnetization (QTM) and quantum phase interference. The union of classical and quantum behavior in these nanomaterials makes SMMs ideal candidates for high-density information storage and quantum computing. However, environmental coupling factors (nuclear spins, phonons, neighboring molecules) must be minimized if such applications are ever to be fully realized. The focus of this work is making small structural changes in well-known manganese SMMs in order to drastically enhance the overall magnetic and quantum properties of the system. Well-isolated molecules of high crystalline quality should lead to well-defined energetic and spectral properties as well. An advantage of SMMs over bulk magnetic materials is that they can be chemically altered from a "bottom-up" approach providing a synthetic tool for tuning magnetic properties. This systematic approach is utilized in the work presented herein by incorporating bulky ligands and/or counterions to "isolate" the magnetic core of [Mn4] dicubane SMMs. Reducing intermolecular interactions in the crystal lattice (neighboring molecules, solvate molecules, dipolar interactions) is an important step toward developing viable quantum computing devices. Detailed bulk magnetic studies as well as single crystal magnetization hysteresis and high-frequency EPR studies on these sterically-isolated complexes show enhanced, and sometimes even unexpected, quantum dynamics. The importance of intra- and intermolecular interactions remains a common theme throughout this work, extending to other SMMs of various topology including

  16. Single Molecule Electrochemical Detection in Aqueous Solutions and Ionic Liquids.

    Science.gov (United States)

    Byers, Joshua C; Paulose Nadappuram, Binoy; Perry, David; McKelvey, Kim; Colburn, Alex W; Unwin, Patrick R

    2015-10-20

    Single molecule electrochemical detection (SMED) is an extremely challenging aspect of electroanalytical chemistry, requiring unconventional electrochemical cells and measurements. Here, SMED is reported using a "quad-probe" (four-channel probe) pipet cell, fabricated by depositing carbon pyrolytically into two diagonally opposite barrels of a laser-pulled quartz quadruple-barreled pipet and filling the open channels with electrolyte solution, and quasi-reference counter electrodes. A meniscus forms at the end of the probe covering the two working electrodes and is brought into contact with a substrate working electrode surface. In this way, a nanogap cell is produced whereby the two carbon electrodes in the pipet can be used to promote redox cycling of an individual molecule with the substrate. Anticorrelated currents generated at the substrate and tip electrodes, at particular distances (typically tens of nanometers), are consistent with the detection of single molecules. The low background noise realized in this droplet format opens up new opportunities in single molecule electrochemistry, including the use of ionic liquids, as well as aqueous solution, and the quantitative assessment and analysis of factors influencing redox cycling currents, due to a precisely known gap size.

  17. 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

    In vitro selection of aptamers that recognize small organic molecules has proven difficult, in part due to the challenge of immobilizing small molecules on solid supports for SELEX (Systematic Evolution of Ligands by Exponential Enrichment). This study describes the implementation of RNA Capture......-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...

  18. Investigation of polyelectrolyte desorption by single molecule force spectroscopy

    International Nuclear Information System (INIS)

    Friedsam, C; Seitz, M; Gaub, H E

    2004-01-01

    Single molecule force spectroscopy has evolved into a powerful method for the investigation of intra- and intermolecular interactions at the level of individual molecules. Many examples, including the investigation of the dynamic properties of complex biological systems as well as the properties of covalent bonds or intermolecular transitions within individual polymers, are reported in the literature. The technique has recently been extended to the systematic investigation of desorption processes of individual polyelectrolyte molecules adsorbed on generic surfaces. The stable covalent attachment of polyelectrolyte molecules to the AFM-tip provides the possibility of performing long-term measurements with the same set of molecules and therefore allows the in situ observation of the impact of environmental changes on the adsorption behaviour of individual molecules. Different types of interactions, e.g. electrostatic or hydrophobic interactions, that determine the adsorption process could be identified and characterized. The experiments provided valuable details that help to understand the nature and the properties of non-covalent interactions, which is helpful with regard to biological systems as well as for technical applications. Apart from this, desorption experiments can be utilized to characterize the properties of surfaces or polymer coatings. Therefore they represent a versatile tool that can be further developed in terms of various aspects

  19. Transition paths in single-molecule force spectroscopy.

    Science.gov (United States)

    Cossio, Pilar; Hummer, Gerhard; Szabo, Attila

    2018-03-28

    In a typical single-molecule force spectroscopy experiment, the ends of the molecule of interest are connected by long polymer linkers to a pair of mesoscopic beads trapped in the focus of two laser beams. At constant force load, the total extension, i.e., the end-to-end distance of the molecule plus linkers, is measured as a function of time. In the simplest systems, the measured extension fluctuates about two values characteristic of folded and unfolded states, with occasional transitions between them. We have recently shown that molecular (un)folding rates can be recovered from such trajectories, with a small linker correction, as long as the characteristic time of the bead fluctuations is shorter than the residence time in the unfolded (folded) state. Here, we show that accurate measurements of the molecular transition path times require an even faster apparatus response. Transition paths, the trajectory segments in which the molecule (un)folds, are properly resolved only if the beads fluctuate more rapidly than the end-to-end distance of the molecule. Therefore, over a wide regime, the measured rates may be meaningful but not the transition path times. Analytic expressions for the measured mean transition path times are obtained for systems diffusing anisotropically on a two-dimensional free energy surface. The transition path times depend on the properties both of the molecule and of the pulling device.

  20. Single-Molecule Counting of Point Mutations by Transient DNA Binding

    Science.gov (United States)

    Su, Xin; Li, Lidan; Wang, Shanshan; Hao, Dandan; Wang, Lei; Yu, Changyuan

    2017-03-01

    High-confidence detection of point mutations is important for disease diagnosis and clinical practice. Hybridization probes are extensively used, but are hindered by their poor single-nucleotide selectivity. Shortening the length of DNA hybridization probes weakens the stability of the probe-target duplex, leading to transient binding between complementary sequences. The kinetics of probe-target binding events are highly dependent on the number of complementary base pairs. Here, we present a single-molecule assay for point mutation detection based on transient DNA binding and use of total internal reflection fluorescence microscopy. Statistical analysis of single-molecule kinetics enabled us to effectively discriminate between wild type DNA sequences and single-nucleotide variants at the single-molecule level. A higher single-nucleotide discrimination is achieved than in our previous work by optimizing the assay conditions, which is guided by statistical modeling of kinetics with a gamma distribution. The KRAS c.34 A mutation can be clearly differentiated from the wild type sequence (KRAS c.34 G) at a relative abundance as low as 0.01% mutant to WT. To demonstrate the feasibility of this method for analysis of clinically relevant biological samples, we used this technology to detect mutations in single-stranded DNA generated from asymmetric RT-PCR of mRNA from two cancer cell lines.

  1. Spin-Spin Cross Relaxation in Single-Molecule Magnets

    Science.gov (United States)

    Wernsdorfer, W.; Bhaduri, S.; Tiron, R.; Hendrickson, D. N.; Christou, G.

    2002-10-01

    The one-body tunnel picture of single-molecule magnets (SMMs) is not always sufficient to explain the measured tunnel transitions. An improvement to the picture is proposed by including also two-body tunnel transitions such as spin-spin cross relaxation (SSCR) which are mediated by dipolar and weak superexchange interactions between molecules. A Mn4 SMM is used as a model system. At certain external fields, SSCRs lead to additional quantum resonances which show up in hysteresis loop measurements as well-defined steps. A simple model is used to explain quantitatively all observed transitions.

  2. Action spectroscopy for single-molecule reactions - Experiments and theory

    Science.gov (United States)

    Kim, Y.; Motobayashi, K.; Frederiksen, T.; Ueba, H.; Kawai, M.

    2015-05-01

    We review several representative experimental results of action spectroscopy (AS) of single molecules on metal surfaces using a scanning tunneling microscope (STM) by M. Kawai's group over last decade. The experimental procedures to observe STM-AS are described. A brief description of a low-temperature STM and experimental setup are followed by key experimental techniques of how to determine an onset bias voltage of a reaction and how to measure a current change associated with reactions and finally how to observe AS for single molecule reactions. The experimental results are presented for vibrationally mediated chemical transformation of trans-2-butene to 1.3-butadiene molecule and rotational motion of a single cis-2-butene molecule among four equivalent orientations on Pd(1 1 0). The AS obtained from the motion clearly detects more vibrational modes than inelastic electron tunneling spectroscopy with an STM. AS is demonstrated as a useful and novel single molecule vibrational spectroscopy. The AS for a lateral hopping of water dimer on Pt(1 1 1) is presented as an example of novelty. Several distinct vibrational modes are detected as the thresholds in the AS. The assignment of the vibrational modes determined from the analysis of the AS is made from a view of the adsorption geometry of hydrogen-bond donor or acceptor molecules in water dimer. A generic theory of STM-AS, i.e., a reaction rate or yield as a function of bias voltage, is presented using a single adsorbate resonance model for single molecule reactions induced by the inelastic tunneling current. Formulas for the reaction rate R (V) and Y (V) , i.e., reaction yield per electron Y (V) = eR (V) / I are derived. It provides a versatile framework to analyze any vibrationally mediated reactions of single adsorbates on metal surfaces. Numerical examples are presented to demonstrate generic features of the vibrational generation rate and Y (V) at different levels of approximations and to show how the effective

  3. Fluorescently-tagged human eIF3 for single-molecule spectroscopy.

    Science.gov (United States)

    Johnson, Alex G; Petrov, Alexey N; Fuchs, Gabriele; Majzoub, Karim; Grosely, Rosslyn; Choi, Junhong; Puglisi, Joseph D

    2018-01-25

    Human translation initiation relies on the combined activities of numerous ribosome-associated eukaryotic initiation factors (eIFs). The largest factor, eIF3, is an ∼800 kDa multiprotein complex that orchestrates a network of interactions with the small 40S ribosomal subunit, other eIFs, and mRNA, while participating in nearly every step of initiation. How these interactions take place during the time course of translation initiation remains unclear. Here, we describe a method for the expression and affinity purification of a fluorescently-tagged eIF3 from human cells. The tagged eIF3 dodecamer is structurally intact, functions in cell-based assays, and interacts with the HCV IRES mRNA and the 40S-IRES complex in vitro. By tracking the binding of single eIF3 molecules to the HCV IRES RNA with a zero-mode waveguides-based instrument, we show that eIF3 samples both wild-type IRES and an IRES that lacks the eIF3-binding region, and that the high-affinity eIF3-IRES interaction is largely determined by slow dissociation kinetics. The application of single-molecule methods to more complex systems involving eIF3 may unveil dynamics underlying mRNA selection and ribosome loading during human translation initiation. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. Automation of a single-DNA molecule stretching device

    DEFF Research Database (Denmark)

    Sørensen, Kristian Tølbøl; Lopacinska, Joanna M.; Tommerup, Niels

    2015-01-01

    We automate the manipulation of genomic-length DNA in a nanofluidic device based on real-time analysis of fluorescence images. In our protocol, individual molecules are picked from a microchannel and stretched with pN forces using pressure driven flows. The millimeter-long DNA fragments free...... flowing in micro- and nanofluidics emit low fluorescence and change shape, thus challenging the image analysis for machine vision. We demonstrate a set of image processing steps that increase the intrinsically low signal-to-noise ratio associated with single-molecule fluorescence microscopy. Furthermore......, we demonstrate how to estimate the length of molecules by continuous real-time image stitching and how to increase the effective resolution of a pressure controller by pulse width modulation. The sequence of image-processing steps addresses the challenges of genomic-length DNA visualization; however...

  5. Vibrationally coupled electron transport through single-molecule junctions

    Energy Technology Data Exchange (ETDEWEB)

    Haertle, Rainer

    2012-04-26

    Single-molecule junctions are among the smallest electric circuits. They consist of a molecule that is bound to a left and a right electrode. With such a molecular nanocontact, the flow of electrical currents through a single molecule can be studied and controlled. Experiments on single-molecule junctions show that a single molecule carries electrical currents that can even be in the microampere regime. Thereby, a number of transport phenomena have been observed, such as, for example, diode- or transistor-like behavior, negative differential resistance and conductance switching. An objective of this field, which is commonly referred to as molecular electronics, is to relate these transport phenomena to the properties of the molecule in the contact. To this end, theoretical model calculations are employed, which facilitate an understanding of the underlying transport processes and mechanisms. Thereby, one has to take into account that molecules are flexible structures, which respond to a change of their charge state by a profound reorganization of their geometrical structure or may even dissociate. It is thus important to understand the interrelation between the vibrational degrees of freedom of a singlemolecule junction and the electrical current flowing through the contact. In this thesis, we investigate vibrational effects in electron transport through singlemolecule junctions. For these studies, we calculate and analyze transport characteristics of both generic and first-principles based model systems of a molecular contact. To this end, we employ a master equation and a nonequilibrium Green's function approach. Both methods are suitable to describe this nonequilibrium transport problem and treat the interactions of the tunneling electrons on the molecular bridge non-perturbatively. This is particularly important with respect to the vibrational degrees of freedom, which may strongly interact with the tunneling electrons. We show in detail that the resulting

  6. Single vesicle biochips for ultra-miniaturized nanoscale fluidics and single molecule bioscience

    DEFF Research Database (Denmark)

    Christensen, Andreas Lauge; Lohr, Christina; Christensen, Sune M.

    2013-01-01

    , their fabrication via controlled self-assembly, and their characterization using fluorescence microscopy. We also highlight their applications in selected fields such as nanofluidics and single molecule bioscience. Despite their great potential for improved biocompatibility, extreme miniaturization and high...

  7. Localization microscopy: mapping cellular dynamics with single molecules.

    Science.gov (United States)

    Nelson, A J; Hess, S T

    2014-04-01

    Resolution describes the smallest details within a sample that can be recovered by a microscope lens system. For optical microscopes detecting visible light, diffraction limits the resolution to ∼200-250 nm. In contrast, localization measures the position of an isolated object using its image. Single fluorescent molecules can be localized with an uncertainty of a few tens of nanometres, and in some cases less than one nanometre. Superresolution fluorescence localization microscopy (SRFLM) images and localizes fluorescent molecules in a sample. By controlling the visibility of the fluorescent molecules with light, it is possible to cause a sparse subset of the tags to fluoresce and be spatially separated from each other. A movie is acquired with a camera, capturing images of many sets of visible fluorescent tags over a period of time. The movie is then analysed by a computer whereby all of the single molecules are independently measured, and their positions are recorded. When the coordinates of a sufficient number of molecules are collected, an image can be rendered by plotting the coordinates of the localized molecules. The spatial resolution of these rendered images can be better than 20 nm, roughly an order of magnitude better than the diffraction limited resolution. The invention of SRFLM has led to an explosion of related techniques. Through the use of specialized optics, the fluorescent signal can be split into multiple detection channels. These channels can capture additional information such as colour (emission wavelength), orientation and three-dimensional position of the detected molecules. Measurement of the colour of the detected fluorescence can allow researchers to distinguish multiple types of fluorescent tags and to study the interaction between multiple molecules of interest. Three-dimensional imaging and determination of molecular orientations offer insight into structural organization of the sample. SRFLM is compatible with living samples and

  8. Detecting single DNA molecule interactions with optical microcavities (Presentation Recording)

    Science.gov (United States)

    Vollmer, Frank

    2015-09-01

    Detecting molecules and their interactions lies at the heart of all biosensor devices, which have important applications in health, environmental monitoring and biomedicine. Achieving biosensing capability at the single molecule level is, moreover, a particularly important goal since single molecule biosensors would not only operate at the ultimate detection limit by resolving individual molecular interactions, but they could also monitor biomolecular properties which are otherwise obscured in ensemble measurements. For example, a single molecule biosensor could resolve the fleeting interaction kinetics between a molecule and its receptor, with immediate applications in clinical diagnostics. We have now developed a label-free biosensing platform that is capable of monitoring single DNA molecules and their interaction kinetics[1], hence achieving an unprecedented sensitivity in the optical domain, Figure 1. We resolve the specific contacts between complementary oligonucleotides, thereby detecting DNA strands with less than 2.4 kDa molecular weight. Furthermore we can discern strands with single nucleotide mismatches by monitoring their interaction kinetics. Our device utilizes small glass microspheres as optical transducers[1,2, 3], which are capable of increasing the number of interactions between a light beam and analyte molecules. A prism is used to couple the light beam into the microsphere. Ourr biosensing approach resolves the specific interaction kinetics between single DNA fragments. The optical transducer is assembled in a simple three-step protocol, and consists of a gold nanorod attached to a glass microsphere, where the surface of the nanorod is further modified with oligonucleotide receptors. The interaction kinetics of an oligonucleotide receptor with DNA fragments in the surrounding aqueous solution is monitored at the single molecule level[1]. The light remains confined inside the sphere where it is guided by total internal reflections along a

  9. Simple test system for single molecule recognition force microscopy

    International Nuclear Information System (INIS)

    Riener, Christian K.; Stroh, Cordula M.; Ebner, Andreas; Klampfl, Christian; Gall, Alex A.; Romanin, Christoph; Lyubchenko, Yuri L.; Hinterdorfer, Peter; Gruber, Hermann J.

    2003-01-01

    We have established an easy-to-use test system for detecting receptor-ligand interactions on the single molecule level using atomic force microscopy (AFM). For this, avidin-biotin, probably the best characterized receptor-ligand pair, was chosen. AFM sensors were prepared containing tethered biotin molecules at sufficiently low surface concentrations appropriate for single molecule studies. A biotin tether, consisting of a 6 nm poly(ethylene glycol) (PEG) chain and a functional succinimide group at the other end, was newly synthesized and covalently coupled to amine-functionalized AFM tips. In particular, PEG 800 diamine was glutarylated, the mono-adduct NH 2 -PEG-COOH was isolated by ion exchange chromatography and reacted with biotin succinimidylester to give biotin-PEG-COOH which was then activated as N-hydroxysuccinimide (NHS) ester to give the biotin-PEG-NHS conjugate which was coupled to the aminofunctionalized AFM tip. The motional freedom provided by PEG allows for free rotation of the biotin molecule on the AFM sensor and for specific binding to avidin which had been adsorbed to mica surfaces via electrostatic interactions. Specific avidin-biotin recognition events were discriminated from nonspecific tip-mica adhesion by their typical unbinding force (∼40 pN at 1.4 nN/s loading rate), unbinding length (<13 nm), the characteristic nonlinear force-distance relation of the PEG linker, and by specific block with excess of free d-biotin. The convenience of the test system allowed to evaluate, and compare, different methods and conditions of tip aminofunctionalization with respect to specific binding and nonspecific adhesion. It is concluded that this system is well suited as calibration or start-up kit for single molecule recognition force microscopy

  10. New Antifouling Platform Characterized by Single-Molecule Imaging

    Science.gov (United States)

    2015-01-01

    Antifouling surfaces have been widely studied for their importance in medical devices and industry. Antifouling surfaces mostly achieved by methoxy-poly(ethylene glycol) (mPEG) have shown biomolecular adsorption less than 1 ng/cm2 which was measured by surface analytical tools such as surface plasmon resonance (SPR) spectroscopy, quartz crystal microbalance (QCM), or optical waveguide lightmode (OWL) spectroscopy. Herein, we utilize a single-molecule imaging technique (i.e., an ultimate resolution) to study antifouling properties of functionalized surfaces. We found that about 600 immunoglobulin G (IgG) molecules are adsorbed. This result corresponds to ∼5 pg/cm2 adsorption, which is far below amount for the detection limit of the conventional tools. Furthermore, we developed a new antifouling platform that exhibits improved antifouling performance that shows only 78 IgG molecules adsorbed (∼0.5 pg/cm2). The antifouling platform consists of forming 1 nm TiO2 thin layer, on which peptidomimetic antifouling polymer (PMAP) is robustly anchored. The unprecedented antifouling performance can potentially revolutionize a variety of research fields such as single-molecule imaging, medical devices, biosensors, and others. PMID:24503420

  11. Light-Induced Switching of Tunable Single-Molecule Junctions

    KAUST Repository

    Sendler, Torsten

    2015-04-16

    A major goal of molecular electronics is the development and implementation of devices such as single-molecular switches. Here, measurements are presented that show the controlled in situ switching of diarylethene molecules from their nonconductive to conductive state in contact to gold nanoelectrodes via controlled light irradiation. Both the conductance and the quantum yield for switching of these molecules are within a range making the molecules suitable for actual devices. The conductance of the molecular junctions in the opened and closed states is characterized and the molecular level E 0, which dominates the current transport in the closed state, and its level broadening Γ are identified. The obtained results show a clear light-induced ring forming isomerization of the single-molecule junctions. Electron withdrawing side-groups lead to a reduction of conductance, but do not influence the efficiency of the switching mechanism. Quantum chemical calculations of the light-induced switching processes correlate these observations with the fundamentally different low-lying electronic states of the opened and closed forms and their comparably small modification by electron-withdrawing substituents. This full characterization of a molecular switch operated in a molecular junction is an important step toward the development of real molecular electronics devices.

  12. Machine learning approach for single molecule localisation microscopy.

    Science.gov (United States)

    Colabrese, Silvia; Castello, Marco; Vicidomini, Giuseppe; Del Bue, Alessio

    2018-04-01

    Single molecule localisation (SML) microscopy is a fundamental tool for biological discoveries; it provides sub-diffraction spatial resolution images by detecting and localizing "all" the fluorescent molecules labeling the structure of interest. For this reason, the effective resolution of SML microscopy strictly depends on the algorithm used to detect and localize the single molecules from the series of microscopy frames. To adapt to the different imaging conditions that can occur in a SML experiment, all current localisation algorithms request, from the microscopy users, the choice of different parameters. This choice is not always easy and their wrong selection can lead to poor performance. Here we overcome this weakness with the use of machine learning. We propose a parameter-free pipeline for SML learning based on support vector machine (SVM). This strategy requires a short supervised training that consists in selecting by the user few fluorescent molecules (∼ 10-20) from the frames under analysis. The algorithm has been extensively tested on both synthetic and real acquisitions. Results are qualitatively and quantitatively consistent with the state of the art in SML microscopy and demonstrate that the introduction of machine learning can lead to a new class of algorithms competitive and conceived from the user point of view.

  13. The elastic theory of a single DNA molecule

    Indian Academy of Sciences (India)

    the other hand, if there is a negative torsional stress, a pulling force as small as 0.3 pN can distort the native structure of DNA considerably [5,6]. Related to the latter, there has been recent progress in understanding the force–extension curves of single-stranded DNA. (ssDNA) and RNA [7–12]. Many distinct transitions have ...

  14. The Conformational Dynamics of Cas9 Governing DNA Cleavage Are Revealed by Single-Molecule FRET

    Directory of Open Access Journals (Sweden)

    Mengyi Yang

    2018-01-01

    Full Text Available Summary: Off-target binding and cleavage by Cas9 pose major challenges in its application. How the conformational dynamics of Cas9 govern its nuclease activity under on- and off-target conditions remains largely unknown. Here, using intra-molecular single-molecule fluorescence resonance energy transfer measurements, we revealed that Cas9 in apo, sgRNA-bound, and dsDNA/sgRNA-bound forms spontaneously transits among three major conformational states, mainly reflecting significant conformational mobility of the catalytic HNH domain. We also uncovered surprising long-range allosteric communication between the HNH domain and the RNA/DNA heteroduplex at the PAM-distal end to ensure correct positioning of the catalytic site, which demonstrated that a unique proofreading mechanism served as the last checkpoint before DNA cleavage. Several Cas9 residues were likely to mediate the allosteric communication and proofreading step. Modulating interactions between Cas9 and heteroduplex at the PAM-distal end by introducing mutations on these sites provides an alternative route to improve and optimize the CRISPR/Cas9 toolbox. : Yang et al. revealed significant conformational dynamics of Cas9 at global and local scales using single-molecule FRET. They uncovered surprising long-range allosteric communication between the HNH nuclease domain and the RNA/DNA heteroduplex at the PAM-distal end that serves as a proofreading checkpoint to govern the nuclease activity and specificity of Cas9. Keywords: CRISPR, Cas9, single-molecule, FRET, conformational dynamics, proofreading, off-target, allosteric communication, genome editing

  15. Nonequilibrium Energetics of a Single F1-ATPase Molecule

    OpenAIRE

    Toyabe, Shoichi; Watanabe-Nakayama, Takahiro; Okamoto, Tetsuaki; Kudo, Seishi; Muneyuki, Eiro

    2010-01-01

    Molecular motors drive mechanical motions utilizing the free energy liberated from chemical reactions such as ATP hydrolysis. Although it is essential to know the efficiency of this free energy transduction, it has been a challenge due to the system's microscopic scale. Here, we evaluate the single-molecule energetics of a rotary molecular motor, F1-ATPase, by applying a recently derived nonequilibrium equality together with an electrorotation method. We show that the sum of the heat flow thr...

  16. Berry-phase blockade in single-molecule magnets

    OpenAIRE

    Gonzalez, Gabriel; Leuenberger, Michael N.

    2006-01-01

    We formulate the problem of electron transport through a single-molecule magnet (SMM) in the Coulomb blockade regime taking into account topological interference effects for the tunneling of the large spin of a SMM. The interference originates from spin Berry phases associated with different tunneling paths. We show that in the case of incoherent spin states it is essential to place the SMM between oppositely spin-polarized source and drain leads in order to detect the spin tunneling in the s...

  17. Dysprosium Acetylacetonato Single-Molecule Magnet Encapsulated in Carbon Nanotubes

    Directory of Open Access Journals (Sweden)

    Ryo Nakanishi

    2016-12-01

    Full Text Available Dy single-molecule magnets (SMMs, which have several potential uses in a variety of applications, such as quantum computing, were encapsulated in multi-walled carbon nanotubes (MWCNTs by using a capillary method. Encapsulation was confirmed by using transmission electron microscopy (TEM. In alternating current magnetic measurements, the magnetic susceptibilities of the Dy acetylacetonato complexes showed clear frequency dependence even inside the MWCNTs, meaning that this hybrid can be used as magnetic materials in devices.

  18. Viruses and Tetraspanins: Lessons from Single Molecule Approaches

    Science.gov (United States)

    Dahmane, Selma; Rubinstein, Eric; Milhiet, Pierre-Emmanuel

    2014-01-01

    Tetraspanins are four-span membrane proteins that are widely distributed in multi-cellular organisms and involved in several infectious diseases. They have the unique property to form a network of protein-protein interaction within the plasma membrane, due to the lateral associations with one another and with other membrane proteins. Tracking tetraspanins at the single molecule level using fluorescence microscopy has revealed the membrane behavior of the tetraspanins CD9 and CD81 in epithelial cell lines, providing a first dynamic view of this network. Single molecule tracking highlighted that these 2 proteins can freely diffuse within the plasma membrane but can also be trapped, permanently or transiently, in tetraspanin-enriched areas. More recently, a similar strategy has been used to investigate tetraspanin membrane behavior in the context of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) infection. In this review we summarize the main results emphasizing the relationship in terms of membrane partitioning between tetraspanins, some of their partners such as Claudin-1 and EWI-2, and viral proteins during infection. These results will be analyzed in the context of other membrane microdomains, stressing the difference between raft and tetraspanin-enriched microdomains, but also in comparison with virus diffusion at the cell surface. New advanced single molecule techniques that could help to further explore tetraspanin assemblies will be also discussed. PMID:24800676

  19. Single-Molecule Photocurrent at a Metal-Molecule-Semiconductor Junction.

    Science.gov (United States)

    Vezzoli, Andrea; Brooke, Richard J; Higgins, Simon J; Schwarzacher, Walther; Nichols, Richard J

    2017-11-08

    We demonstrate here a new concept for a metal-molecule-semiconductor nanodevice employing Au and GaAs contacts that acts as a photodiode. Current-voltage traces for such junctions are recorded using a STM, and the "blinking" or "I(t)" method is used to record electrical behavior at the single-molecule level in the dark and under illumination, with both low and highly doped GaAs samples and with two different types of molecular bridge: nonconjugated pentanedithiol and the more conjugated 1,4-phenylene(dimethanethiol). Junctions with highly doped GaAs show poor rectification in the dark and a low photocurrent, while junctions with low doped GaAs show particularly high rectification ratios in the dark (>10 3 for a 1.5 V bias potential) and a high photocurrent in reverse bias. In low doped GaAs, the greater thickness of the depletion layer not only reduces the reverse bias leakage current, but also increases the volume that contributes to the photocurrent, an effect amplified by the point contact geometry of the junction. Furthermore, since photogenerated holes tunnel to the metal electrode assisted by the HOMO of the molecular bridge, the choice of the latter has a strong influence on both the steady state and transient metal-molecule-semiconductor photodiode response. The control of junction current via photogenerated charge carriers adds new functionality to single-molecule nanodevices.

  20. Excitonic Coupling in Linear and Trefoil Trimer Perylenediimide Molecules Probed by Single-Molecule Spectroscopy

    KAUST Repository

    Yoo, Hyejin

    2012-10-25

    Perylenediimide (PDI) molecules are promising building blocks for photophysical studies of electronic interactions within multichromophore arrays. Such PDI arrays are important materials for fabrication of molecular nanodevices such as organic light-emitting diodes, organic semiconductors, and biosensors because of their high photostability, chemical and physical inertness, electron affinity, and high tinctorial strength over the entire visible spectrum. In this work, PDIs have been organized into linear (L3) and trefoil (T3) trimer molecules and investigated by single-molecule fluorescence microscopy to probe the relationship between molecular structures and interchromophoric electronic interactions. We found a broad distribution of coupling strengths in both L3 and T3 and hence strong/weak coupling between PDI units by monitoring spectral peak shifts in single-molecule fluorescence spectra upon sequential photobleaching of each constituent chromophore. In addition, we used a wide-field defocused imaging technique to resolve heterogeneities in molecular structures of L3 and T3 embedded in a PMMA polymer matrix. A systematic comparison between the two sets of experimental results allowed us to infer the correlation between intermolecular interactions and molecular structures. Our results show control of the PDI intermolecular interactions using suitable multichromophoric structures. © 2012 American Chemical Society.

  1. Capture, unfolding, and detection of individual tRNA molecules using a nanopore device

    Directory of Open Access Journals (Sweden)

    Andrew M Smith

    2015-06-01

    Full Text Available Transfer RNAs (tRNA are the most common RNA molecules in cells and have critical roles as both translators of the genetic code and regulators of protein synthesis. As such, numerous methods have focused on studying tRNA abundance and regulation, with the most widely used methods being RNA-seq and microarrays. Though revolutionary to transcriptomics, these assays are limited by an inability to encode tRNA modifications in the requisite cDNA. These modifications are abundant in tRNA and critical to their function. Here we describe proof-of-concept experiments where individual tRNA molecules are examined as linear strands using a biological nanopore. This method utilizes an enzymatically ligated synthetic DNA adapter to concentrate tRNA at the lipid bilayer of the nanopore device and efficiently denature individual tRNA molecules as they are pulled through the α-hemolysin (α-HL nanopore. Additionally, the DNA adapter provides a loading site for ϕ29 DNA polymerase (ϕ29 DNAP, which acts as a brake on the translocating tRNA. This increases the dwell time of adapted tRNA in the nanopore, allowing us to identify the region of the nanopore signal that is produced by the translocating tRNA itself. Using adapter-modified E. coli tRNAfMet and tRNALys, we show that the nanopore signal during controlled translocation is dependent on the identity of the tRNA. This confirms that adapter-modified tRNA can translocate end-to-end through nanopores and provides the foundation for future work in direct sequencing of individual transfer RNA with a nanopore-based device.

  2. Capture, Unfolding, and Detection of Individual tRNA Molecules Using a Nanopore Device

    Science.gov (United States)

    Smith, Andrew M.; Abu-Shumays, Robin; Akeson, Mark; Bernick, David L.

    2015-01-01

    Transfer RNAs (tRNA) are the most common RNA molecules in cells and have critical roles as both translators of the genetic code and regulators of protein synthesis. As such, numerous methods have focused on studying tRNA abundance and regulation, with the most widely used methods being RNA-seq and microarrays. Though revolutionary to transcriptomics, these assays are limited by an inability to encode tRNA modifications in the requisite cDNA. These modifications are abundant in tRNA and critical to their function. Here, we describe proof-of-concept experiments where individual tRNA molecules are examined as linear strands using a biological nanopore. This method utilizes an enzymatically ligated synthetic DNA adapter to concentrate tRNA at the lipid bilayer of the nanopore device and efficiently denature individual tRNA molecules, as they are pulled through the α-hemolysin (α-HL) nanopore. Additionally, the DNA adapter provides a loading site for ϕ29 DNA polymerase (ϕ29 DNAP), which acts as a brake on the translocating tRNA. This increases the dwell time of adapted tRNA in the nanopore, allowing us to identify the region of the nanopore signal that is produced by the translocating tRNA itself. Using adapter-modified Escherichia coli tRNAfMet and tRNALys, we show that the nanopore signal during controlled translocation is dependent on the identity of the tRNA. This confirms that adapter-modified tRNA can translocate end-to-end through nanopores and provide the foundation for future work in direct sequencing of individual transfer RNA with a nanopore-based device. PMID:26157798

  3. Covalent stabilization of a small molecule-RNA complex.

    Science.gov (United States)

    Peacock, Hayden; Bachu, Radhika; Beal, Peter A

    2011-09-01

    We demonstrate covalent bond formation between an RNA aptamer containing a cysteamine-tethered nucleobase and helix-threading peptides (HTPs) containing α-bromoacetamide N-termini. The reaction is high yielding and inhibited by a DNA strand Watson-Crick complementary to the aptamer sequence indicating covalent reaction is dependent on the high affinity HTP-binding site present in the folded aptamer. These results are important for future structural studies of HTP-RNA complexes and methods for the discovery of new high affinity analogs via covalent tethering strategies. Copyright © 2011 Elsevier Ltd. All rights reserved.

  4. Heterobifunctional crosslinkers for tethering single ligand molecules to scanning probes

    International Nuclear Information System (INIS)

    Riener, Christian K.; Kienberger, Ferry; Hahn, Christoph D.; Buchinger, Gerhard M.; Egwim, Innocent O.C.; Haselgruebler, Thomas; Ebner, Andreas; Romanin, Christoph; Klampfl, Christian; Lackner, Bernd; Prinz, Heino; Blaas, Dieter; Hinterdorfer, Peter; Gruber, Hermann J.

    2003-01-01

    Single molecule recognition force microscopy (SMRFM) is a versatile atomic force microscopy (AFM) method to probe specific interactions of cognitive molecules on the single molecule level. It allows insights to be gained into interaction potentials and kinetic barriers and is capable of mapping interaction sites with nm positional accuracy. These applications require a ligand to be attached to the AFM tip, preferably by a distensible poly(ethylene glycol) (PEG) chain between the measuring tip and the ligand molecule. The PEG chain greatly facilitates specific binding of the ligand to immobile receptor sites on the sample surface. The present study contributes to tip-PEG-ligand tethering in three ways: (i) a convenient synthetic route was found to prepare NH 2 -PEG-COOH which is the key intermediate for long heterobifunctional crosslinkers; (ii) a variety of heterobifunctional PEG derivatives for tip-PEG-ligand linking were prepared from NH 2 -PEG-COOH; (iii) in particular, a new PEG crosslinker with one thiol-reactive end and one terminal nitrilotriacetic acid (NTA) group was synthesized and successfully used to tether His 6 -tagged protein molecules to AFM tips via noncovalent NTA-Ni 2+ -His 6 bridges. The new crosslinker was applied to link a recombinant His 6 -tagged fragment of the very-low density lipoprotein receptor to the AFM tip whereupon specific docking to the capsid of human rhinovirus particles was observed by force microscopy. In a parallel study, the specific interaction of the small GTPase Ran with the nuclear import receptor importin β1 was studied in detail by SMRFM, using the new crosslinker to link His 6 -tagged Ran to the measuring tip [Nat. Struct. Biol. (2003), 10, 553-557

  5. Single-molecule studies of unconventional motor protein myosin VI

    Science.gov (United States)

    Kim, HyeongJun

    Myosin VI is one of the myosin superfamily members that are actin-based molecular motors. It has received special attention due to its distinct features as compared to other myosins, such as its opposite directionality and a much larger step size than expected given the length of its "leg". This dissertation presents the author.s graduate work of several single-molecule studies on myosin VI. Special attention was paid to some of myosin VI.s tail domains that consist of proximal tail (PT), medial tail (MT), distal tail (DT) domains and cargo-binding domain (CBD). The functional form of myosin VI in cells is still under debate. Although full length myosin VI proteins in cytosolic extracts of cells were monomers from earlier studies, there are several reasons why it is now believed that myosin VI could exist as a dimer. If this is true and dimerization occurs, the next logical question would be which parts of myosin VI are dimerization regions? One model claimed that the CBD is the sole dimerization region. A competing model claimed that there must be another region that could be involved in dimerization, based on their observation that a construct without the CBD could still dimerize. Our single-molecule experiment with progressively truncated myosin VI constructs showed that the MT domain is a dimerization region, supporting the latter model. Additional single-molecule experiments and molecular dynamics (MD) simulation done with our collaborators suggest that electrostatic salt bridges formed between positive and negative amino acid residues are mainly responsible for the MT domain dimerization. After resolving this, we are left with another important question which is how myosin VI can take such a large step. Recent crystal structure showed that one of the tail domains preceding the MT domain, called the PT domain, is a three-helix bundle. The most easily conceivable way might be an unfolding of the three-helix bundle upon dimerization, allowing the protein to

  6. Intrinsic Properties of tRNA Molecules as Deciphered via Bayesian Network and Distribution Divergence Analysis

    Directory of Open Access Journals (Sweden)

    Sergio Branciamore

    2018-02-01

    Full Text Available The identity/recognition of tRNAs, in the context of aminoacyl tRNA synthetases (and other molecules, is a complex phenomenon that has major implications ranging from the origins and evolution of translation machinery and genetic code to the evolution and speciation of tRNAs themselves to human mitochondrial diseases to artificial genetic code engineering. Deciphering it via laboratory experiments, however, is difficult and necessarily time- and resource-consuming. In this study, we propose a mathematically rigorous two-pronged in silico approach to identifying and classifying tRNA positions important for tRNA identity/recognition, rooted in machine learning and information-theoretic methodology. We apply Bayesian Network modeling to elucidate the structure of intra-tRNA-molecule relationships, and distribution divergence analysis to identify meaningful inter-molecule differences between various tRNA subclasses. We illustrate the complementary application of these two approaches using tRNA examples across the three domains of life, and identify and discuss important (informative positions therein. In summary, we deliver to the tRNA research community a novel, comprehensive methodology for identifying the specific elements of interest in various tRNA molecules, which can be followed up by the corresponding experimental work and/or high-resolution position-specific statistical analyses.

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

    Indian Academy of Sciences (India)

    2012-06-25

    Jun 25, 2012 ... diseases particularly in viral infections like HIV, AIDS and hepatitis C has led to growing interest in RNA as a potential target for therapeutic intervention (Gallego and Varani 2001;. Foloppe et al. 2006; Liu et al. 2008; Fulle and Gohlke 2010). Furthermore, the recent discovery of a number of micro-.

  8. Single molecule transcription factor dynamics in the syncytial Drosophila embryo

    Science.gov (United States)

    Darzacq, Xavier

    During early development in the Drosophila embryo, cell fates are determined over the course of just 2 hours with exquisite spatio-temoral precision. One of the key regulators of this process is the transcription factor Bicoid which forms a concentration gradient across the long axis of the embryo. Although Bicoids' primary role is activation at the anterior, where concentrations are highest, it is also known to play a role in the posterior where there are only 100s of molecules per nucleus. Understanding how Bicoid can find its target at such low concentrations has remained intractable, largely due to the inability to perform single molecule imaging in the context of the developing embryo. Here we use lattice light sheet microscopy to overcome the technical barriers of sample thickness and auto-fluorescence to characterize the single molecule dynamics of Bicoid. We find that off-rates do not vary across the embryo and that instead the on-rates are modulated through the formation of clusters that enrich local concentration. This data is contrary to the current concentration dependent model of Bicoid function since local concentration within the nucleus is now a regulated parameter and suggests a previously unknown mechanism for regulation at extremely low concentrations.

  9. A thin permeable-membrane device for single-molecule manipulation.

    Science.gov (United States)

    Park, Chang-Young; Jacobson, David R; Nguyen, Dan T; Willardson, Sam; Saleh, Omar A

    2016-01-01

    Single-molecule manipulation instruments have unparalleled abilities to interrogate the structure and elasticity of single biomolecules. Key insights are derived by measuring the system response in varying solution conditions; yet, typical solution control strategies require imposing a direct fluid flow on the measured biomolecule that perturbs the high-sensitivity measurement and/or removes interacting molecules by advection. An alternate approach is to fabricate devices that permit solution changes by diffusion of the introduced species through permeable membranes, rather than by direct solution flow through the sensing region. Prior implementations of permeable-membrane devices are relatively thick, disallowing their use in apparatus that require the simultaneous close approach of external instrumentation from two sides, as occurs in single-molecule manipulation devices like the magnetic tweezer. Here, we describe the construction and use of a thin microfluidic device appropriate for single-molecule studies. We create a flow cell of only ∼500 μm total thickness by sandwiching glass coverslips around a thin plastic gasket and then create permeable walls between laterally separated channels in situ through photo-induced cross-linking of poly(ethylene glycol) diacrylate hydrogels. We show that these membranes permit passage of ions and small molecules (thus permitting solution equilibration in the absence of direct flow), but the membranes block the passage of larger biomolecules (thus retaining precious samples). Finally, we demonstrate the suitability of the device for high-resolution magnetic-tweezer experiments by measuring the salt-dependent folding of a single RNA hairpin under force.

  10. A thin permeable-membrane device for single-molecule manipulation

    Science.gov (United States)

    Park, Chang-Young; Jacobson, David R.; Nguyen, Dan T.; Willardson, Sam; Saleh, Omar A.

    2016-01-01

    Single-molecule manipulation instruments have unparalleled abilities to interrogate the structure and elasticity of single biomolecules. Key insights are derived by measuring the system response in varying solution conditions; yet, typical solution control strategies require imposing a direct fluid flow on the measured biomolecule that perturbs the high-sensitivity measurement and/or removes interacting molecules by advection. An alternate approach is to fabricate devices that permit solution changes by diffusion of the introduced species through permeable membranes, rather than by direct solution flow through the sensing region. Prior implementations of permeable-membrane devices are relatively thick, disallowing their use in apparatus that require the simultaneous close approach of external instrumentation from two sides, as occurs in single-molecule manipulation devices like the magnetic tweezer. Here, we describe the construction and use of a thin microfluidic device appropriate for single-molecule studies. We create a flow cell of only ˜500 μm total thickness by sandwiching glass coverslips around a thin plastic gasket and then create permeable walls between laterally separated channels in situ through photo-induced cross-linking of poly(ethylene glycol) diacrylate hydrogels. We show that these membranes permit passage of ions and small molecules (thus permitting solution equilibration in the absence of direct flow), but the membranes block the passage of larger biomolecules (thus retaining precious samples). Finally, we demonstrate the suitability of the device for high-resolution magnetic-tweezer experiments by measuring the salt-dependent folding of a single RNA hairpin under force.

  11. Single Molecule Cluster Analysis Identifies Signature Dynamic Conformations along the Splicing Pathway

    Science.gov (United States)

    Blanco, Mario R.; Martin, Joshua S.; Kahlscheuer, Matthew L.; Krishnan, Ramya; Abelson, John; Laederach, Alain; Walter, Nils G.

    2016-01-01

    The spliceosome is the dynamic RNA-protein machine responsible for faithfully splicing introns from precursor messenger RNAs (pre-mRNAs). Many of the dynamic processes required for the proper assembly, catalytic activation, and disassembly of the spliceosome as it acts on its pre-mRNA substrate remain poorly understood, a challenge that persists for many biomolecular machines. Here, we developed a fluorescence-based Single Molecule Cluster Analysis (SiMCAn) tool to dissect the manifold conformational dynamics of a pre-mRNA through the splicing cycle. By clustering common dynamic behaviors derived from selectively blocked splicing reactions, SiMCAn was able to identify signature conformations and dynamic behaviors of multiple ATP-dependent intermediates. In addition, it identified a conformation adopted late in splicing by a 3′ splice site mutant, invoking a mechanism for substrate proofreading. SiMCAn presents a novel framework for interpreting complex single molecule behaviors that should prove widely useful for the comprehensive analysis of a plethora of dynamic cellular machines. PMID:26414013

  12. Kondo effect in single-molecule magnet transistors

    Science.gov (United States)

    Gonzalez, Gabriel; Leuenberger, Michael; Mucciolo, Eduardo

    2009-03-01

    We present a careful and thorough microscopic derivation of the anisotropic Kondo Hamiltonian for single-molecule magnet (SMM) transistors. When the molecule is strongly coupled to metallic leads, we show that by applying a transverse magnetic field it is possible to topologically induce or quench the Kondo effect in the conductance of a SMM with either an integer or a half-integer spin S>1/2. This topological Kondo effect is due to the Berry-phase interference between multiple quantum tunneling paths of the spin. We calculate the renormalized Berry-phase oscillations of the two Kondo peaks as a function of a transverse magnetic field by means of the poor man's scaling approach. We illustrate our findings with the SMM Ni4, which we propose as a possible candidate for the experimental observation of the conductance oscillations.

  13. Robust Magnetic Properties of a Sublimable Single-Molecule Magnet.

    Science.gov (United States)

    Kiefl, Evan; Mannini, Matteo; Bernot, Kevin; Yi, Xiaohui; Amato, Alex; Leviant, Tom; Magnani, Agnese; Prokscha, Thomas; Suter, Andreas; Sessoli, Roberta; Salman, Zaher

    2016-06-28

    The organization of single-molecule magnets (SMMs) on surfaces via thermal sublimation is a prerequisite for the development of future devices for spintronics exploiting the richness of properties offered by these magnetic molecules. However, a change in the SMM properties due to the interaction with specific surfaces is usually observed. Here we present a rare example of an SMM system that can be thermally sublimated on gold surfaces while maintaining its intact chemical structure and magnetic properties. Muon spin relaxation and ac susceptibility measurements are used to demonstrate that, unlike other SMMs, the magnetic properties of this system in thin films are very similar to those in the bulk, throughout the full volume of the film, including regions near the metal and vacuum interfaces. These results exhibit the robustness of chemical and magnetic properties of this complex and provide important clues for the development of nanostructures based on SMMs.

  14. Low-temperature phonoemissive tunneling rates in single molecule magnets

    Science.gov (United States)

    Liu, Yun; Garg, Anupam

    2016-03-01

    Tunneling between the two lowest energy levels of single molecule magnets with Ising type anisotropy, accompanied by the emission or absorption of phonons, is considered. Quantitatively accurate calculations of the rates for such tunneling are performed for a model Hamiltonian especially relevant to the best studied example, Fe8. Two different methods are used: high-order perturbation theory in the spin-phonon interaction and the non-Ising-symmetric parts of the spin Hamiltonian, and a novel semiclassical approach based on spin-coherent-state-path-integral instantons. The methods are found to be in good quantitative agreement with other, and consistent with previous approaches to the problem. The implications of these results for magnetization of molecular solids of these molecules are discussed briefly.

  15. Single molecule study of a processivity clamp sliding on DNA

    Energy Technology Data Exchange (ETDEWEB)

    Laurence, T A; Kwon, Y; Johnson, A; Hollars, C; O?Donnell, M; Camarero, J A; Barsky, D

    2007-07-05

    Using solution based single molecule spectroscopy, we study the motion of the polIII {beta}-subunit DNA sliding clamp ('{beta}-clamp') on DNA. Present in all cellular (and some viral) forms of life, DNA sliding clamps attach to polymerases and allow rapid, processive replication of DNA. In the absence of other proteins, the DNA sliding clamps are thought to 'freely slide' along the DNA; however, the abundance of positively charged residues along the inner surface may create favorable electrostatic contact with the highly negatively charged DNA. We have performed single-molecule measurements on a fluorescently labeled {beta}-clamp loaded onto freely diffusing plasmids annealed with fluorescently labeled primers of up to 90 bases. We find that the diffusion constant for 1D diffusion of the {beta}-clamp on DNA satisfies D {le} 10{sup -14} cm{sup 2}/s, much slower than the frictionless limit of D = 10{sup -10} cm{sup 2}/s. We find that the {beta} clamp remains at the 3-foot end in the presence of E. coli single-stranded binding protein (SSB), which would allow for a sliding clamp to wait for binding of the DNA polymerase. Replacement of SSB with Human RP-A eliminates this interaction; free movement of sliding clamp and poor binding of clamp loader to the junction allows sliding clamp to accumulate on DNA. This result implies that the clamp not only acts as a tether, but also a placeholder.

  16. Characterizing single-molecule FRET dynamics with probability distribution analysis.

    Science.gov (United States)

    Santoso, Yusdi; Torella, Joseph P; Kapanidis, Achillefs N

    2010-07-12

    Probability distribution analysis (PDA) is a recently developed statistical tool for predicting the shapes of single-molecule fluorescence resonance energy transfer (smFRET) histograms, which allows the identification of single or multiple static molecular species within a single histogram. We used a generalized PDA method to predict the shapes of FRET histograms for molecules interconverting dynamically between multiple states. This method is tested on a series of model systems, including both static DNA fragments and dynamic DNA hairpins. By fitting the shape of this expected distribution to experimental data, the timescale of hairpin conformational fluctuations can be recovered, in good agreement with earlier published results obtained using different techniques. This method is also applied to studying the conformational fluctuations in the unliganded Klenow fragment (KF) of Escherichia coli DNA polymerase I, which allows both confirmation of the consistency of a simple, two-state kinetic model with the observed smFRET distribution of unliganded KF and extraction of a millisecond fluctuation timescale, in good agreement with rates reported elsewhere. We expect this method to be useful in extracting rates from processes exhibiting dynamic FRET, and in hypothesis-testing models of conformational dynamics against experimental data.

  17. Bright photoactivatable fluorophores for single-molecule imaging.

    Science.gov (United States)

    Grimm, Jonathan B; English, Brian P; Choi, Heejun; Muthusamy, Anand K; Mehl, Brian P; Dong, Peng; Brown, Timothy A; Lippincott-Schwartz, Jennifer; Liu, Zhe; Lionnet, Timothée; Lavis, Luke D

    2016-12-01

    Small-molecule fluorophores are important tools for advanced imaging experiments. We previously reported a general method to improve small, cell-permeable fluorophores which resulted in the azetidine-containing 'Janelia Fluor' (JF) dyes. Here, we refine and extend the utility of these dyes by synthesizing photoactivatable derivatives that are compatible with live-cell labeling strategies. Once activated, these derived compounds retain the superior brightness and photostability of the JF dyes, enabling improved single-particle tracking and facile localization microscopy experiments.

  18. Tunneling anisotropic magnetoresistance in single-molecule magnet junctions

    Science.gov (United States)

    Xie, Haiqing; Wang, Qiang; Jiao, Hujun; Liang, J.-Q.

    2012-08-01

    We theoretically investigate quantum transport through single-molecule magnet (SMM) junctions with ferromagnetic and normal-metal leads in the sequential regime. The current obtained by means of the rate-equation gives rise to the tunneling anisotropic magnetoresistance (TAMR), which varies with the angle between the magnetization direction of ferromagnetic lead and the easy axis of SMM. The angular dependence of TAMR can serve as a probe to determine experimentally the easy axis of SMM. Moreover, it is demonstrated that both the magnitude and the sign of TAMR are tunable by the bias voltage, suggesting a new spin-valve device with only one magnetic electrode in molecular spintronics.

  19. Berry-Phase Blockade in Single-Molecule Magnets

    Science.gov (United States)

    González, Gabriel; Leuenberger, Michael N.

    2007-06-01

    We formulate the problem of electron transport through a single-molecule magnet (SMM) in the Coulomb blockade regime taking into account topological interference effects for the tunneling of the large spin of a SMM. The interference originates from spin Berry phases associated with different tunneling paths. We show that, in the case of incoherent spin states, it is essential to place the SMM between oppositely spin-polarized source and drain leads in order to detect the spin tunneling in the stationary current, which exhibits topological zeros as a function of the transverse magnetic field.

  20. Nonequilibrium Energetics of Single Molecule Motor, Kinesin-1

    Science.gov (United States)

    Ariga, Takayuki; Tomishige, Michio; Mizuno, Daisuke

    2018-02-01

    Molecular motors are nonequilibrium open systems that convert chemical energy to mechanical work. Here we investigate the nonequilibrium energetics of a single molecule kinesin by measuring the motion of an attached probe particle and its response to external forces with optical tweezers. The sum of the heat dissipation estimated from the violation of the fluctuation-response relation and the output power was inconsistent with the input free energy rate, implying that internal dissipation is dominant. By using a two-state Markov model, we discuss the energy flow of the kinesin motor.

  1. Single-molecule chemical reactions on DNA origami

    DEFF Research Database (Denmark)

    Voigt, Niels Vinther; Tørring, Thomas; Rotaru, Alexandru

    2010-01-01

    DNA nanotechnology and particularly DNA origami, in which long, single-stranded DNA molecules are folded into predetermined shapes, can be used to form complex self-assembled nanostructures. Although DNA itself has limited chemical, optical or electronic functionality, DNA nanostructures can serve...... on a DNA origami scaffold by atomic force microscopy. The high yields and chemoselectivities of successive cleavage and bond-forming reactions observed in these experiments demonstrate the feasibility of post-assembly chemical modification of DNA nanostructures and their potential use as locally...

  2. The Single-Molecule Centroid Localization Algorithm Improves the Accuracy of Fluorescence Binding Assays.

    Science.gov (United States)

    Hua, Boyang; Wang, Yanbo; Park, Seongjin; Han, Kyu Young; Singh, Digvijay; Kim, Jin H; Cheng, Wei; Ha, Taekjip

    2018-03-13

    Here, we demonstrate that the use of the single-molecule centroid localization algorithm can improve the accuracy of fluorescence binding assays. Two major artifacts in this type of assay, i.e., nonspecific binding events and optically overlapping receptors, can be detected and corrected during analysis. The effectiveness of our method was confirmed by measuring two weak biomolecular interactions, the interaction between the B1 domain of streptococcal protein G and immunoglobulin G and the interaction between double-stranded DNA and the Cas9-RNA complex with limited sequence matches. This analysis routine requires little modification to common experimental protocols, making it readily applicable to existing data and future experiments.

  3. Identifying fluorescently labeled single molecules in image stacks using machine learning.

    Science.gov (United States)

    Rifkin, Scott A

    2011-01-01

    In the past several years, a host of new technologies have made it possible to visualize single molecules within cells and organisms (Raj et al., Nat Methods 5:877-879, 2008; Paré et al., Curr Biol 19:2037-2042, 2009; Lu and Tsourkas, Nucleic Acids Res 37:e100, 2009; Femino et al., Science 280:585-590, 1998; Rodriguez et al., Semin Cell Dev Biol 18:202-208, 2007; Betzig et al., Science 313:1642-1645, 2006; Rust et al., Nat Methods 3:793-796, 2006; Fusco et al., Curr Biol 13:161-167, 2003). Many of these are based on fluorescence, either fluorescent proteins or fluorescent dyes coupled to a molecule of interest. In many applications, the fluorescent signal is limited to a few pixels, which poses a classic signal processing problem: how can actual signal be distinguished from background noise? In this chapter, I present a MATLAB (MathWorks (2010) MATLAB. Retrieved from http://www.mathworks.com) software suite designed to work with these single-molecule visualization technologies (Rifkin (2010) spotFinding Suite. http://www.biology.ucsd.edu/labs/rifkin/software.html). It takes images or image stacks from a fluorescence microscope as input and outputs locations of the molecules. Although the software was developed for the specific application of identifying single mRNA transcripts in fixed specimens, it is more general than this and can be used and/or customized for other applications that produce localized signals embedded in a potentially noisy background. The analysis pipeline consists of the following steps: (a) create a gold-standard dataset, (b) train a machine-learning algorithm to classify image features as signal or noise depending upon user defined statistics, (c) run the machine-learning algorithm on a new dataset to identify mRNA locations, and (d) visually inspect and correct the results.

  4. Non-fluorescent Quenchers to Correlate Single-Molecule Conformational and Compositional Dynamics

    Science.gov (United States)

    Chen, Jin; Tsai, Albert; Petrov, Alexey; Puglisi, Joseph D.

    2015-01-01

    Single-molecule Förster resonance energy transfer (smFRET) is a powerful method to study the conformational dynamics of a biomolecule in real-time. However, studying how interacting ligands correlate with and regulate the conformational dynamics of the biomolecule is extremely challenging due to the availability of a limited number of fluorescent dyes with both high quantum yield and minimal spectral overlap. Here, we report the use of a non-fluorescent quencher (Black Hole Quencher, BHQ) as an acceptor for smFRET. Using a Cy3/BHQ pair, we accurately follow conformational changes of the ribosome during elongation in real time. We demonstrate the application of single-color FRET to correlate the conformational dynamics of the ribosome with the compositional dynamics of tRNA. We use the normal Cy5 FRET acceptor to observe arrival of a fluorescently-labeled tRNA with a concomitant transition of the ribosome from the locked to the unlocked conformation. Our results illustrate the potential of non-fluorescent quenchers in single-molecule correlation studies. PMID:22428667

  5. Real-time tRNA transit on single translating ribosomes at codon resolution

    Science.gov (United States)

    Uemura, Sotaro; Aitken, Colin Echeverría; Korlach, Jonas; Flusberg, Benjamin A.; Turner, Stephen W.; Puglisi, Joseph D.

    2015-01-01

    Translation by the ribosome occurs by a complex mechanism involving the coordinated interaction of multiple nucleic acid and protein ligands. Here we have used zero-mode waveguides (ZMWs) and sophisticated detection instrumentation to allow real-time observation of translation at physiologically-relevant (μM) ligand concentrations. Translation at each codon is monitored by stable binding of tRNAs – labeled with distinct fluorophores – to translating ribosomes, allowing direct detection of the identity of tRNA molecules bound to the ribosome, and therefore, the underlying mRNA sequence. We observe the transit of tRNAs on single translating ribosomes and have determined the number of tRNA molecules simultaneously bound to the ribosome, at each codon of an mRNA. Our results show that ribosomes are only briefly occupied by two tRNAs and that release of deacylated tRNA from the E site is uncoupled from binding of A-site tRNA and occurs rapidly after translocation. The methods outlined here have broad application to the study of mRNA sequences, and the mechanism and regulation of translation. PMID:20393556

  6. Complexities due to single-stranded RNA during antibody detection of genomic rna:dna hybrids.

    Science.gov (United States)

    Zhang, Zheng Z; Pannunzio, Nicholas R; Hsieh, Chih-Lin; Yu, Kefei; Lieber, Michael R

    2015-04-08

    Long genomic R-loops in eukaryotes were first described at the immunoglobulin heavy chain locus switch regions using bisulfite sequencing and functional studies. A mouse monoclonal antibody called S9.6 has been used for immunoprecipitation (IP) to identify R-loops, based on the assumption that it is specific for RNA:DNA over other nucleic acid duplexes. However, recent work has demonstrated that a variable domain of S9.6 binds AU-rich RNA:RNA duplexes with a KD that is only 5.6-fold weaker than for RNA:DNA duplexes. Most IP protocols do not pre-clear the genomic nucleic acid with RNase A to remove free RNA. Fold back of ssRNA can readily generate RNA:RNA duplexes that may bind the S9.6 antibody, and adventitious binding of RNA may also create short RNA:DNA regions. Here we investigate whether RNase A is needed to obtain reliable IP with S9.6. As our test locus, we chose the most well-documented site for kilobase-long mammalian genomic R-loops, the immunoglobulin heavy chain locus (IgH) class switch regions. The R-loops at this locus can be induced by using cytokines to stimulate transcription from germline transcript promoters. We tested IP using S9.6 with and without various RNase treatments. The RNase treatments included RNase H to destroy the RNA in an RNA:DNA duplex and RNase A to destroy single-stranded (ss) RNA to prevent it from binding S9.6 directly (as duplex RNA) and to prevent the ssRNA from annealing to the genome, resulting in adventitious RNA:DNA hybrids. We find that optimal detection of RNA:DNA duplexes requires removal of ssRNA using RNase A. Without RNase A treatment, known regions of R-loop formation containing RNA:DNA duplexes can not be reliably detected. With RNase A treatment, a signal can be detected over background, but only within a limited 2 or 3-fold range, even with a stable kilobase-long genomic R-loop. Any use of the S9.6 antibody must be preceded by RNase A treatment to remove free ssRNA that may compete for the S9.6 binding by

  7. Magnetic Quantum Tunneling and Symmetry in Single Molecule Magnets

    Science.gov (United States)

    Kent, Andrew D.

    2003-03-01

    We have studied the symmetry of magnetic quantum tunneling (MQT) in single molecule magnets (SMMs) using a micro-Hall effect magnetometer and high field vector superconducting magnet system. In the most widely studied SMM, Mn12-acetate, an average crystal 4-fold symmetry in the magnetic response is shown to be due to local molecular environments of 2-fold symmetry that are rotated by 90 degrees with respect to one another. We attribute this to ligand disorder that leads to local rhombic distortions, a model first proposed by Cornia et al. based on x-ray diffraction data [1]. We have magnetically distilled a Mn12-acetate crystal to study a subset of these lower (2-fold) site symmetry molecules and present evidence for a spin-parity effect consistent with a local 2-fold symmetry [2]. These results highlight the importance of subtle changes in molecule environment in modulating magnetic anisotropy and MQT. [1] Cornia et al. Phys. Rev. Lett. 89, 257201 (2002) [2] E. del Barco, A. D. Kent, E. Rumberger, D. H. Hendrickson, G. Christou, submitted for publication (2002) and Europhys. Lett. 60, 768 (2002)

  8. Single molecule transistor based nanopore for the detection of nicotine

    Science.gov (United States)

    Ray, S. J.

    2014-12-01

    A nanopore based detection methodology was proposed and investigated for the detection of Nicotine. This technique uses a Single Molecular Transistor working as a nanopore operational in the Coulomb Blockade regime. When the Nicotine molecule is pulled through the nanopore area surrounded by the Source(S), Drain (D), and Gate electrodes, the charge stability diagram can detect the presence of the molecule and is unique for a specific molecular structure. Due to the weak coupling between the different electrodes which is set by the nanopore size, the molecular energy states stay almost unaffected by the electrostatic environment that can be realised from the charge stability diagram. Identification of different orientation and position of the Nicotine molecule within the nanopore area can be made from specific regions of overlap between different charge states on the stability diagram that could be used as an electronic fingerprint for detection. This method could be advantageous and useful to detect the presence of Nicotine in smoke which is usually performed using chemical chromatography techniques.

  9. Single molecule transistor based nanopore for the detection of nicotine

    Energy Technology Data Exchange (ETDEWEB)

    Ray, S. J., E-mail: ray.sjr@gmail.com [Institute of Materials Science, Technical University of Darmstadt, Alarich-Weiss-Str. 2, 64287 Darmstadt (Germany)

    2014-12-28

    A nanopore based detection methodology was proposed and investigated for the detection of Nicotine. This technique uses a Single Molecular Transistor working as a nanopore operational in the Coulomb Blockade regime. When the Nicotine molecule is pulled through the nanopore area surrounded by the Source(S), Drain (D), and Gate electrodes, the charge stability diagram can detect the presence of the molecule and is unique for a specific molecular structure. Due to the weak coupling between the different electrodes which is set by the nanopore size, the molecular energy states stay almost unaffected by the electrostatic environment that can be realised from the charge stability diagram. Identification of different orientation and position of the Nicotine molecule within the nanopore area can be made from specific regions of overlap between different charge states on the stability diagram that could be used as an electronic fingerprint for detection. This method could be advantageous and useful to detect the presence of Nicotine in smoke which is usually performed using chemical chromatography techniques.

  10. Single-Molecule Electrochemical Gating in Ionic Liquids

    DEFF Research Database (Denmark)

    Kay, Nicola J.; Higgins, Simon J.; Jeppesen, Jan O.

    2012-01-01

    The single-molecular conductance of a redox active molecular bridge has been studied in an electrochemical single-molecule transistor configuration in a room-temperature ionic liquid (RTIL). The redox active pyrrolo-tetrathiafulvalene (pTTF) moiety was attached to gold contacts at both ends through...... and decreases again as the second redox process is passed. This is described as an “off–on–off–on–off” conductance switching behavior. This molecular conductance vs electrochemical potential relation could be modeled well as a sequential two-step charge transfer process with full or partial vibrational...... relaxation. Using this view, reorganization energies of ∼1.2 eV have been estimated for both the first and second redox transitions for the pTTF bridge in the 1-butyl-3-methylimidazolium trifluoromethanesulfonate (BMIOTf) ionic liquid environment. By contrast, in aqueous environments, a much smaller...

  11. Enhancing Single Molecule Imaging in Optofluidics and Microfluidics

    Directory of Open Access Journals (Sweden)

    Andreas E. Vasdekis

    2011-08-01

    Full Text Available Microfluidics and optofluidics have revolutionized high-throughput analysis and chemical synthesis over the past decade. Single molecule imaging has witnessed similar growth, due to its capacity to reveal heterogeneities at high spatial and temporal resolutions. However, both resolution types are dependent on the signal to noise ratio (SNR of the image. In this paper, we review how the SNR can be enhanced in optofluidics and microfluidics. Starting with optofluidics, we outline integrated photonic structures that increase the signal emitted by single chromophores and minimize the excitation volume. Turning then to microfluidics, we review the compatible functionalization strategies that reduce noise stemming from non-specific interactions and architectures that minimize bleaching and blinking.

  12. Photoinduced nuclear spin conversion of methyl groups of single molecules

    International Nuclear Information System (INIS)

    Sigl, A.

    2007-01-01

    A methyl group is an outstanding quantum system due to its special symmetry properties. The threefold rotation around one of its bond is isomorphic to the group of even permutations of the remaining protons, a property which imposes severe quantum restrictions on the system, for instance a strict correlation of rotational states with nuclear spin states. The resulting long lifetimes of the rotational tunneling states of the methyl group can be exploited for applying certain high resolution optical techniques, like hole burning or single molecule spectroscopy to optically switch the methyl group from one tunneling state to another therebye changing the nuclear spin of the protons. One goal of the thesis was to perform this switching in single methyl groups. To this end the methyl group was attached to a chromophoric system, in the present case terrylene, which is well suited for single molecule spectroscopy as well as for hole burning. Experiments were performed with the bare terrylene molecule in a hexadecane lattice which served as a reference system, with alphamethyl terrylene and betamethyl terrylene, both embedded in hexadecane, too. A single molecular probe is a highly sensitive detector for dynamic lattice instabilities. Already the bare terrylene probe showed a wealth of interesting local dynamic effects of the hexadecane lattice which could be well acounted for by the assumption of two nearly degenerate sites with rather different optical and thermal properties, all of which could be determined in a quantitative fashion. As to the methylated terrylene systems, the experiments verified that for betamethyl terrylene it is indeed possible to measure rotational tunneling events in single methyl groups. However, the spectral patterns obtained was much more complicated than expected pointing to the presence of three spectroscopically different methyl groups. In order to achieve a definite assignement, molecular mechanics simulations of the terrylene probes in the

  13. Compact quantum dots for single-molecule imaging.

    Science.gov (United States)

    Smith, Andrew M; Nie, Shuming

    2012-10-09

    Single-molecule imaging is an important tool for understanding the mechanisms of biomolecular function and for visualizing the spatial and temporal heterogeneity of molecular behaviors that underlie cellular biology (1-4). To image an individual molecule of interest, it is typically conjugated to a fluorescent tag (dye, protein, bead, or quantum dot) and observed with epifluorescence or total internal reflection fluorescence (TIRF) microscopy. While dyes and fluorescent proteins have been the mainstay of fluorescence imaging for decades, their fluorescence is unstable under high photon fluxes necessary to observe individual molecules, yielding only a few seconds of observation before complete loss of signal. Latex beads and dye-labeled beads provide improved signal stability but at the expense of drastically larger hydrodynamic size, which can deleteriously alter the diffusion and behavior of the molecule under study. Quantum dots (QDs) offer a balance between these two problematic regimes. These nanoparticles are composed of semiconductor materials and can be engineered with a hydrodynamically compact size with exceptional resistance to photodegradation (5). Thus in recent years QDs have been instrumental in enabling long-term observation of complex macromolecular behavior on the single molecule level. However these particles have still been found to exhibit impaired diffusion in crowded molecular environments such as the cellular cytoplasm and the neuronal synaptic cleft, where their sizes are still too large (4,6,7). Recently we have engineered the cores and surface coatings of QDs for minimized hydrodynamic size, while balancing offsets to colloidal stability, photostability, brightness, and nonspecific binding that have hindered the utility of compact QDs in the past (8,9). The goal of this article is to demonstrate the synthesis, modification, and characterization of these optimized nanocrystals, composed of an alloyed HgxCd1-xSe core coated with an

  14. High sensitivity fluorescent single particle and single molecule detection apparatus and method

    Science.gov (United States)

    Mathies, Richard A.; Peck, Konan; Stryer, Lubert

    1990-01-01

    Apparatus is described for ultrasensitive detection of single fluorescent particles down to the single fluorescent molecule limit in a fluid or on a substrate comprising means for illuminating a predetermined volume of the fluid or area of the substrate whereby to emit light including background light from the fluid and burst of photons from particles residing in the area. The photon burst is detected in real time to generate output representative signal. The signal is received and the burst of energy from the fluorescent particles is distinguished from the background energy to provide an indication of the number, location or concentration of the particles or molecules.

  15. Analyzing Single Molecule Localization Microscopy Data Using Cubic Splines.

    Science.gov (United States)

    Babcock, Hazen P; Zhuang, Xiaowei

    2017-04-03

    The resolution of super-resolution microscopy based on single molecule localization is in part determined by the accuracy of the localization algorithm. In most published approaches to date this localization is done by fitting an analytical function that approximates the point spread function (PSF) of the microscope. However, particularly for localization in 3D, analytical functions such as a Gaussian, which are computationally inexpensive, may not accurately capture the PSF shape leading to reduced fitting accuracy. On the other hand, analytical functions that can accurately capture the PSF shape, such as those based on pupil functions, can be computationally expensive. Here we investigate the use of cubic splines as an alternative fitting approach. We demonstrate that cubic splines can capture the shape of any PSF with high accuracy and that they can be used for fitting the PSF with only a 2-3x increase in computation time as compared to Gaussian fitting. We provide an open-source software package that measures the PSF of any microscope and uses the measured PSF to perform 3D single molecule localization microscopy analysis with reasonable accuracy and speed.

  16. Dual-Colored DNA Comb Polymers for Single Molecule Rheology

    Science.gov (United States)

    Mai, Danielle; Marciel, Amanda; Schroeder, Charles

    2014-03-01

    We report the synthesis and characterization of branched biopolymers for single molecule rheology. In our work, we utilize a hybrid enzymatic-synthetic approach to graft ``short'' DNA branches to ``long'' DNA backbones, thereby producing macromolecular DNA comb polymers. The branches and backbones are synthesized via polymerase chain reaction with chemically modified deoxyribonucleotides (dNTPs): ``short'' branches consist of Cy5-labeled dNTPs and a terminal azide group, and ``long'' backbones contain dibenzylcyclooctyne-modified (DBCO) dNTPs. In this way, we utilize strain-promoted, copper-free cycloaddition ``click'' reactions for facile grafting of azide-terminated branches at DBCO sites along backbones. Copper-free click reactions are bio-orthogonal and nearly quantitative when carried out under mild conditions. Moreover, comb polymers can be labeled with an intercalating dye (e.g., YOYO) for dual-color fluorescence imaging. We characterized these materials using gel electrophoresis, HPLC, and optical microscopy, with atomic force microscopy in progress. Overall, DNA combs are suitable for single molecule dynamics, and in this way, our work holds the potential to improve our understanding of topologically complex polymer melts and solutions.

  17. Single-molecule mechanics of protein-labelled DNA handles

    Directory of Open Access Journals (Sweden)

    Vivek S. Jadhav

    2016-01-01

    Full Text Available DNA handles are often used as spacers and linkers in single-molecule experiments to isolate and tether RNAs, proteins, enzymes and ribozymes, amongst other biomolecules, between surface-modified beads for nanomechanical investigations. Custom DNA handles with varying lengths and chemical end-modifications are readily and reliably synthesized en masse, enabling force spectroscopic measurements with well-defined and long-lasting mechanical characteristics under physiological conditions over a large range of applied forces. Although these chemically tagged DNA handles are widely used, their further individual modification with protein receptors is less common and would allow for additional flexibility in grabbing biomolecules for mechanical measurements. In-depth information on reliable protocols for the synthesis of these DNA–protein hybrids and on their mechanical characteristics under varying physiological conditions are lacking in literature. Here, optical tweezers are used to investigate different protein-labelled DNA handles in a microfluidic environment under different physiological conditions. Digoxigenin (DIG-dsDNA-biotin handles of varying sizes (1000, 3034 and 4056 bp were conjugated with streptavidin or neutravidin proteins. The DIG-modified ends of these hybrids were bound to surface-modified polystyrene (anti-DIG beads. Using different physiological buffers, optical force measurements showed consistent mechanical characteristics with long dissociation times. These protein-modified DNA hybrids were also interconnected in situ with other tethered biotinylated DNA molecules. Electron-multiplying CCD (EMCCD imaging control experiments revealed that quantum dot–streptavidin conjugates at the end of DNA handles remain freely accessible. The experiments presented here demonstrate that handles produced with our protein–DNA labelling procedure are excellent candidates for grasping single molecules exposing tags suitable for molecular

  18. Topoisomerase I as a Biomarker: Detection of Activity at the Single Molecule Level

    DEFF Research Database (Denmark)

    Proszek, Joanna; Roy, Amit; Jakobsen, Ann-Katrine

    2014-01-01

    of hTopI cleavage-religation activity at the single molecule level, may be used to detect posttranslational enzymatic differences influencing CPT response. These differences cannot be detected by analysis of hTopI gene copy number, mRNA amount, or protein amount, and only become apparent upon measuring......Human topoisomerase I (hTopI) is an essential cellular enzyme. The enzyme is often upregulated in cancer cells, and it is a target for chemotherapeutic drugs of the camptothecin (CPT) family. Response to CPT-based treatment is dependent on hTopI activity, and reduction in activity, and mutations...... in hTopI have been reported to result in CPT resistance. Therefore, hTOPI gene copy number, mRNA level, protein amount, and enzyme activity have been studied to explain differences in cellular response to CPT. We show that Rolling Circle Enhanced Enzyme Activity Detection (REEAD), allowing measurement...

  19. Atomic force microscope observation of branching in single transcript molecules derived from human cardiac muscle

    Science.gov (United States)

    Reed, Jason; Hsueh, Carlin; Mishra, Bud; Gimzewski, James K.

    2008-09-01

    We have used an atomic force microscope to examine a clinically derived sample of single-molecule gene transcripts, in the form of double-stranded cDNA, (c: complementary) obtained from human cardiac muscle without the use of polymerase chain reaction (PCR) amplification. We observed a log-normal distribution of transcript sizes, with most molecules being in the range of 0.4-7.0 kilobase pairs (kb) or 130-2300 nm in contour length, in accordance with the expected distribution of mRNA (m: messenger) sizes in mammalian cells. We observed novel branching structures not previously known to exist in cDNA, and which could have profound negative effects on traditional analysis of cDNA samples through cloning, PCR and DNA sequencing.

  20. 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

  1. Post-transcriptional bursting in genes regulated by small RNA molecules

    Science.gov (United States)

    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.

  2. 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...

  3. Single-cell sequencing of the small-RNA transcriptome.

    Science.gov (United States)

    Faridani, Omid R; Abdullayev, Ilgar; Hagemann-Jensen, Michael; Schell, John P; Lanner, Fredrik; Sandberg, Rickard

    2016-12-01

    Little is known about the heterogeneity of small-RNA expression as small-RNA profiling has so far required large numbers of cells. Here we present a single-cell method for small-RNA sequencing and apply it to naive and primed human embryonic stem cells and cancer cells. Analysis of microRNAs and fragments of tRNAs and small nucleolar RNAs (snoRNAs) reveals the potential of microRNAs as markers for different cell types and states.

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

    Science.gov (United States)

    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.

  5. Tetramethylammonium-filled protein nanopore for single-molecule analysis.

    Science.gov (United States)

    Wang, Ying; Yao, Fujun; Kang, Xiao-feng

    2015-10-06

    Nanopore technology, as the simplest and most inexpensive single-molecule tool, is being intensively developed. In nanopore stochastic sensing, KCl and NaCl have traditionally been employed as pore-filled electrolytes for recording the change of ion conductance in nanopores triggered by analyte translocation through the pore. However, some challenges limit its further advance. Here we used tetramethylammonium (TMA) chloride, instead of KCl, as a novel analysis system for nanopores. Some unique nanopore characteristics were observed: (1) The stability of the planar lipid bilayer for embedding the protein pores was elevated at least 6 times. (2) The TMA-Cl system could effectively reduce the noise of single-channel recording. (3) It was easy to control the insertion of protein pores into the lipid bilayer, and the formed single nanopore could last for a sufficiently long time. (4) TMA-Cl could be used as a DNA speed bump in the nanopore to slow DNA translocation speed. (5) The capacity of the nanopore capture of DNA (capture rate) increased significantly and simultaneously increased the translocation time of DNA in the pore. (6) The TMA-filled nanopore could discriminate between various polynucleotides.

  6. Simultaneous Multiplexed Measurement of RNA and Proteins in Single Cells

    Directory of Open Access Journals (Sweden)

    Spyros Darmanis

    2016-01-01

    Full Text Available Significant advances have been made in methods to analyze genomes and transcriptomes of single cells, but to fully define cell states, proteins must also be accessed as central actors defining a cell’s phenotype. Methods currently used to analyze endogenous protein expression in single cells are limited in specificity, throughput, or multiplex capability. Here, we present an approach to simultaneously and specifically interrogate large sets of protein and RNA targets in lysates from individual cells, enabling investigations of cell functions and responses. We applied our method to investigate the effects of BMP4, an experimental therapeutic agent, on early-passage glioblastoma cell cultures. We uncovered significant heterogeneity in responses to treatment at levels of RNA and protein, with a subset of cells reacting in a distinct manner to BMP4. Moreover, we found overall poor correlation between protein and RNA at the level of single cells, with proteins more accurately defining responses to treatment.

  7. Photothermal cantilever actuation for fast single-molecule force spectroscopy

    Science.gov (United States)

    Stahl, Stefan W.; Puchner, Elias M.; Gaub, Hermann E.

    2009-07-01

    Photothermal cantilever excitation provides a fast and easy to implement means to control the deflection of standard atomic force microscopy cantilevers. Minute heat pulses yield deflections on the order of several tens of nanometers or when the deflection is kept constant, forces of several hundreds of piconewton can be applied. In our case these pulses resulted in less than 1 K temperature changes at the sample position. Here we present and characterize the implementation of photothermal actuation for single-molecule force-spectroscopy experiments. When molecules are stretched under force-clamp conditions, fast control cycles that re-establish the pulling force after the rupture of molecular domains are essential for detecting the complete unfolding pattern with high precision. By combining the fast response of photothermal cantilever excitation with a conventional piezoactuator, a fast force-clamp with high accuracy and large working distances is reached. Simple feedback mechanisms and standard cantilever geometries lead to step response times of less than 90 μs, which is more than one order of magnitude faster than those of conventional force-clamp systems that are based only on piezo feedback. We demonstrate the fast and accurate performance of the setup by unfolding a protein construct consisting of one green fluorescent protein and eight surrounding immunoglobulin domains at constant force.

  8. Mechanisms of Cellular Proteostasis: Insights from Single-Molecule Approaches

    Science.gov (United States)

    Bustamante, Carlos J.; Kaiser, Christian M.; Maillard, Rodrigo A.; Goldman, Daniel H.; Wilson, Christian A.M.

    2015-01-01

    Cells employ a variety of strategies to maintain proteome homeostasis. Beginning during protein biogenesis, the translation machinery and a number of molecular chaperones promote correct de novo folding of nascent proteins even before synthesis is complete. Another set of molecular chaperones helps to maintain proteins in their functional, native state. Polypeptides that are no longer needed or pose a threat to the cell, such as misfolded proteins and aggregates, are removed in an efficient and timely fashion by ATP-dependent proteases. In this review, we describe how applications of single-molecule manipulation methods, in particular optical tweezers, are shedding new light on the molecular mechanisms of quality control during the life cycles of proteins. PMID:24895851

  9. Quantum turnstile operation of single-molecule magnets

    International Nuclear Information System (INIS)

    Moldoveanu, V; Dinu, I V; Tanatar, B; Moca, C P

    2015-01-01

    The time-dependent transport through single-molecule magnets coupled to magnetic or non-magnetic electrodes is studied in the framework of the generalized master equation method. We investigate the transient regime induced by the periodic switching of the source and drain contacts. If the electrodes have opposite magnetizations the quantum turnstile operation allows the stepwise writing of intermediate excited states. In turn, the transient currents provide a way to read these states. Within our approach we take into account both the uniaxial and transverse anisotropy. The latter may induce additional quantum tunneling processes which affect the efficiency of the proposed read-and-write scheme. An equally weighted mixture of molecular spin states can be prepared if one of the electrodes is ferromagnetic. (paper)

  10. Spin interactions in Graphene-Single Molecule Magnets Hybrids

    Science.gov (United States)

    Cervetti, Christian; Rettori, Angelo; Pini, Maria Gloria; Cornia, Andrea; Repollés, Aña; Luis, Fernando; Rauschenbach, Stephan; Dressel, Martin; Kern, Klaus; Burghard, Marko; Bogani, Lapo

    2014-03-01

    Graphene is a potential component of novel spintronics devices owing to its long spin diffusion length. Besides its use as spin-transport channel, graphene can be employed for the detection and manipulation of molecular spins. This requires an appropriate coupling between the sheets and the single molecular magnets (SMM). Here, we present a comprehensive characterization of graphene-Fe4 SMM hybrids. The Fe4 clusters are anchored non-covalently to the graphene following a diffusion-limited assembly and can reorganize into random networks when subjected to slightly elevated temperature. Molecules anchored on graphene sheets show unaltered static magnetic properties, whilst the quantum dynamics is profoundly modulated. Interaction with Dirac fermions becomes the dominant spin-relaxation channel, with observable effects produced by graphene phonons and reduced dipolar interactions. Coupling to graphene drives the spins over Villain's threshold, allowing the first observation of strongly-perturbative tunneling processes. Preliminary spin-transport experiments at low-temperature are further presented.

  11. Quantum Tunneling of Magnetization in Trigonal Single-Molecule Magnets

    Science.gov (United States)

    Liu, Junjie; Del Barco, Enrique; Hill, Stephen

    2012-02-01

    We perform a numerical analysis of the quantum tunneling of magnetization (QTM) that occurs in a spin S = 6 single-molecule magnet (SMM) with idealized C3 symmetry. The deconstructive points in the QTM are located by following the Berry-phase interference (BPI) oscillations. We find that the O4^3 (=12[Sz,S+^3 +S-^3 ]) operator unfreezes odd-k QTM resonances and generates three-fold patterns of BPI minima in all resonances, including k = 0! This behavior cannot be reproduced with operators that possess even rotational symmetry about the quantization axis. We find also that the k = 0 BPI minima shift away from zero longitudinal field. The wider implications of these results will be discussed in terms of the QTM behavior observed in other SMMs.

  12. A Low Spin Manganese(IV) Nitride Single Molecule Magnet.

    Science.gov (United States)

    Ding, Mei; Cutsail, George E; Aravena, Daniel; Amoza, Martín; Rouzières, Mathieu; Dechambenoit, Pierre; Losovyj, Yaroslav; Pink, Maren; Ruiz, Eliseo; Clérac, Rodolphe; Smith, Jeremy M

    2016-09-01

    Structural, spectroscopic and magnetic methods have been used to characterize the tris(carbene)borate compound PhB(MesIm) 3 Mn≡N as a four-coordinate manganese(IV) complex with a low spin ( S = 1/2) configuration. The slow relaxation of the magnetization in this complex, i.e. its single-molecule magnet (SMM) properties, is revealed under an applied dc field. Multireference quantum mechanical calculations indicate that this SMM behavior originates from an anisotropic ground doublet stabilized by spin-orbit coupling. Consistent theoretical and experiment data show that the resulting magnetization dynamics in this system is dominated by ground state quantum tunneling, while its temperature dependence is influenced by Raman relaxation.

  13. Spin thermoelectric effects in organic single-molecule devices

    Energy Technology Data Exchange (ETDEWEB)

    Wang, H.L.; Wang, M.X.; Qian, C.; Hong, X.K.; Zhang, D.B.; Liu, Y.S.; Yang, X.F., E-mail: xfyang@cslg.edu.cn

    2017-05-25

    Highlights: • A stronger spin thermoelectric performance in a polyacetylene device is observed. • For the antiferromagnetic (AFM) ordering, a transport gap is opened. Thus the thermoelectric effects are largely enhanced. - Abstract: The spin thermoelectric performance of a polyacetylene chain bridging two zigzag graphene nanoribbons (ZGNRs) is investigated based on first principles method. Two different edge spin arrangements in ZGNRs are considered. For ferromagnetic (FM) ordering, transmission eigenstates with different spin indices distributed below and above Fermi level are observed, leading directly to a strong spin thermoelectric effect in a wide temperature range. With the edge spins arranged in the antiferromagnetic (AFM) ordering, an obvious transport gap appears in the system, which greatly enhances the thermoelectric effects. The presence of a small spin splitting also induces a spin thermoelectric effect greater than the charge thermoelectric effect in certain temperature range. In general, the single-molecule junction exhibits the potential to be used for the design of perfect thermospin devices.

  14. Nanofabrication of SERS Substrates for Single/Few Molecules Detection

    KAUST Repository

    Melino, Gianluca

    2015-05-04

    Raman spectroscopy is among the most widely employed methods to investigate the properties of materials in several fields of study. Evolution in materials science allowed us to fabricate suitable substrates, at the nanoscale, capable to enhance the electromagnetic field of the signals coming from the samples which at this range turn out to be in most cases singles or a few molecules. This particular variation of the classical technique is called SERS (Surface Enanched Raman Spectroscopy). In this work, the enhancement of the electromagnetic field is obtained by manipulation of the optical properties of metals with respect to their size. By using electroless deposition (bottom up technique), gold and silver nanoparticles were deposited in nanostructured patterns obtained on silicon wafers by means of electron beam lithography (top down technique). Rhodamine 6G in aqueous solution at extremely low concentration (10-8 M) was absorbed on the resultant dimers and the collection of the Raman spectra demonstrated the high efficiency of the substrates.

  15. Single-molecule studies of DNA transcription using atomic force microscopy

    International Nuclear Information System (INIS)

    Billingsley, Daniel J; Crampton, Neal; Thomson, Neil H; Bonass, William A; Kirkham, Jennifer

    2012-01-01

    Atomic force microscopy (AFM) can detect single biomacromolecules with a high signal-to-noise ratio on atomically flat biocompatible support surfaces, such as mica. Contrast arises from the innate forces and therefore AFM does not require imaging contrast agents, leading to sample preparation that is relatively straightforward. The ability of AFM to operate in hydrated environments, including humid air and aqueous buffers, allows structure and function of biological and biomolecular systems to be retained. These traits of the AFM are ensuring that it is being increasingly used to study deoxyribonucleic acid (DNA) structure and DNA–protein interactions down to the secondary structure level. This report focuses in particular on reviewing the applications of AFM to the study of DNA transcription in reductionist single-molecule bottom-up approaches. The technique has allowed new insights into the interactions between ribonucleic acid (RNA) polymerase to be gained and enabled quantification of some aspects of the transcription process, such as promoter location, DNA wrapping and elongation. More recently, the trend is towards studying the interactions of more than one enzyme operating on a single DNA template. These methods begin to reveal the mechanics of gene expression at the single-molecule level and will enable us to gain greater understanding of how the genome is transcribed and translated into the proteome. (topical review)

  16. Single particle labeling of RNA virus in live cells.

    Science.gov (United States)

    Liu, Xiaohui; Ouyang, Ting; Ouyang, Hongsheng; Ren, Linzhu

    2017-06-02

    Real-time and visual tracking of viral infection is crucial for elucidating the infectious and pathogenesis mechanisms. To track the virus successfully, an efficient labeling method is necessary. In this review, we first discuss the practical labeling techniques for virus tracking in live cells. We then describe the current knowledge of interactions between RNA viruses (especially influenza viruses, immunodeficiency viruses, and Flaviviruses) and host cellular structures, obtained using single particle labeling techniques combined with real-time fluorescence microscopy. Single particle labeling provides an easy system for understanding the RNA virus life cycle. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. The LncRNA Connectivity Map: Using LncRNA Signatures to Connect Small Molecules, LncRNAs, and Diseases.

    Science.gov (United States)

    Yang, Haixiu; Shang, Desi; Xu, Yanjun; Zhang, Chunlong; Feng, Li; Sun, Zeguo; Shi, Xinrui; Zhang, Yunpeng; Han, Junwei; Su, Fei; Li, Chunquan; Li, Xia

    2017-07-27

    Well characterized the connections among diseases, long non-coding RNAs (lncRNAs) and drugs are important for elucidating the key roles of lncRNAs in biological mechanisms in various biological states. In this study, we constructed a database called LNCmap (LncRNA Connectivity Map), available at http://www.bio-bigdata.com/LNCmap/ , to establish the correlations among diseases, physiological processes, and the action of small molecule therapeutics by attempting to describe all biological states in terms of lncRNA signatures. By reannotating the microarray data from the Connectivity Map database, the LNCmap obtained 237 lncRNA signatures of 5916 instances corresponding to 1262 small molecular drugs. We provided a user-friendly interface for the convenient browsing, retrieval and download of the database, including detailed information and the associations of drugs and corresponding affected lncRNAs. Additionally, we developed two enrichment analysis methods for users to identify candidate drugs for a particular disease by inputting the corresponding lncRNA expression profiles or an associated lncRNA list and then comparing them to the lncRNA signatures in our database. Overall, LNCmap could significantly improve our understanding of the biological roles of lncRNAs and provide a unique resource to reveal the connections among drugs, lncRNAs and diseases.

  18. Developing DNA nanotechnology using single-molecule fluorescence.

    Science.gov (United States)

    Tsukanov, Roman; Tomov, Toma E; Liber, Miran; Berger, Yaron; Nir, Eyal

    2014-06-17

    CONSPECTUS: An important effort in the DNA nanotechnology field is focused on the rational design and manufacture of molecular structures and dynamic devices made of DNA. As is the case for other technologies that deal with manipulation of matter, rational development requires high quality and informative feedback on the building blocks and final products. For DNA nanotechnology such feedback is typically provided by gel electrophoresis, atomic force microscopy (AFM), and transmission electron microscopy (TEM). These analytical tools provide excellent structural information; however, usually they do not provide high-resolution dynamic information. For the development of DNA-made dynamic devices such as machines, motors, robots, and computers this constitutes a major problem. Bulk-fluorescence techniques are capable of providing dynamic information, but because only ensemble averaged information is obtained, the technique may not adequately describe the dynamics in the context of complex DNA devices. The single-molecule fluorescence (SMF) technique offers a unique combination of capabilities that make it an excellent tool for guiding the development of DNA-made devices. The technique has been increasingly used in DNA nanotechnology, especially for the analysis of structure, dynamics, integrity, and operation of DNA-made devices; however, its capabilities are not yet sufficiently familiar to the community. The purpose of this Account is to demonstrate how different SMF tools can be utilized for the development of DNA devices and for structural dynamic investigation of biomolecules in general and DNA molecules in particular. Single-molecule diffusion-based Förster resonance energy transfer and alternating laser excitation (sm-FRET/ALEX) and immobilization-based total internal reflection fluorescence (TIRF) techniques are briefly described and demonstrated. To illustrate the many applications of SMF to DNA nanotechnology, examples of SMF studies of DNA hairpins and

  19. 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

  20. Single Protein Molecule Mapping with Magnetic Atomic Force Microscopy

    Science.gov (United States)

    Moskalenko, Andriy V.; Yarova, Polina L.; Gordeev, Sergey N.; Smirnov, Sergey V.

    2010-01-01

    Abstract Understanding the structural organization and distribution of proteins in biological cells is of fundamental importance in biomedical research. The use of conventional fluorescent microscopy for this purpose is limited due to its relatively low spatial resolution compared to the size of a single protein molecule. Atomic force microscopy (AFM), on the other hand, allows one to achieve single-protein resolution by scanning the cell surface using a specialized ligand-coated AFM tip. However, because this method relies on short-range interactions, it is limited to the detection of binding sites that are directly accessible to the AFM tip. We developed a method based on magnetic (long-range) interactions and applied it to investigate the structural organization and distribution of endothelin receptors on the surface of smooth muscle cells. Endothelin receptors were labeled with 50-nm superparamagnetic microbeads and then imaged with magnetic AFM. Considering its high spatial resolution and ability to “see” magnetically labeled proteins at a distance of up to 150 nm, this approach may become an important tool for investigating the dynamics of individual proteins both on the cell membrane and in the submembrane space. PMID:20141762

  1. Single molecule atomic force microscopy and force spectroscopy of chitosan.

    Science.gov (United States)

    Kocun, Marta; Grandbois, Michel; Cuccia, Louis A

    2011-02-01

    Atomic force microscopy (AFM) and AFM-based force spectroscopy was used to study the desorption of individual chitosan polymer chains from substrates with varying chemical composition. AFM images of chitosan adsorbed onto a flat mica substrate show elongated single strands or aggregated bundles. The aggregated state of the polymer is consistent with the high level of flexibility and mobility expected for a highly positively charged polymer strand. Conversely, the visualization of elongated strands indicated the presence of stabilizing interactions with the substrate. Surfaces with varying chemical composition (glass, self-assembled monolayer of mercaptoundecanoic acid/decanethiol and polytetrafluoroethylene (PTFE)) were probed with chitosan modified AFM tips and the corresponding desorption energies, calculated from plateau-like features, were attributed to the desorption of individual polymer strands. Desorption energies of 2.0±0.3×10(-20)J, 1.8±0.3×10(-20)J and 3.5±0.3×10(-20)J were obtained for glass, SAM of mercaptoundecanoic/dodecanethiol and PTFE, respectively. These single molecule level results can be used as a basis for investigating chitosan and chitosan-based materials for biomaterial applications. Copyright © 2010 Elsevier B.V. All rights reserved.

  2. Single Molecule Kinetics of ENTH Binding to Lipid Membranes

    Energy Technology Data Exchange (ETDEWEB)

    Rozovsky, Sharon [Univ. of Delaware, Newark, DE (United States); Forstner, Martin B. [Syracuse Univ., NY (United States); Sondermann, Holger [Cornell Univ., Ithaca, NY (United States); Groves, Jay T. [Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

    2012-04-03

    Transient recruitment of proteins to membranes is a fundamental mechanism by which the cell exerts spatial and temporal control over proteins’ localization and interactions. Thus, the specificity and the kinetics of peripheral proteins’ membrane residence are an attribute of their function. In this article, we describe the membrane interactions of the interfacial epsin N-terminal homology (ENTH) domain with its target lipid phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P2). The direct visualization and quantification of interactions of single ENTH molecules with supported lipid bilayers is achieved using total internal reflection fluorescence microscopy (TIRFM) with a time resolution of 13 ms. This enables the recording of the kinetic behavior of ENTH interacting with membranes with physiologically relevant concentrations of PtdIns(4,5)P2 despite the low effective binding affinity. Subsequent single fluorophore tracking permits us to build up distributions of residence times and to measure ENTH dissociation rates as a function of membrane composition. In addition, due to the high time resolution, we are able to resolve details of the motion of ENTH associated with a simple, homogeneous membrane. In this case ENTH’s diffusive transport appears to be the result of at least three different diffusion processes.

  3. Photon-Induced Magnetization Reversal in Single Molecule Magnets

    Science.gov (United States)

    Bal, Mustafa

    2005-03-01

    Single-molecule magnets (SMM) have been the subject of intensive research for more than a decade now because of their unique properties such as macroscopic quantum tunneling. Recent work in this area is focused on whether SMM are potential qubits, as proposed theoretically [1]. We use continuous millimeter wave radiation to manipulate the populations of the energy levels of a single crystal molecular magnet Fe8 [2]. When radiation is in resonance with the transitions between energy levels, the steady state magnetization exhibits dips. As expected, the magnetic field locations of these dips vary linearly with the radiation frequency. We will describe our experimental results, which provide a lower bound of 0.17 ns for transverse relaxation time. Transitions between excited states are found even though these states have negligible population at the experimental temperature. We find evidence that the sample heating is significant when the resonance condition is satisfied. Recent experiments are concentrated on the spin dynamics of Fe8 induced by pulsed radiation and results of these studies will also be presented. [1] Leuenberger, M. N. and Loss, D., Nature 410, 789 (2001). [2] M. Bal et al., Phys. Rev. B 70, 100408(R) (2004).

  4. Single-strand DNA molecule translocation through nanoelectrode gaps

    International Nuclear Information System (INIS)

    Zhao Xiongce; Payne, Christina M; Cummings, Peter T; Lee, James W

    2007-01-01

    Molecular dynamics simulations were performed to investigate the translocation of single-strand DNA through nanoscale electrode gaps under the action of a constant driving force. The application behind this theoretical study is a proposal to use nanoelectrodes as a screening gap as part of a rapid genomic sequencing device. Preliminary results from a series of simulations using various gap widths and driving forces suggest that the narrowest electrode gap that a single-strand DNA can pass is ∼1.5 nm. The minimum force required to initiate the translocation within nanoseconds is ∼0.3 nN. Simulations using DNA segments of various lengths indicate that the minimum initiation force is insensitive to the length of DNA. However, the average threading velocity of DNA varies appreciably from short to long DNA segments. We attribute such variation to the different nature of drag force experienced by the short and long DNA segments in the environment. It is found that DNA molecules deform significantly to fit in the shape of the nanogap during the translocation

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

    Directory of Open Access Journals (Sweden)

    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

  6. 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.

  7. 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

  8. Computational and Experimental Insight Into Single-Molecule Piezoelectric Materials

    Science.gov (United States)

    Marvin, Christopher Wayne

    Piezoelectric materials allow for the harvesting of ambient waste energy from the environment. Producing lightweight, highly responsive materials is a challenge for this type of material, requiring polymer, foam, or bio-inspired materials. In this dissertation, I explore the origin of the piezoelectric effect in single molecules through density functional theory (DFT), analyze the piezoresponse of bio-inspired peptidic materials through the use of atomic and piezoresponse force microscopy (AFM and PFM), and develop a novel class of materials combining flexible polyurethane foams and non-piezoelectric, polar dopants. For the DFT calculations, functional group, regiochemical, and heteroatom derivatives of [6]helicene were examined for their influence on the piezoelectric response. An aza[6]helicene derivative was found to have a piezoelectric response (108 pm/V) comparable to ceramics such as lead zirconium titanate (200+ pm/V). These computed materials have the possibility to compete with current field-leading piezomaterials such as lead zirconium titanate (PZT), zinc oxide (ZnO), and polyvinylidene difluoride (PVDF) and its derivatives. The use of AFM/PFM allows for the demonstration of the piezoelectric effect of the selfassembled monolayer (SAM) peptidic systems. Through PFM, the influence that the helicity and sequence of the peptide has on the overall response of the molecule can be analyzed. Finally, development of a novel class of piezoelectrics, the foam-based materials, expands the current understanding of the qualities required for a piezoelectric material from ceramic and rigid materials to more flexible, organic materials. Through the exploration of these novel types of piezoelectric materials, new design rules and figures of merit have been developed.

  9. Force feedback effects on single molecule hopping and pulling experiments

    Science.gov (United States)

    Rico-Pasto, M.; Pastor, I.; Ritort, F.

    2018-03-01

    Single-molecule experiments with optical tweezers have become an important tool to study the properties and mechanisms of biological systems, such as cells and nucleic acids. In particular, force unzipping experiments have been used to extract the thermodynamics and kinetics of folding and unfolding reactions. In hopping experiments, a molecule executes transitions between the unfolded and folded states at a preset value of the force [constant force mode (CFM) under force feedback] or trap position [passive mode (PM) without feedback] and the force-dependent kinetic rates extracted from the lifetime of each state (CFM) and the rupture force distributions (PM) using the Bell-Evans model. However, hopping experiments in the CFM are known to overestimate molecular distances and folding free energies for fast transitions compared to the response time of the feedback. In contrast, kinetic rate measurements from pulling experiments have been mostly done in the PM while the CFM is seldom implemented in pulling protocols. Here, we carry out hopping and pulling experiments in a short DNA hairpin in the PM and CFM at three different temperatures (6 °C, 25 °C, and 45 °C) exhibiting largely varying kinetic rates. As expected, we find that equilibrium hopping experiments in the CFM and PM perform well at 6 °C (where kinetics are slow), whereas the CFM overestimates molecular parameters at 45 °C (where kinetics are fast). In contrast, nonequilibrium pulling experiments perform well in both modes at all temperatures. This demonstrates that the same kind of feedback algorithm in the CFM leads to more reliable determination of the folding reaction parameters in irreversible pulling experiments.

  10. Calix[4]arene Based Single-Molecule Magnets

    Energy Technology Data Exchange (ETDEWEB)

    Karotsis, Georgios; Teat, Simon J.; Wernsdorfer, Wolfgang; Piligkos, Stergios; Dalgarno, Scott J.; Brechin, Euan K.

    2009-06-04

    Single-molecule magnets (SMMs) have been the subject of much interest in recent years because their molecular nature and inherent physical properties allow the crossover between classical and quantum physics to be observed. The macroscopic observation of quantum phenomena - tunneling between different spin states, quantum interference between tunnel paths - not only allows scientists to study quantum mechanical laws in great detail, but also provides model systems with which to investigate the possible implementation of spin-based solid state qubits and molecular spintronics. The isolation of small, simple SMMs is therefore an exciting prospect. To date almost all SMMs have been made via the self-assembly of 3d metal ions in the presence of bridging/chelating organic ligands. However, very recently an exciting new class of SMMs, based on 3d metal clusters (or single lanthanide ions) housed within polyoxometalates, has appeared. These types of molecule, in which the SMM is completely encapsulated within (or shrouded by) a 'protective' organic or inorganic sheath have much potential for design and manipulation: for example, for the removal of unwanted dipolar interactions, the introduction of redox activity, or to simply aid functionalization for surface grafting. Calix[4]arenes are cyclic (typically bowl-shaped) polyphenols that have been used extensively in the formation of versatile self-assembled supramolecular structures. Although many have been reported, p-{sup t}But-calix[4]arene and calix[4]arene (TBC4 and C4 respectively, Figure 1A) are frequently encountered due to (a) synthetic accessibility, and (b) vast potential for alteration at either the upper or lower rim of the macrocyclic framework. Within the field of supramolecular chemistry, TBC4 is well known for interesting polymorphic behavior and phase transformations within anti-parallel bi-layer arrays, while C4 often forms self-included trimers. The polyphenolic nature of calix[n]arenes (where

  11. Single molecules in soft matter : a study of biomolecular conformation, heterogeneity and plasmon enhanced fluorescence

    NARCIS (Netherlands)

    Yuan, Haifeng

    2013-01-01

    We study the dynamics of single molecules and individual gold nanorods in glycerol at variable temperatures. We demonstrate temperature-cycle microscopy on FRET-labeled polyproline and double-stranded DNA molecules to access micro-second dynamics of single molecules, and reveal the influences of

  12. A versatile optical microscope for time-dependent single-molecule and single-particle spectroscopy.

    Science.gov (United States)

    Li, Hao; Yang, Haw

    2018-03-28

    This work reports the design and implementation of a multi-function optical microscope for time-dependent spectroscopy on single molecules and single nanoparticles. It integrates the now-routine single-object measurements into one standalone platform so that no reconfiguration is needed when switching between different types of sample or spectroscopy modes. The illumination modes include evanescent field through total internal reflection, dark-field illumination, and epi-excitation onto a diffraction-limited spot suitable for confocal detection. The detection modes include spectrally resolved line imaging, wide-field imaging with dual-color capability, and two-color single-element photon-counting detection. The switch between different spectroscopy and data acquisition modes is fully automated and executed through computer programming. The capability of this microscope is demonstrated through selected proof-of-principle experiments.

  13. A versatile optical microscope for time-dependent single-molecule and single-particle spectroscopy

    Science.gov (United States)

    Li, Hao; Yang, Haw

    2018-03-01

    This work reports the design and implementation of a multi-function optical microscope for time-dependent spectroscopy on single molecules and single nanoparticles. It integrates the now-routine single-object measurements into one standalone platform so that no reconfiguration is needed when switching between different types of sample or spectroscopy modes. The illumination modes include evanescent field through total internal reflection, dark-field illumination, and epi-excitation onto a diffraction-limited spot suitable for confocal detection. The detection modes include spectrally resolved line imaging, wide-field imaging with dual-color capability, and two-color single-element photon-counting detection. The switch between different spectroscopy and data acquisition modes is fully automated and executed through computer programming. The capability of this microscope is demonstrated through selected proof-of-principle experiments.

  14. Monitoring and Manipulating Motions of Single Molecules/Nanoparticles

    Science.gov (United States)

    Chen, Fang

    -nanometer pores in theory. We then experimentally studied nanoparticles diffusing on membrane filters containing 200 nm polyethyleneglycol- or C18-modified pores. Using STED microscopy, we resolved for the first time how small particles are retained by the pores. Trapping by the pore entrances rather than adsorption is responsible for the retention. Further studies on C18-modified pores showed consistency in Gibbs free energy about the retention process. In addition, in order to understand how nanoparticles interact with the surface when they are forced to be on, or very close to, the surface, we studied nanosecond rotation dynamics of gold nanorods with one end attached on the surface. We found that the nanorod motion is dominated by van der Waals interaction-induced immobilization rather Brownian rotational diffusion as previously thought. The actual rotation, during which the nanorod transits from one immobilized state to the other, slows down by 50 times. The second part of the research is the collaboration with Tour's group in Rice University. The ultimate goal is to use light to drive a motorized nanocar at ambient conditions. To fulfill this goal, we first studied the moving kinetics of adamantane-wheeled nanocars on hydroxylated and PEG-modified surfaces using single molecule fluorescence microscopy. We found that nanocars' diffusion slows down on solid surface over time, which is possibly caused by the increased hydrophobicity of the substrate surface due to the adsorbates from the air. A sticky-spots model was proposed to explain the observed slowing down. To find out whether a light-activatable motor works when it is incorporated into a nanocar, we carefully designed a series of molecules containing a regular motor, a slow motor, a nonunidirectional motor, and no motor. We found that a fast unidirectional rotating motor enhanced the diffusion of the molecule in solution upon UV-illumination. Detailed analysis suggested that the unimolecular submersible nanomachine (USN

  15. Kinetic analysis of single molecule FRET transitions without trajectories

    Science.gov (United States)

    Schrangl, Lukas; Göhring, Janett; Schütz, Gerhard J.

    2018-03-01

    Single molecule Förster resonance energy transfer (smFRET) is a popular tool to study biological systems that undergo topological transitions on the nanometer scale. smFRET experiments typically require recording of long smFRET trajectories and subsequent statistical analysis to extract parameters such as the states' lifetimes. Alternatively, analysis of probability distributions exploits the shapes of smFRET distributions at well chosen exposure times and hence works without the acquisition of time traces. Here, we describe a variant that utilizes statistical tests to compare experimental datasets with Monte Carlo simulations. For a given model, parameters are varied to cover the full realistic parameter space. As output, the method yields p-values which quantify the likelihood for each parameter setting to be consistent with the experimental data. The method provides suitable results even if the actual lifetimes differ by an order of magnitude. We also demonstrated the robustness of the method to inaccurately determine input parameters. As proof of concept, the new method was applied to the determination of transition rate constants for Holliday junctions.

  16. A single molecule study of cellulase hydrolysis of crystalline cellulose

    Science.gov (United States)

    Liu, Yu-San; Luo, Yonghua; Baker, John O.; Zeng, Yining; Himmel, Michael E.; Smith, Steve; Ding, Shi-You

    2010-02-01

    Cellobiohydrolase-I (CBH I), a processive exoglucanase secreted by Trichoderma reesei, is one of the key enzyme components in a commercial cellulase mixture currently used for processing biomass to biofuels. CBH I contains a family 7 glycoside hydrolase catalytic module, a family 1 carbohydrate-binding module (CBM), and a highlyglycosylated linker peptide. It has been proposed that the CBH I cellulase initiates the hydrolysis from the reducing end of one cellulose chain and successively cleaves alternate β-1,4-glycosidic bonds to release cellobiose as its principal end product. The role each module of CBH I plays in the processive hydrolysis of crystalline cellulose has yet to be convincingly elucidated. In this report, we use a single-molecule approach that combines optical (Total Internal Reflection Fluorescence microscopy, or TIRF-M) and non-optical (Atomic Force Microscopy, or AFM) imaging techniques to analyze the molecular motion of CBM tagged with green fluorescence protein (GFP), and to investigate the surface structure of crystalline cellulose and changes made in the structure by CBM and CBH I. The preliminary results have revealed a confined nanometer-scale movement of the TrCBM1-GFP bound to cellulose, and decreases in cellulose crystal size as well as increases in surface roughness during CBH I hydrolysis of crystalline cellulose.

  17. Fluorescence molecule counting for single-molecule studies in crowded environment of living cells without and with broken ergodicity.

    Science.gov (United States)

    Földes-Papp, Zeno; Baumann, Gerd

    2011-05-01

    We present a new approach to distinguish between non-ergodic and ergodic behavior. Performing ensemble averaging in a subpopulation of individual molecules leads to a mean value that can be similar to the mean value obtained in an ergodic system. The averaging is carried out by minimizing the variation between the sum of the temporal averaged mean square deviation of the simulated data with respect to the logarithmic scaling behavior of the subpopulation. For this reason, we first introduce a kind of Continuous Time Random Walks (CTRW), which we call Limited Continuous Time Random Walks (LCTRW) on fractal support. The random waiting time distributions are sampled at points which fulfill the condition N <1, where N is the Poisson probability of finding a single molecule in the femtoliter-sized observation volume ΔV at the single-molecule level. Given a subpopulation of different single molecules of the same kind, the ratio T/ T(m) between the measurement time T and the meaningful time T(m), which is the time for observing just one and the same single molecule, is the experimentally accessible quantity that allows to compare different molecule numbers in the subpopulation. In addition, the mean square displacement traveled by the molecule during the time t is determined by an upper limit of the geometric dimension of the living cell or its nucleus.

  18. Investigation on Single-Molecule Junctions Based on Current–Voltage Characteristics

    Directory of Open Access Journals (Sweden)

    Yuji Isshiki

    2018-02-01

    Full Text Available The relationship between the current through an electronic device and the voltage across its terminals is a current–voltage characteristic (I–V that determine basic device performance. Currently, I–V measurement on a single-molecule scale can be performed using break junction technique, where a single molecule junction can be prepared by trapping a single molecule into a nanogap between metal electrodes. The single-molecule I–Vs provide not only the device performance, but also reflect information on energy dispersion of the electronic state and the electron-molecular vibration coupling in the junction. This mini review focuses on recent representative studies on I–Vs of the single molecule junctions that cover investigation on the single-molecule diode property, the molecular vibration, and the electronic structure as a form of transmission probability, and electronic density of states, including the spin state of the single-molecule junctions. In addition, thermoelectronic measurements based on I–Vs and identification of the charged carriers (i.e., electrons or holes are presented. The analysis in the single-molecule I–Vs provides fundamental and essential information for a better understanding of the single-molecule science, and puts the single molecule junction to more practical use in molecular devices.

  19. 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.

  20. Simultaneous delivery of hydrophobic small molecules and siRNA using Sterosomes to direct mesenchymal stem cell differentiation for bone repair.

    Science.gov (United States)

    Cui, Zhong-Kai; Sun, Justin A; Baljon, Jessalyn J; Fan, Jiabing; Kim, Soyon; Wu, Benjamin M; Aghaloo, Tara; Lee, Min

    2017-08-01

    The use of small molecular drugs with gene manipulation offers synergistic therapeutic efficacy by targeting multiple signaling pathways for combined treatment. Stimulation of mesenchymal stem cells (MSCs) with osteoinductive small molecule phenamil combined with suppression of noggin is a promising therapeutic strategy that increases bone morphogenetic protein (BMP) signaling and bone repair. Our cationic Sterosome formulated with stearylamine (SA) and cholesterol (Chol) is an attractive co-delivery system that not only forms stable complexes with small interfering RNA (siRNA) molecules but also solubilizes hydrophobic small molecules in a single vehicle, for directing stem cell differentiation. Herein, we demonstrate the ability of SA/Chol Sterosomes to simultaneously deliver hydrophobic small molecule phenamil and noggin-directed siRNA to enhance osteogenic differentiation of MSCs both in in vitro two- and three-dimensional settings as well as in a mouse calvarial defect model. These results suggest a novel liposomal platform to simultaneously deliver therapeutic genes and small molecules for combined therapy. Application of phenamil, a small molecular bone morphogenetic protein (BMP) stimulator, combined with suppression of natural BMP antagonists such as noggin is a promising therapeutic strategy to enhance bone regeneration. Here, we present a novel strategy to co-deliver hydrophobic small molecule phenamil and noggin-targeted siRNA via cationic Sterosomes formed with stearylamine (SA) and high content of cholesterol (Chol) to enhance osteogenesis and bone repair. SA/Chol Sterosomes demonstrated high phenamil encapsulation efficiency, supported sustained release of encapsulated drugs, and significantly reduced drug dose requirements to induce osteogenic differentiation of mesenchymal stem cells (MSCs). Simultaneous deliver of phenamil and noggin siRNA in a single vehicle synergistically enhanced MSC osteogenesis and calvarial bone repair. This study suggests

  1. Electrochemical Control of Single-Molecule Conductance by Fermi- Level Tuning and Conjugation Switching

    DEFF Research Database (Denmark)

    Baghernejad, Masoud; Zhao, Xiaotao; Ørnsø, Kristian Baruël

    2014-01-01

    Controlling charge transport through a single molecule connected to metallic electrodes remains one of the most fundamental challenges of nanoelectronics. Here we use electrochemical gating to reversibly tune the conductance of two different organic molecules, both containing anthraquinone (AQ...

  2. The Conformational Dynamics of Cas9 Governing DNA Cleavage Are Revealed by Single-Molecule FRET.

    Science.gov (United States)

    Yang, Mengyi; Peng, Sijia; Sun, Ruirui; Lin, Jingdi; Wang, Nan; Chen, Chunlai

    2018-01-09

    Off-target binding and cleavage by Cas9 pose major challenges in its application. How the conformational dynamics of Cas9 govern its nuclease activity under on- and off-target conditions remains largely unknown. Here, using intra-molecular single-molecule fluorescence resonance energy transfer measurements, we revealed that Cas9 in apo, sgRNA-bound, and dsDNA/sgRNA-bound forms spontaneously transits among three major conformational states, mainly reflecting significant conformational mobility of the catalytic HNH domain. We also uncovered surprising long-range allosteric communication between the HNH domain and the RNA/DNA heteroduplex at the PAM-distal end to ensure correct positioning of the catalytic site, which demonstrated that a unique proofreading mechanism served as the last checkpoint before DNA cleavage. Several Cas9 residues were likely to mediate the allosteric communication and proofreading step. Modulating interactions between Cas9 and heteroduplex at the PAM-distal end by introducing mutations on these sites provides an alternative route to improve and optimize the CRISPR/Cas9 toolbox. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  3. 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

  4. Single molecule studies of DNA packaging by bacteriophages

    Science.gov (United States)

    Fuller, Derek Nathan

    The DNA packaging dynamics of bacteriophages φ29, gamma, and T4 were studied at the single molecule level using a dual trap optical tweezers. Also, a method for producing long DNA molecules by PCR for optical tweezers studies of protein DNA interactions is presented and thoroughly characterized. This DNA preparation technique provided DNA samples for the φ29 and T4 studies. In the studies of φ29, the role of charge was investigated by varying the ionic conditions of the packaging buffer. Ionic conditions in which the DNA charge was highly screened due to divalent and trivalent cations showed the lowest resistance to packaging of the DNA to high density. This confirmed the importance of counterions in shielding the DNA interstrand repulsion when packaged to high density. While the ionic nature of the packaging buffer had a strong effect on packaging velocities, there was no clear trend between the counterion-screened charge of the DNA and the maximum packaging velocity. The packaging studies of lambda and T4 served as systems for comparative studies with φ29. Each system showed similarities to the φ29 system and unique differences. Both the lambda and T4 packaging motors were capable of generating forces in excess of 50 pN and showed remarkably high processivity, similar to φ29. However, dynamic structural transitions were observed with lambda that are not observed with φ29. The packaging of the lambda genome showed capsid expansion at approximately 30 percent of the genome packaged and capsid rupture at 90 percent of the genome packaged in the absence of capsid stabilizing protein gpD. Unique to the T4 packaging motor, packaging dynamics showed a remarkable amount of variability in velocities. This variability was seen both within individual packaging phages and from one phage to the next. This is possibly due to different conformational states of the packaging machinery. Additionally, lambda and T4 had average packaging velocities under minimal load of 600

  5. 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.

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

    Science.gov (United States)

    Kaplinski, Lauris; Scheler, Ott; Parkel, Sven; Palta, Priit; Toome, Kadri; Kurg, Ants; Remm, Maido

    2010-04-28

    The hybridization of synthetic Streptococcus pneumoniae tmRNA on a detection microarray is slow at 34 degrees C resulting in low signal intensities. 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 degrees 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. 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.

  7. 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.

  8. BOBA FRET: bootstrap-based analysis of single-molecule FRET data.

    Directory of Open Access Journals (Sweden)

    Sebastian L B König

    Full Text Available Time-binned single-molecule Förster resonance energy transfer (smFRET experiments with surface-tethered nucleic acids or proteins permit to follow folding and catalysis of single molecules in real-time. Due to the intrinsically low signal-to-noise ratio (SNR in smFRET time traces, research over the past years has focused on the development of new methods to extract discrete states (conformations from noisy data. However, limited observation time typically leads to pronounced cross-sample variability, i.e., single molecules display differences in the relative population of states and the corresponding conversion rates. Quantification of cross-sample variability is necessary to perform statistical testing in order to assess whether changes observed in response to an experimental parameter (metal ion concentration, the presence of a ligand, etc. are significant. However, such hypothesis testing has been disregarded to date, precluding robust biological interpretation. Here, we address this problem by a bootstrap-based approach to estimate the experimental variability. Simulated time traces are presented to assess the robustness of the algorithm in conjunction with approaches commonly used in thermodynamic and kinetic analysis of time-binned smFRET data. Furthermore, a pair of functionally important sequences derived from the self-cleaving group II intron Sc.ai5γ (d3'EBS1/IBS1 is used as a model system. Through statistical hypothesis testing, divalent metal ions are shown to have a statistically significant effect on both thermodynamic and kinetic aspects of their interaction. The Matlab source code used for analysis (bootstrap-based analysis of smFRET data, BOBA FRET, as well as a graphical user interface, is available via http://www.aci.uzh.ch/rna/.

  9. Electrons, Photons, and Force: Quantitative Single-Molecule Measurements from Physics to Biology

    Science.gov (United States)

    2011-01-01

    Single-molecule measurement techniques have illuminated unprecedented details of chemical behavior, including observations of the motion of a single molecule on a surface, and even the vibration of a single bond within a molecule. Such measurements are critical to our understanding of entities ranging from single atoms to the most complex protein assemblies. We provide an overview of the strikingly diverse classes of measurements that can be used to quantify single-molecule properties, including those of single macromolecules and single molecular assemblies, and discuss the quantitative insights they provide. Examples are drawn from across the single-molecule literature, ranging from ultrahigh vacuum scanning tunneling microscopy studies of adsorbate diffusion on surfaces to fluorescence studies of protein conformational changes in solution. PMID:21338175

  10. A comparison of single molecule and amplification based sequencing of cancer transcriptomes.

    Directory of Open Access Journals (Sweden)

    Lee T Sam

    2011-03-01

    Full Text Available The second wave of next generation sequencing technologies, referred to as single-molecule sequencing (SMS, carries the promise of profiling samples directly without employing polymerase chain reaction steps used by amplification-based sequencing (AS methods. To examine the merits of both technologies, we examine mRNA sequencing results from single-molecule and amplification-based sequencing in a set of human cancer cell lines and tissues. We observe a characteristic coverage bias towards high abundance transcripts in amplification-based sequencing. A larger fraction of AS reads cover highly expressed genes, such as those associated with translational processes and housekeeping genes, resulting in relatively lower coverage of genes at low and mid-level abundance. In contrast, the coverage of high abundance transcripts plateaus off using SMS. Consequently, SMS is able to sequence lower- abundance transcripts more thoroughly, including some that are undetected by AS methods; however, these include many more mapping artifacts. A better understanding of the technical and analytical factors introducing platform specific biases in high throughput transcriptome sequencing applications will be critical in cross platform meta-analytic studies.

  11. Single-molecule transport across an individual biomimetic nuclear pore complex

    Science.gov (United States)

    Kowalczyk, Stefan W.; Kapinos, Larisa; Blosser, Timothy R.; Magalhães, Tomás; van Nies, Pauline; Lim, Roderick Y. H.; Dekker, Cees

    2011-07-01

    Nuclear pore complexes regulate the selective exchange of RNA and proteins across the nuclear envelope in eukaryotic cells. Biomimetic strategies offer new opportunities to investigate this remarkable transport phenomenon. Here, we show selective transport of proteins across individual biomimetic nuclear pore complexes at the single-molecule level. Each biomimetic complex is constructed by covalently tethering either Nup98 or Nup153 (phenylalanine-glycine (FG) nucleoporins) to a solid-state nanopore. Individual translocation events are monitored using ionic current measurements with sub-millisecond temporal resolution. Transport receptors (Impβ) proceed with a dwell time of ~2.5 ms for both Nup98- and Nup153-coated pores, whereas the passage of non-specific proteins is strongly inhibited with different degrees of selectivity. For pores up to ~25 nm in diameter, Nups form a dense and low-conducting barrier, whereas they adopt a more open structure in larger pores. Our biomimetic nuclear pore complex provides a quantitative platform for studying nucleocytoplasmic transport phenomena at the single-molecule level in vitro.

  12. 48-spot single-molecule FRET setup with periodic acceptor excitation

    Science.gov (United States)

    Ingargiola, Antonino; Segal, Maya; Gulinatti, Angelo; Rech, Ivan; Labanca, Ivan; Maccagnani, Piera; Ghioni, Massimo; Weiss, Shimon; Michalet, Xavier

    2018-03-01

    Single-molecule Förster resonance energy transfer (smFRET) allows measuring distances between donor and acceptor fluorophores on the 3-10 nm range. Solution-based smFRET allows measurement of binding-unbinding events or conformational changes of dye-labeled biomolecules without ensemble averaging and free from surface perturbations. When employing dual (or multi) laser excitation, smFRET allows resolving the number of fluorescent labels on each molecule, greatly enhancing the ability to study heterogeneous samples. A major drawback to solution-based smFRET is the low throughput, which renders repetitive measurements expensive and hinders the ability to study kinetic phenomena in real-time. Here we demonstrate a high-throughput smFRET system that multiplexes acquisition by using 48 excitation spots and two 48-pixel single-photon avalanche diode array detectors. The system employs two excitation lasers allowing separation of species with one or two active fluorophores. The performance of the system is demonstrated on a set of doubly labeled double-stranded DNA oligonucleotides with different distances between donor and acceptor dyes along the DNA duplex. We show that the acquisition time for accurate subpopulation identification is reduced from several minutes to seconds, opening the way to high-throughput screening applications and real-time kinetics studies of enzymatic reactions such as DNA transcription by bacterial RNA polymerase.

  13. Single molecule DNA interaction kinetics of retroviral nucleic acid chaperone proteins

    Science.gov (United States)

    Williams, Mark

    2010-03-01

    Retroviral nucleocapsid (NC) proteins are essential for several viral replication processes including specific genomic RNA packaging and reverse transcription. The nucleic acid chaperone activity of NC facilitates the latter process. In this study, we use single molecule biophysical methods to quantify the DNA interactions of wild type and mutant human immunodeficiency virus type 1 (HIV-1) NC and Gag and human T-cell leukemia virus type 1 (HTLV-1) NC. We find that the nucleic acid interaction properties of these proteins differ significantly, with HIV-1 NC showing rapid protein binding kinetics, significant duplex destabilization, and strong DNA aggregation, all properties that are critical components of nucleic acid chaperone activity. In contrast, HTLV-1 NC exhibits significant destabilization activity but extremely slow DNA interaction kinetics and poor aggregating capability, which explains why HTLV-1 NC is a poor nucleic acid chaperone. To understand these results, we developed a new single molecule method for quantifying protein dissociation kinetics, and applied this method to probe the DNA interactions of wild type and mutant HIV-1 and HTLV-1 NC. We find that mutations to aromatic and charged residues strongly alter the proteins' nucleic acid interaction kinetics. Finally, in contrast to HIV-1 NC, HIV-1 Gag, the nucleic acid packaging protein that contains NC as a domain, exhibits relatively slow binding kinetics, which may negatively impact its ability to act as a nucleic acid chaperone.

  14. Lab-on-a-chip technologies for single-molecule studies.

    Science.gov (United States)

    Zhao, Yanhui; Chen, Danqi; Yue, Hongjun; French, Jarrod B; Rufo, Joseph; Benkovic, Stephen J; Huang, Tony Jun

    2013-06-21

    Recent developments on various lab-on-a-chip techniques allow miniaturized and integrated devices to perform on-chip single-molecule studies. Fluidic-based platforms that utilize unique microscale fluidic behavior are capable of conducting single-molecule experiments with high sensitivities and throughputs, while biomolecular systems can be studied on-chip using techniques such as DNA curtains, magnetic tweezers, and solid-state nanopores. The advances of these on-chip single-molecule techniques lead to next-generation lab-on-a-chip devices, such as DNA transistors, and single-molecule real-time (SMRT) technology for rapid and low-cost whole genome DNA sequencing. In this Focus article, we will discuss some recent successes in the development of lab-on-a-chip techniques for single-molecule studies and expound our thoughts on the near future of on-chip single-molecule studies.

  15. Quantitative affinity electrophoresis of RNA-small molecule interactions by cross-linking the ligand to acrylamide.

    Science.gov (United States)

    Boodram, Sherry N; McCann, Lucas C; Organ, Michael G; Johnson, Philip E

    2013-11-15

    We show that the affinity electrophoresis analysis of RNA-small molecule interactions can be made quantifiable by cross-linking the ligand to the gel matrix. Using an RNA-aminoglycoside model system to verify our method, we attached an acryloyl chloride molecule to the aminoglycosides paromomycin and neomycin B to synthesize an acrylamide-aminoglycoside monomer. This molecule was then used as a component in gel polymerization for affinity electrophoresis, covalently attaching an aminoglycoside molecule to the gel matrix. To test RNA binding to the cross-linked aminoglycosides, we used the aminoglycoside binding RNA molecule derived from thymidylate synthase messenger RNA (mRNA) that contains a C-C mismatch. Binding is indicated by the difference in RNA mobility between gels with cross-linked ligand, with ligand embedded during polymerization, and with no ligand present. Critically, the predicted straight line relationship between the reciprocal of the relative migration of the RNA and the ligand concentration is obtained when using cross-linked aminoglycosides, whereas a straight line is not obtained using embedded aminoglycosides. Average apparent dissociation constants are determined from the slope of the line from these plots. This method allows an easy quantitative comparison between different nucleic acid molecules for a small molecule ligand. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. Efficient unfolding pattern recognition in single molecule force spectroscopy data

    Directory of Open Access Journals (Sweden)

    Labudde Dirk

    2011-06-01

    Full Text Available Abstract Background Single-molecule force spectroscopy (SMFS is a technique that measures the force necessary to unfold a protein. SMFS experiments generate Force-Distance (F-D curves. A statistical analysis of a set of F-D curves reveals different unfolding pathways. Information on protein structure, conformation, functional states, and inter- and intra-molecular interactions can be derived. Results In the present work, we propose a pattern recognition algorithm and apply our algorithm to datasets from SMFS experiments on the membrane protein bacterioRhodopsin (bR. We discuss the unfolding pathways found in bR, which are characterised by main peaks and side peaks. A main peak is the result of the pairwise unfolding of the transmembrane helices. In contrast, a side peak is an unfolding event in the alpha-helix or other secondary structural element. The algorithm is capable of detecting side peaks along with main peaks. Therefore, we can detect the individual unfolding pathway as the sequence of events labeled with their occurrences and co-occurrences special to bR's unfolding pathway. We find that side peaks do not co-occur with one another in curves as frequently as main peaks do, which may imply a synergistic effect occurring between helices. While main peaks co-occur as pairs in at least 50% of curves, the side peaks co-occur with one another in less than 10% of curves. Moreover, the algorithm runtime scales well as the dataset size increases. Conclusions Our algorithm satisfies the requirements of an automated methodology that combines high accuracy with efficiency in analyzing SMFS datasets. The algorithm tackles the force spectroscopy analysis bottleneck leading to more consistent and reproducible results.

  17. Single Molecule Study of DNA Organization and Recombination

    Science.gov (United States)

    Xiao, Botao

    We have studied five projects related to DNA organization and recombination using mainly single molecule force-spectroscopy and statistical tools. First, HU is one of the most abundant DNA-organizing proteins in bacterial chromosomes and participates in gene regulation. We report experiments that study the dependence of DNA condensation by HU on force, salt and HU concentration. A first important result is that at physiological salt levels, HU only bends DNA, resolving a previous paradox of why a chromosome-compacting protein should have a DNA-stiffening function. A second major result is quantitative demonstration of strong dependencies of HU-DNA dissociation on both salt concentration and force. Second, we have used a thermodynamic Maxwell relation to count proteins driven off large DNAs by tension, an effect important to understanding DNA organization. Our results compare well with estimates of numbers of proteins HU and Fis in previous studies. We have also shown that a semi-flexible polymer model describes our HU experimental data well. The force-dependent binding suggests mechano-chemical mechanisms for gene regulation. Third, the elusive role of protein H1 in chromatin has been clarified with purified H1 and Xenopus extracts. We find that H1 compacts DNA by both bending and looping. Addition of H1 enhances chromatin formation and maintains the plasticity of the chromatin. Fourth, the topology and mechanics of DNA twisting are critical to DNA organization and recombination. We have systematically measured DNA extension as a function of linking number density from 0.08 to -2 with holding forces from 0.2 to 2.4 pN. Unlike previous proposals, the DNA extension decreases with negative linking number. Finally, DNA recombination is a dynamic process starting from enzyme-DNA binding. We report that the Int-DBD domain of lambda integrase binds to DNA without compaction at low Int-DBD concentration. High concentration of Int-DBD loops DNA below a threshold force

  18. Nonlinear and Nonsymmetric Single-Molecule Electronic Properties Towards Molecular Information Processing.

    Science.gov (United States)

    Tamaki, Takashi; Ogawa, Takuji

    2017-09-05

    This review highlights molecular design for nonlinear and nonsymmetric single-molecule electronic properties such as rectification, negative differential resistance, and switching, which are important components of future single-molecule information processing devices. Perspectives on integrated "molecular circuits" are also provided. Nonlinear and nonsymmetric single-molecule electronics can be designed by utilizing (1) asymmetric molecular cores, (2) asymmetric anchoring groups, (3) an asymmetric junction environment, and (4) asymmetric electrode materials. This review mainly focuses on the design of molecular cores.

  19. Development of new microscope unit for single molecule spectroscopy under various ambient conditions

    International Nuclear Information System (INIS)

    Yamada, T; Kaji, T; Ueda, R; Otomo, A

    2013-01-01

    This paper introduces techniques we previously developed for single molecule spectroscopy and continues on to describe our studies on dipole orientation imaging of single molecules under various ambient conditions. In these studies, we successfully obtained defocused images of single perylene diimide (PDI) molecules under air, high-vacuum, and pure N 2 gas conditions by utilizing the advantages of our new microscope unit. The studies are positioned as one of the important applications of our microscope unit for single molecule spectroscopy. We expect a wide range of applications for this unit for various microscope measurements for many types of materials.

  20. Ordered array of CoPc-vacancies filled with single-molecule rotors

    Science.gov (United States)

    Xie, Zheng-Bo; Wang, Ya-Li; Tao, Min-Long; Sun, Kai; Tu, Yu-Bing; Yuan, Hong-Kuan; Wang, Jun-Zhong

    2018-05-01

    We report the highly ordered array of CoPc-vacancies and the single-molecule rotors inside the vacancies. When CoPc molecules are deposited on Cd(0001) at low-temperature, three types of molecular vacancies appeared randomly in the CoPc monolayer. Annealing the sample to higher temperature leads to the spontaneous phase separation and self-organized arrangement of the vacancies. Highly ordered arrays of two-molecule vacancies and single-molecule vacancies have been obtained. In particular, there is a rotating CoPc molecule inside each single-molecule vacancy, which constitutes the array of single-molecule rotors. These results provide a new routine to fabricate the nano-machines on a large scale.

  1. Stereoelectronic Effect-Induced Conductance Switching in Aromatic Chain Single-Molecule Junctions.

    Science.gov (United States)

    Xin, Na; Wang, Jinying; Jia, Chuancheng; Liu, Zitong; Zhang, Xisha; Yu, Chenmin; Li, Mingliang; Wang, Shuopei; Gong, Yao; Sun, Hantao; Zhang, Guanxin; Liu, Zhirong; Zhang, Guangyu; Liao, Jianhui; Zhang, Deqing; Guo, Xuefeng

    2017-02-08

    Biphenyl, as the elementary unit of organic functional materials, has been widely used in electronic and optoelectronic devices. However, over decades little has been fundamentally understood regarding how the intramolecular conformation of biphenyl dynamically affects its transport properties at the single-molecule level. Here, we establish the stereoelectronic effect of biphenyl on its electrical conductance based on the platform of graphene-molecule single-molecule junctions, where a specifically designed hexaphenyl aromatic chain molecule is covalently sandwiched between nanogapped graphene point contacts to create stable single-molecule junctions. Both theoretical and temperature-dependent experimental results consistently demonstrate that phenyl twisting in the aromatic chain molecule produces different microstates with different degrees of conjugation, thus leading to stochastic switching between high- and low-conductance states. These investigations offer new molecular design insights into building functional single-molecule electrical devices.

  2. Three-dimensional Nanowire Structures for Ultra-Fast Separation of DNA, Protein and RNA Molecules

    Science.gov (United States)

    Rahong, Sakon; Yasui, Takao; Yanagida, Takeshi; Nagashima, Kazuki; Kanai, Masaki; Meng, Gang; He, Yong; Zhuge, Fuwei; Kaji, Noritada; Kawai, Tomoji; Baba, Yoshinobu

    2015-01-01

    Separation and analysis of biomolecules represent crucial processes for biological and biomedical engineering development; however, separation resolution and speed for biomolecules analysis still require improvements. To achieve separation and analysis of biomolecules in a short time, the use of highly-ordered nanostructures fabricated by top-down or bottom-up approaches have been proposed. Here, we reported on the use of three-dimensional (3D) nanowire structures embedded in microchannels fabricated by a bottom-up approach for ultrafast separation of small biomolecules, such as DNA, protein, and RNA molecules. The 3D nanowire structures could analyze a mixture of DNA molecules (50–1000 bp) within 50 s, a mixture of protein molecules (20–340 kDa) within 5 s, and a mixture of RNA molecules (100–1000 bases) within 25 s. And, we could observe the electrophoretic mobility difference of biomolecules as a function of molecular size in the 3D nanowire structures. Since the present methodology allows users to control the pore size of sieving materials by varying the number of cycles for nanowire growth, the 3D nanowire structures have a good potential for use as alternatives for other sieving materials. PMID:26073192

  3. Forces and conductances in a single-molecule bipyridine junction

    DEFF Research Database (Denmark)

    Stadler, Robert; Thygesen, Kristian Sommer; Jacobsen, Karsten Wedel

    2005-01-01

    Inspired by recent measurements of forces and conductances of bipyridine nanojunctions, we have performed density functional theory calculations of structure and electron transport in a bipyridine molecule attached between gold electrodes for seven different contact geometries. The calculations...

  4. Single-molecule stochastic times in a reversible bimolecular reaction.

    Science.gov (United States)

    Keller, Peter; Valleriani, Angelo

    2012-08-28

    In this work, we consider the reversible reaction between reactants of species A and B to form the product C. We consider this reaction as a prototype of many pseudobiomolecular reactions in biology, such as for instance molecular motors. We derive the exact probability density for the stochastic waiting time that a molecule of species A needs until the reaction with a molecule of species B takes place. We perform this computation taking fully into account the stochastic fluctuations in the number of molecules of species B. We show that at low numbers of participating molecules, the exact probability density differs from the exponential density derived by assuming the law of mass action. Finally, we discuss the condition of detailed balance in the exact stochastic and in the approximate treatment.

  5. Probing the local environment of a single OPE3 molecule using inelastic tunneling electron spectroscopy

    NARCIS (Netherlands)

    Frisenda, R.; Perrin, M.L.; Van der Zant, H.S.J.

    2015-01-01

    We study single-molecule oligo(phenylene ethynylene)dithiol junctions by means of inelastic electron tunneling spectroscopy (IETS). The molecule is contacted with gold nano-electrodes formed with the mechanically controllable break junction technique. We record the IETS spectrum of the molecule from

  6. Rational design of single-molecule magnets: a supramolecular approach.

    Science.gov (United States)

    Glaser, Thorsten

    2011-01-07

    Since the discovery that Mn(12)OAc acts as a single-molecule magnet (SMM), an increasing number of transition metal complexes have been demonstrated to behave as SMMs. The signature of a SMM is a slow relaxation of the magnetization at low temperatures accompanied by a magnetic hysteresis. The origin of SMM behaviour is the existence of an appreciable thermal barrier U for spin-reversal called magnetic anisotropy barrier which is related to the combination of a large total spin ground state (S(t)) and an easy-axis magnetic anisotropy. The extensive research on Mn(12)OAc and other SMMs has established more prerequisites for a rational development of new SMMs besides the high-spin ground state and the magnetic anisotropy: the symmetry should be at least C(3) to minimize the quantum tunneling of the magnetization through the anisotropy barrier but lower than cubic to avoid the cancellation of the local anisotropies upon projection onto the spin ground state. Based on these prerequisites, we have designed the ligand triplesalen which combines the phloroglucinol bridging unit for high spin ground states by the spin-polarization mechanism with a salen-like ligand environment for single-site magnetic anisotropies by a strong tetragonal ligand field. The C(3) symmetric, trinuclear complexes of the triplesalen ligand (talen(t-Bu(2)))(6-) exhibit a strong ligand folding resulting in an overall bowl-shaped molecular structure. This ligand folding preorganizes the axial coordination sites of the metal salen subunits for the complementary binding of three facial nitrogen atoms of a hexacyanometallate unit. This leads to a high driving force for the formation of heptanuclear complexes [M(t)(6)M(c)](n+) by the assembly of three molecular building blocks. Attractive van der Waals interactions of the tert-butyl phenyl units of two triplesalen trinuclear building blocks increase the driving force. In this respect, we have been able to synthesize the isostructural series [Mn(III)(6

  7. 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.

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

    Science.gov (United States)

    Scheler, Ott; Kaplinski, Lauris; Glynn, Barry; Palta, Priit; Parkel, Sven; Toome, Kadri; Maher, Majella; Barry, Thomas; Remm, Maido; Kurg, Ants

    2011-02-28

    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. 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. The described technological solution and both its separate components SLICSel and NASBA-microarray technology independently are applicative for many different areas of microbial diagnostics.

  9. 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.

  10. 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.

  11. Studies of G-quadruplex DNA structures at the single molecule level

    DEFF Research Database (Denmark)

    Kragh, Sofie Louise

    2015-01-01

    Folding of G-quaduplex structures adopted by the human telomeric repeat is here studied by single molecule FRET microscopy. This method allows for the investigation of G-quadruplex structures and their conformational dynamic. Telomeres are located at the ends of our chromosomes and end in a single...... with human telomeric repeat adopt several different G-quadruplex conformations in the presence of K+ ions. G-quadruplexes inhibit telomerase activity and are therefore potential targets for anti-cancer drugs, which can be small molecule ligands capable of stabilizing G-quadruplex structures. Understanding...... range. FRET spectroscopy can be performed on an ensemble of molecules, or on the single molecule level. In single molecule FRET experiments it is possible to follow the behaviour in time for each molecule independently, allowing insight into both dynamically and statistically heterogeneous molecular...

  12. Voltage-Driven Conformational Switching with Distinct Raman Signature in a Single-Molecule Junction.

    Science.gov (United States)

    Bi, Hai; Palma, Carlos-Andres; Gong, Yuxiang; Hasch, Peter; Elbing, Mark; Mayor, Marcel; Reichert, Joachim; Barth, Johannes V

    2018-04-11

    Precisely controlling well-defined, stable single-molecule junctions represents a pillar of single-molecule electronics. Early attempts to establish computing with molecular switching arrays were partly challenged by limitations in the direct chemical characterization of metal-molecule-metal junctions. While cryogenic scanning probe studies have advanced the mechanistic understanding of current- and voltage-induced conformational switching, metal-molecule-metal conformations are still largely inferred from indirect evidence. Hence, the development of robust, chemically sensitive techniques is instrumental for advancement in the field. Here we probe the conformation of a two-state molecular switch with vibrational spectroscopy, while simultaneously operating it by means of the applied voltage. Our study emphasizes measurements of single-molecule Raman spectra in a room-temperature stable single-molecule switch presenting a signal modulation of nearly 2 orders of magnitude.

  13. Electrochemistry and bioelectrochemistry towards the single-molecule level: Theoretical notions and systems

    DEFF Research Database (Denmark)

    Zhang, Jingdong; Chi, Qijin; Albrecht, Tim

    2005-01-01

    Surface structures controlled at the nanometer and single-molecule levels, with functions crucially determined by interfacial electron transfer (ET) are broadly reported in recent years, with different kinds of electrochemically controlled nanoscale/single molecule systems. One is the broad class...... tunnelling spectroscopic (STS) features. Mapping of redox metalloproteins from the three major classes, i.e. blue copper proteins, heme proteins, and iron-sulfur proteins, at the monolayer and single-molecule levels have also been achieved. In situ STM and spectroscopy of redox molecules and biomolecules...

  14. Conserved linear dynamics of single-molecule Brownian motion

    Science.gov (United States)

    Serag, Maged F.; Habuchi, Satoshi

    2017-06-01

    Macromolecular diffusion in homogeneous fluid at length scales greater than the size of the molecule is regarded as a random process. The mean-squared displacement (MSD) of molecules in this regime increases linearly with time. Here we show that non-random motion of DNA molecules in this regime that is undetectable by the MSD analysis can be quantified by characterizing the molecular motion relative to a latticed frame of reference. Our lattice occupancy analysis reveals unexpected sub-modes of motion of DNA that deviate from expected random motion in the linear, diffusive regime. We demonstrate that a subtle interplay between these sub-modes causes the overall diffusive motion of DNA to appear to conform to the linear regime. Our results show that apparently random motion of macromolecules could be governed by non-random dynamics that are detectable only by their relative motion. Our analytical approach should advance broad understanding of diffusion processes of fundamental relevance.

  15. Conserved linear dynamics of single-molecule Brownian motion

    KAUST Repository

    Serag, Maged F.

    2017-06-06

    Macromolecular diffusion in homogeneous fluid at length scales greater than the size of the molecule is regarded as a random process. The mean-squared displacement (MSD) of molecules in this regime increases linearly with time. Here we show that non-random motion of DNA molecules in this regime that is undetectable by the MSD analysis can be quantified by characterizing the molecular motion relative to a latticed frame of reference. Our lattice occupancy analysis reveals unexpected sub-modes of motion of DNA that deviate from expected random motion in the linear, diffusive regime. We demonstrate that a subtle interplay between these sub-modes causes the overall diffusive motion of DNA to appear to conform to the linear regime. Our results show that apparently random motion of macromolecules could be governed by non-random dynamics that are detectable only by their relative motion. Our analytical approach should advance broad understanding of diffusion processes of fundamental relevance.

  16. Single-molecule-sensitive fluorescence resonance energy transfer in freely-diffusing attoliter droplets

    Energy Technology Data Exchange (ETDEWEB)

    Rahmanseresht, Sheema; Ramos, Kieran P.; Gamari, Ben D.; Goldner, Lori S., E-mail: lgoldner@physics.umass.edu [Department of Physics, University of Massachusetts, Amherst, Massachusetts 01003 (United States); Milas, Peker [Department of Neuroscience, University of Wisconsin, Madison, Wisconsin 53705 (United States)

    2015-05-11

    Fluorescence resonance energy transfer (FRET) from individual, dye-labeled RNA molecules confined in freely-diffusing attoliter-volume aqueous droplets is carefully compared to FRET from unconfined RNA in solution. The use of freely-diffusing droplets is a remarkably simple and high-throughput technique that facilitates a substantial increase in signal-to-noise for single-molecular-pair FRET measurements. We show that there can be dramatic differences between FRET in solution and in droplets, which we attribute primarily to an altered pH in the confining environment. We also demonstrate that a sufficient concentration of a non-ionic surfactant mitigates this effect and restores FRET to its neutral-pH solution value. At low surfactant levels, even accounting for pH, we observe differences between the distribution of FRET values in solution and in droplets which remain unexplained. Our results will facilitate the use of nanoemulsion droplets as attoliter volume reactors for use in biophysical and biochemical assays, and also in applications such as protein crystallization or nanoparticle synthesis, where careful attention to the pH of the confined phase is required.

  17. Aligned deposition and electrical measurements on single DNA molecules

    DEFF Research Database (Denmark)

    Eidelshtein, Gennady; Kotlyar, Alexander; Hashemi, Mohtadin

    2015-01-01

    . Due to its positive charge, the avidin attached to one end of the DNA anchors the complex to negatively charged substrates. Subsequent drying with a directional gas flow yields DNA molecules perfectly aligned on the surface. In the avidin–DNA complex only the avidin moiety is strongly and irreversibly......A reliable method of deposition of aligned individual dsDNA molecules on mica, silicon, and micro/nanofabricated circuits is presented. Complexes of biotinylated double stranded poly(dG)–poly(dC) DNA with avidin were prepared and deposited on mica and silicon surfaces in the absence of Mg2+ ions...

  18. Handling and Sensing of Single Enzyme Molecules: From Fluorescence Detection towards Nanoscale Electrical Measurements

    DEFF Research Database (Denmark)

    Mathwig, Klaus; Chi, Qijin; Lemay, Serge G.

    2016-01-01

    advances in all-electrical single enzyme studies with a focus on recent micro- and nanofluidic tools, which offer new ways of handling and studying small numbers of molecules or even single enzyme molecules. We particularly emphasize nanofluidic devices, which enable the integration of electrochemical...

  19. Injection molded nanofluidic chips: Fabrication method and functional tests using single-molecule DNA experiments

    DEFF Research Database (Denmark)

    Utko, Pawel; Persson, Karl Fredrik; Kristensen, Anders

    2011-01-01

    We demonstrate that fabrication of nanofluidic systems can be greatly simplified by injection molding of polymers. We functionally test our devices by single-molecule DNA experiments in nanochannels.......We demonstrate that fabrication of nanofluidic systems can be greatly simplified by injection molding of polymers. We functionally test our devices by single-molecule DNA experiments in nanochannels....

  20. Electric-Field Control of Interfering Transport Pathways in a Single-Molecule Anthraquinone Transistor

    NARCIS (Netherlands)

    Koole, Max; Thijssen, Jos M.; Valkenier, Hennie; Hummelen, Jan C.; van der Zant, Herre S. J.

    It is understood that molecular conjugation plays an important role in charge transport through single-molecule junctions. Here, we investigate electron transport through an anthraquinone based single-molecule three-terminal device. With the use of an electric-field induced by a gate electrode, the

  1. Electrochemical detection of single molecules using abiotic nanopores having electrically tunable dimensions

    Energy Technology Data Exchange (ETDEWEB)

    Sansinena, Jose-Maria; Redondo, Antonio; Olazabal, Virginia; Hoffbauer, Mark A.

    2017-09-12

    A barrier structure for use in an electrochemical stochastic membrane sensor for single molecule detection. The sensor is based upon inorganic nanopores having electrically tunable dimensions. The inorganic nanopores are formed from inorganic materials and an electrically conductive polymer. Methods of making the barrier structure and sensing single molecules using the barrier structure are also described.

  2. Single-molecule analysis of DNA replication in Xenopus egg extracts

    NARCIS (Netherlands)

    Yardimci, Hasan; Loveland, Anna B.; van Oijen, Antoine M.; Walter, Johannes C.; Mechali, Marcel

    The recent advent in single-molecule imaging and manipulation methods has made a significant impact on the understanding of molecular mechanisms underlying many essential cellular processes. Single-molecule techniques such as electron microscopy and DNA fiber assays have been employed to study the

  3. Electrochemical detection of single molecules using abiotic nanopores having electrically tunable dimensions

    Science.gov (United States)

    Sansinena, Jose-Maria [Los Alamos, NM; Redondo, Antonio [Los Alamos, NM; Olazabal, Virginia [Los Alamos, NM; Hoffbauer, Mark A [Los Alamos, NM; Akhadov, Elshan A [Los Alamos, NM

    2009-12-29

    A barrier structure for use in an electrochemical stochastic membrane sensor for single molecule detection. The sensor is based upon inorganic nanopores having electrically tunable dimensions. The inorganic nanopores are formed from inorganic materials and an electrically conductive polymer. Methods of making the barrier structure and sensing single molecules using the barrier structure are also described.

  4. Electrochemical detection of single molecules using abiotic nanopores having electrically tunable dimensions

    Energy Technology Data Exchange (ETDEWEB)

    Sansinena, Jose-Maria; Redondo, Antonio; Olazabal, Virginia; Hoffbauer, Mark A.; Akhadov, Elshan A.

    2017-10-31

    A barrier structure for use in an electrochemical stochastic membrane sensor for single molecule detection. The sensor is based upon inorganic nanopores having electrically tunable dimensions. The inorganic nanopores are formed from inorganic materials and an electrically conductive polymer. Methods of making the barrier structure and sensing single molecules using the barrier structure are also described.

  5. Single molecule experimentation in biological physics: exploring the living component of soft condensed matter one molecule at a time.

    Science.gov (United States)

    Harriman, O L J; Leake, M C

    2011-12-21

    The soft matter of biological systems consists of mesoscopic length scale building blocks, composed of a variety of different types of biological molecules. Most single biological molecules are so small that 1 billion would fit on the full-stop at the end of this sentence, but collectively they carry out the vital activities in living cells whose length scale is at least three orders of magnitude greater. Typically, the number of molecules involved in any given cellular process at any one time is relatively small, and so real physiological events may often be dominated by stochastics and fluctuation behaviour at levels comparable to thermal noise, and are generally heterogeneous in nature. This challenging combination of heterogeneity and stochasticity is best investigated experimentally at the level of single molecules, as opposed to more conventional bulk ensemble-average techniques. In recent years, the use of such molecular experimental approaches has become significantly more widespread in research laboratories around the world. In this review we discuss recent experimental approaches in biological physics which can be applied to investigate the living component of soft condensed matter to a precision of a single molecule. © 2011 IOP Publishing Ltd Printed in the UK & the USA

  6. Probing single-molecule electron-hole transfer dynamics at a molecule-NiO semiconductor nanocrystalline interface.

    Science.gov (United States)

    Dhital, Bharat; Rao, Vishal Govind; Lu, H Peter

    2017-07-14

    Interfacial charge transfer dynamics in dye-sensitized NiO nanoparticles are being investigated for photocathodes in p-type dye-sensitized solar cells. In the photoreaction, after fast electron transfer from NiO to a molecule, the recombination of the hole in the nanoparticles with the electron in a reduced molecule plays an important role in the charge separation process and solar energy harvesting. Nevertheless, knowledge of the interfacial charge recombination (CR) rate and its mechanism is still limited due to the complex photoinduced electron and hole dynamics and lack of characterization of the inhomogeneity of the dynamics. Here, we report our work on probing interfacial charge recombination dynamics in Zn(ii)-5,10,15,20-tetra(3-carboxyphenyl)porphyrin (m-ZnTCPP) dye-sensitized NiO nanoparticles by correlating single-molecule fluorescence blinking dynamics with charge transfer dynamics using single-molecule photon-stamping spectroscopy. The correlated analyses of single-molecule fluorescence intensity, lifetime, and blinking reveal the intrinsic distribution and temporal fluctuation of interfacial charge transfer reactivity, which are closely related to site-specific molecular interactions and dynamics.

  7. Detection of a single enzyme molecule based on a solid-state nanopore sensor.

    Science.gov (United States)

    Tan, ShengWei; Gu, DeJian; Liu, Hang; Liu, QuanJun

    2016-04-15

    The nanopore sensor as a high-throughput and low-cost technology can detect a single molecule in a solution. In the present study, relatively large silicon nitride (Si3N4) nanopores with diameters of ∼28 and ∼88 nm were fabricated successfully using a focused Ga ion beam. We have used solid-state nanopores with various sizes to detect the single horseradish peroxidase (HRP) molecule and for the first time analyzed single HRP molecular translocation events. In addition, a real-time monitored single enzyme molecular biochemical reaction and a translocation of the product of enzyme catalysis substrates were investigated by using a Si3N4 nanopore. Our nanopore system showed a high sensitivity in detecting single enzyme molecules and a real-time monitored single enzyme molecular biochemical reaction. This method could also be significant for studying gene expression or enzyme dynamics at the single-molecule level.

  8. Single-Photon Source for Quantum Information Based on Single Dye Molecule Fluorescence in Liquid Crystal Host

    International Nuclear Information System (INIS)

    Lukishova, S.G.; Knox, R.P.; Freivald, P.; McNamara, A.; Boyd, R.W.; Stroud, Jr. C.R.; Schmid, A.W.; Marshall, K.L.

    2006-01-01

    This paper describes a new application for liquid crystals: quantum information technology. A deterministically polarized single-photon source that efficiently produces photons exhibiting antibunching is a pivotal hardware element in absolutely secure quantum communication. Planar-aligned nematic liquid crystal hosts deterministically align the single dye molecules which produce deterministically polarized single (antibunched) photons. In addition, 1-D photonic bandgap cholesteric liquid crystals will increase single-photon source efficiency. The experiments and challenges in the observation of deterministically polarized fluorescence from single dye molecules in planar-aligned glassy nematic-liquid-crystal oligomer as well as photon antibunching in glassy cholesteric oligomer are described for the first time

  9. 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.

  10. Fluorescent labeling of NASBA amplified tmRNA molecules for microarray applications.

    Science.gov (United States)

    Scheler, Ott; Glynn, Barry; Parkel, Sven; Palta, Priit; Toome, Kadri; Kaplinski, Lauris; Remm, Maido; Maher, Majella; Kurg, Ants

    2009-05-15

    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. 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. 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.

  11. 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.

  12. Electrochemistry and bioelectrochemistry towards the single-molecule level: Theoretical notions and systems

    International Nuclear Information System (INIS)

    Zhang Jingdong; Chi Qijin; Albrecht, Tim; Kuznetsov, Alexander M.; Grubb, Mikala; Hansen, Allan G.; Wackerbarth, Hainer; Welinder, Anne C.; Ulstrup, Jens

    2005-01-01

    Surface structures controlled at the nanometer and single-molecule levels, with functions crucially determined by interfacial electron transfer (ET) are broadly reported in recent years, with different kinds of electrochemically controlled nanoscale/single molecule systems. One is the broad class of metallic and semiconductor-based nanoparticles, nano-arrays, nanotubes, and nanopits. Others are based on self-assembled molecular monolayers. The latter extend to bioelectrochemical systems with redox metalloproteins and DNA-based molecules as targets. We overview here some recent achievements in areas of interfacial electrochemical ET systems, mapped to the nanoscale and single-molecule levels. Focus is on both experimental and theoretical studies in our group. Systems addressed are organized monolayers of redox active transition metal complexes, and metalloproteins and metalloenzymes on single-crystal Au(1 1 1)-electrode surfaces. These systems have been investigated by voltammetry, spectroscopy, microcantilever technology, and scanning probe microscopy. A class of Os-complexes has shown suitable as targets for electrochemical in situ scanning tunnelling microscopy (STM), with close to single-molecule scanning tunnelling spectroscopic (STS) features. Mapping of redox metalloproteins from the three major classes, i.e. blue copper proteins, heme proteins, and iron-sulfur proteins, at the monolayer and single-molecule levels have also been achieved. In situ STM and spectroscopy of redox molecules and biomolecules have been supported by new theoretical frames, which extend established theory of interfacial electrochemical ET. The electrochemical nanoscale and single-molecule systems discussed are compared with other recent nanoscale and single-molecule systems with conspicuous device-like properties, particularly unimolecular rectifiers and single-molecule transistors. Both of these show analogies to electrochemical in situ STM features of redox molecules and

  13. Molecules with multiple switching units on a Au(111) surface: self-organization and single-molecule manipulation

    Science.gov (United States)

    Mielke, Johannes; Selvanathan, Sofia; Peters, Maike; Schwarz, Jutta; Hecht, Stefan; Grill, Leonhard

    2012-10-01

    Three different molecules, each containing two azobenzene switching units, were synthesized, successfully deposited onto a Au(111) surface by sublimation and studied by scanning tunneling microscopy at low temperatures. To investigate the influence of electronic coupling between the switching units as well as to the surface, the two azo moieties were connected either via π-conjugated para-phenylene or decoupling meta-phenylene bridges, and the number of tert-butyl groups was varied in the meta-phenylene-linked derivatives. Single molecules were found to be intact after deposition as identified by their characteristic appearance in STM images. Due to their mobility on the Au(111) surface at room temperature, the molecules spontaneously formed self-organized molecular arrangements that reflected their chemical structure. While lateral displacement of the molecules was accomplished by manipulation, trans-cis isomerization processes, typical for azobenzene switches, could not be induced.

  14. Experimental and computational characterization of biological liquid crystals: a review of single-molecule bioassays.

    Science.gov (United States)

    Eom, Kilho; Yang, Jaemoon; Park, Jinsung; Yoon, Gwonchan; Soo Sohn, Young; Park, Shinsuk; Yoon, Dae Sung; Na, Sungsoo; Kwon, Taeyun

    2009-09-10

    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.

  15. Physics of Polymers under Nanoscopic Confinement: a Single Molecule Study

    NARCIS (Netherlands)

    Keshavarz, M.

    2016-01-01

    Physicist Masoumeh Keshavarz studied the thermal motion of a fluorescently labelled, individual “reporter” polymer molecule, surrounded and entangled by a gel of similar but unlabelled polymers. Owing to their extreme length and stiffness, it is possible to follow the shape and the motion of the

  16. Computing magnetic anisotropy constants of single molecule magnets

    Indian Academy of Sciences (India)

    Administrator

    stringent requirements for a molecule to behave as a. SMM. Modelling magnetic anisotropy in these sys- tems becomes necessary for developing new SMMs with desired properties. Magnetic anisotropy of SMMs is computed by treating the anisotropy Hamiltonian as a perturbation over the Heisenberg exchange ...

  17. Single-molecule microscopy using silicone oil immersion objective lenses

    NARCIS (Netherlands)

    Hink, M.

    2012-01-01

    Microscopy techniques capable of detecting individual molecules and providing quantitative data have the potential to offer great biological insight; however, such approaches require the efficient capture of light. Here, Mark Hink explains how the use of new silicone oil immersion objective lenses

  18. Single-molecule experiments in biophysics: Exploring the thermal ...

    Indian Academy of Sciences (India)

    bath by allowing biomolecules to exchange energy with the molecules of the solvent through the breakage ... nonequilibrium process are generated from ATP consumption during the hydrolysis cycle. Often molecular ... A central notion in thermodynamics of small systems is the concept of control para- meter [5]. This is akin ...

  19. Chemical Kinetics at the Single-Molecule Level

    Science.gov (United States)

    Levitus, Marcia

    2011-01-01

    For over a century, chemists have investigated the rates of chemical reactions using experimental conditions involving huge numbers of molecules. As a consequence, the description of the kinetics of the reaction in terms of average values was good enough for all practical purposes. From the pedagogical point of view, such a description misses the…

  20. Alternative types of molecule-decorated atomic chains in Au–CO–Au single-molecule junctions

    Directory of Open Access Journals (Sweden)

    Zoltán Balogh

    2015-06-01

    Full Text Available We investigate the formation and evolution of Au–CO single-molecule break junctions. The conductance histogram exhibits two distinct molecular configurations, which are further investigated by a combined statistical analysis. According to conditional histogram and correlation analysis these molecular configurations show strong anticorrelations with each other and with pure Au monoatomic junctions and atomic chains. We identify molecular precursor configurations with somewhat higher conductance, which are formed prior to single-molecule junctions. According to detailed length analysis two distinct types of molecule-affected chain-formation processes are observed, and we compare these results to former theoretical calculations considering bridge- and atop-type molecular configurations where the latter has reduced conductance due to destructive Fano interference.

  1. Bianthrone in a Single-Molecule Junction: Conductance Switching with a Bistable Molecule Facilitated by Image Charge Effects

    DEFF Research Database (Denmark)

    Bjørnholm, Thomas

    2010-01-01

    Bianthrone is a sterically hindered compound that exists in the form of two nonplanar isomers. Our experimental study of single-molecule junctions with bianthrone reveals persistent switching of electric conductance at low temperatures, which can be reasonably associated with molecular isomerizat...

  2. Electronic Transport in Single Molecule Junctions: Control of the Molecule-Electrode Coupling Through Intramolecular Tunneling Barriers

    DEFF Research Database (Denmark)

    Danilov, Andrey; Kubatkin, Sergey; Kafanov, Sergey

    2008-01-01

    We report on single molecule electron transport measurements of two oligophenylenevinylene (OPV3) derivatives placed in a nanogap between gold (Au) or lead (Pb) electrodes in a field effect transistor device. Both derivatives contain thiol end groups that allow chemical binding to the electrodes...... to sequential tunneling and Coulomb blockade behavior....

  3. A Starting Point for Fluorescence-Based Single-Molecule Measurements in Biomolecular Research

    Directory of Open Access Journals (Sweden)

    Alexander Gust

    2014-09-01

    Full Text Available Single-molecule fluorescence techniques are ideally suited to provide information about the structure-function-dynamics relationship of a biomolecule as static and dynamic heterogeneity can be easily detected. However, what type of single-molecule fluorescence technique is suited for which kind of biological question and what are the obstacles on the way to a successful single-molecule microscopy experiment? In this review, we provide practical insights into fluorescence-based single-molecule experiments aiming for scientists who wish to take their experiments to the single-molecule level. We especially focus on fluorescence resonance energy transfer (FRET experiments as these are a widely employed tool for the investigation of biomolecular mechanisms. We will guide the reader through the most critical steps that determine the success and quality of diffusion-based confocal and immobilization-based total internal reflection fluorescence microscopy. We discuss the specific chemical and photophysical requirements that make fluorescent dyes suitable for single-molecule fluorescence experiments. Most importantly, we review recently emerged photoprotection systems as well as passivation and immobilization strategies that enable the observation of fluorescently labeled molecules under biocompatible conditions. Moreover, we discuss how the optical single-molecule toolkit has been extended in recent years to capture the physiological complexity of a cell making it even more relevant for biological research.

  4. Assembly and dynamics of the U4/U6 di-snRNP by single-molecule FRET

    Science.gov (United States)

    Hardin, John W.; Warnasooriya, Chandani; Kondo, Yasushi; Nagai, Kiyoshi; Rueda, David

    2015-01-01

    In large ribonucleoprotein machines, such as ribosomes and spliceosomes, RNA functions as an assembly scaffold as well as a critical catalytic component. Protein binding to the RNA scaffold can induce structural changes, which in turn modulate subsequent binding of other components. The spliceosomal U4/U6 di-snRNP contains extensively base paired U4 and U6 snRNAs, Snu13, Prp31, Prp3 and Prp4, seven Sm and seven LSm proteins. We have studied successive binding of all protein components to the snRNA duplex during di-snRNP assembly by electrophoretic mobility shift assay and accompanying conformational changes in the U4/U6 RNA 3-way junction by single-molecule FRET. Stems I and II of the duplex were found to co-axially stack in free RNA and function as a rigid scaffold during the entire assembly, but the U4 snRNA 5′ stem-loop adopts alternative orientations each stabilized by Prp31 and Prp3/4 binding accounting for altered Prp3/4 binding affinities in presence of Prp31. PMID:26503251

  5. Understanding the kinetic mechanism of RNA single base pair formation.

    Science.gov (United States)

    Xu, Xiaojun; Yu, Tao; Chen, Shi-Jie

    2016-01-05

    RNA functions are intrinsically tied to folding kinetics. The most elementary step in RNA folding is the closing and opening of a base pair. Understanding this elementary rate process is the basis for RNA folding kinetics studies. Previous studies mostly focused on the unfolding of base pairs. Here, based on a hybrid approach, we investigate the folding process at level of single base pairing/stacking. The study, which integrates molecular dynamics simulation, kinetic Monte Carlo simulation, and master equation methods, uncovers two alternative dominant pathways: Starting from the unfolded state, the nucleotide backbone first folds to the native conformation, followed by subsequent adjustment of the base conformation. During the base conformational rearrangement, the backbone either retains the native conformation or switches to nonnative conformations in order to lower the kinetic barrier for base rearrangement. The method enables quantification of kinetic partitioning among the different pathways. Moreover, the simulation reveals several intriguing ion binding/dissociation signatures for the conformational changes. Our approach may be useful for developing a base pair opening/closing rate model.

  6. Single-molecule diffusion and conformational dynamics by spatial integration of temporal fluctuations

    KAUST Repository

    Serag, Maged F.

    2014-10-06

    Single-molecule localization and tracking has been used to translate spatiotemporal information of individual molecules to map their diffusion behaviours. However, accurate analysis of diffusion behaviours and including other parameters, such as the conformation and size of molecules, remain as limitations to the method. Here, we report a method that addresses the limitations of existing single-molecular localization methods. The method is based on temporal tracking of the cumulative area occupied by molecules. These temporal fluctuations are tied to molecular size, rates of diffusion and conformational changes. By analysing fluorescent nanospheres and double-stranded DNA molecules of different lengths and topological forms, we demonstrate that our cumulative-area method surpasses the conventional single-molecule localization method in terms of the accuracy of determined diffusion coefficients. Furthermore, the cumulative-area method provides conformational relaxation times of structurally flexible chains along with diffusion coefficients, which together are relevant to work in a wide spectrum of scientific fields.

  7. Probing the local environment of a single OPE3 molecule using inelastic tunneling electron spectroscopy.

    Science.gov (United States)

    Frisenda, Riccardo; Perrin, Mickael L; van der Zant, Herre S J

    2015-01-01

    We study single-molecule oligo(phenylene ethynylene)dithiol junctions by means of inelastic electron tunneling spectroscopy (IETS). The molecule is contacted with gold nano-electrodes formed with the mechanically controllable break junction technique. We record the IETS spectrum of the molecule from direct current measurements, both as a function of time and electrode separation. We find that for fixed electrode separation the molecule switches between various configurations, which are characterized by different IETS spectra. Similar variations in the IETS signal are observed during atomic rearrangements upon stretching of the molecular junction. Using quantum chemistry calculations, we identity some of the vibrational modes which constitute a chemical fingerprint of the molecule. In addition, changes can be attributed to rearrangements of the local molecular environment, in particular at the molecule-electrode interface. This study shows the importance of taking into account the interaction with the electrodes when describing inelastic contributions to transport through single-molecule junctions.

  8. Single molecule experiments challenge the strict wave-particle dualism of light.

    Science.gov (United States)

    Greulich, Karl Otto

    2010-01-21

    Single molecule techniques improve our understanding of the photon and light. If the single photon double slit experiment is performed at the "single photon limit" of a multi-atom light source, faint light pulses with more than one photon hamper the interpretation. Single molecules, quantum dots or defect centres in crystals should be used as light source. "Single photon detectors" do not meet their promise-only "photon number resolving single photon detectors" do so. Particularly, the accumulation time argument, the only safe basis for the postulate of a strictly particle like photon, has so far not yet been verified.

  9. Single Molecule Experiments Challenge the Strict Wave-Particle Dualism of Light

    Directory of Open Access Journals (Sweden)

    Karl Otto Greulich

    2010-01-01

    Full Text Available Single molecule techniques improve our understanding of the photon and light. If the single photon double slit experiment is performed at the “single photon limit” of a multi-atom light source, faint light pulses with more than one photon hamper the interpretation. Single molecules, quantum dots or defect centres in crystals should be used as light source. “Single photon detectors” do not meet their promise―only “photon number resolving single photon detectors” do so. Particularly, the accumulation time argument, the only safe basis for the postulate of a strictly particle like photon, has so far not yet been verified.

  10. Single-Molecule Transport at a Rectifying GaAs Contact.

    Science.gov (United States)

    Vezzoli, Andrea; Brooke, Richard J; Ferri, Nicolò; Higgins, Simon J; Schwarzacher, Walther; Nichols, Richard J

    2017-02-08

    In most single- or few-molecule devices, the contact electrodes are simple ohmic resistors. Here we describe a new type of single-molecule device in which metal and semiconductor contact electrodes impart a function, namely, current rectification, which is then modified by a molecule bridging the gap. We study junctions with the structure Au STM tip/X/n-GaAs substrate, where "X" is either a simple alkanedithiol or a conjugated unit bearing thiol/methylthiol contacts, and we detect current jumps corresponding to the attachment and detachment of single molecules. From the magnitudes of the current jumps we can deduce values for the conductance decay constant with molecule length that agree well with values determined from Au/molecule/Au junctions. The ability to impart functionality to a single-molecule device through the properties of the contacts as well as through the properties of the molecule represents a significant extension of the single-molecule electronics "tool-box".

  11. Modulation and Control of Charge Transport Through Single-Molecule Junctions.

    Science.gov (United States)

    Wang, Kun; Xu, Bingqian

    2017-02-01

    The ability to modulate and control charge transport though single-molecule junction devices is crucial to achieving the ultimate goal of molecular electronics: constructing real-world-applicable electronic components from single molecules. This review aims to highlight the progress made in single-molecule electronics, emphasizing the development of molecular junction electronics in recent years. Among many techniques that attempt to wire a molecule to metallic electrodes, the single-molecule break junction (SMBJ) technique is one of the most reliable and tunable experimental platforms for achieving metal-molecule-metal configurations. It also provides great freedom to tune charge transport through the junction. Soon after the SMBJ technique was introduced, it was extensively used to measure the conductances of individual molecules; however, different conductances were obtained for the same molecule, and it proved difficult to interpret this wide distribution of experimental data. This phenomenon was later found to be mainly due to a lack of precise experimental control and advanced data analysis methods. In recent years, researchers have directed considerable effort into advancing the SMBJ technique by gaining a deeper physical understanding of charge transport through single molecules and thus enhancing its potential applicability in functional molecular-scale electronic devices, such as molecular diodes and molecular transistors. In parallel with that research, novel data analysis methods and approaches that enable the discovery of hidden yet important features in the data are being developed. This review discusses various aspects of molecular junction electronics, from the initial goal of molecular electronics, the development of experimental techniques for creating single-molecule junctions and determining single-molecule conductance, to the characterization of functional current-voltage features and the investigation of physical properties other than charge

  12. Understanding the similarity in thermophoresis between single- and double-stranded DNA or RNA

    Science.gov (United States)

    Reichl, Maren; Herzog, Mario; Greiss, Ferdinand; Wolff, Manuel; Braun, Dieter

    2015-06-01

    Thermophoresis is the movement of molecules in a temperature gradient. For aqueous solutions its microscopic basis is debated. Understanding thermophoresis for this case is, however, important since it proved very useful to detect the binding affinity of biomolecules and since thermophoresis could have played an important role in early molecular evolution. Here we discuss why the thermophoresis of single- and double-stranded oligonucleotides - DNA and RNA - is surprisingly similar. This finding is understood by comparing the spherical capacitor model for single-stranded species with the case of a rod-shaped model for double-stranded oligonucleotides. The approach describes thermophoresis of DNA and RNA with fitted effective charges consistent with electrophoresis measurements and explains the similarity between single- and double-stranded species. We could not confirm the sign change for the thermophoresis of single- versus double-stranded DNA in crowded solutions containing polyethylene glycol [Y. T. Maeda, T. Tlusty, and A. Libchaber, Proc. Natl. Acad. Sci. USA 109, 17972 (2012), 10.1073/pnas.1215764109], but find a salt-independent offset while the Debye length dependence still satisfies the capacitor model. Overall, the analysis documents the continuous progress in the microscopic understanding of thermophoresis.

  13. Quantum interference effects at room temperature in OPV-based single-molecule junctions

    DEFF Research Database (Denmark)

    Arroyo, Carlos R.; Frisenda, Riccardo; Moth-Poulsen, Kasper

    2013-01-01

    Interference effects on charge transport through an individual molecule can lead to a notable modulation and suppression on its conductance. In this letter, we report the observation of quantum interference effects occurring at room temperature in single-molecule junctions based on oligo(3......)-phenylenevinylene (OPV3) derivatives, in which the central benzene ring is coupled to either para- or meta-positions. Using the break-junction technique, we find that the conductance for a single meta-OPV3 molecule wired between gold electrodes is one order of magnitude smaller than that of a para-OPV3 molecule...

  14. Single-molecule measurements and dynamical simulations of protein molecules near silicon substrates

    International Nuclear Information System (INIS)

    Hanasaki, Itsuo; Kawano, Satoyuki; Takahashi, Hiroto; Sazaki, Gen; Nakajima, Kazuo

    2008-01-01

    Interactions between protein molecules and inorganic substrates were studied both experimentally and numerically to obtain fundamental insight into the assembly of biomacromolecules for engineering applications. We experimentally traced individual fluorescent-labelled lysozyme (F-lysozyme) molecules, diffusing in the vicinity of interfaces between a protein solution and oxidized Si(1 0 0) and glass plates. The results indicate that diffusion coefficients of F-lysozyme molecules on both substrates are more than three orders of magnitude smaller than those in a bulk solution. The molecular dynamics simulations reveal a drastically diminished diffusion coefficient of lysozyme on the substrates of pure Si(1 1 1) and oxidized Si(1 0 0) with a hydroxy-terminated surface compared with that in bulk solution due to molecular adsorption behaviour on the substrate, which is in good agreement with experimental results. Furthermore, full atomistic description of the behaviour provides detailed information of deformation due to the adsorption process. Lysozyme on pure Si(1 1 1) undergoes substantial deformation whereas that on oxidized Si(1 0 0) does not, which indicates the importance of substrate surface condition to preserve the structure, i.e. functionality of adsorbed biomolecules

  15. Spectrally Resolved and Functional Super-resolution Microscopy via Ultrahigh-Throughput Single-Molecule Spectroscopy.

    Science.gov (United States)

    Yan, Rui; Moon, Seonah; Kenny, Samuel J; Xu, Ke

    2018-03-20

    As an elegant integration of the spatial and temporal dimensions of single-molecule fluorescence, single-molecule localization microscopy (SMLM) overcomes the diffraction-limited resolution barrier of optical microscopy by localizing single molecules that stochastically switch between fluorescent and dark states over time. While this type of super-resolution microscopy (SRM) technique readily achieves remarkable spatial resolutions of ∼10 nm, it typically provides no spectral information. Meanwhile, current scanning-based single-location approaches for mapping the positions and spectra of single molecules are limited by low throughput and are difficult to apply to densely labeled (bio)samples. In this Account, we summarize the rationale, design, and results of our recent efforts toward the integration of the spectral dimension of single-molecule fluorescence with SMLM to achieve spectrally resolved SMLM (SR-SMLM) and functional SRM ( f-SRM). By developing a wide-field scheme for spectral measurement and implementing single-molecule fluorescence on-off switching typical of SMLM, we first showed that in densely labeled (bio)samples it is possible to record the fluorescence spectra and positions of millions of single molecules synchronously within minutes, giving rise to ultrahigh-throughput single-molecule spectroscopy and SR-SMLM. This allowed us to first show statistically that for many dyes, single molecules of the same species exhibit near identical emission in fixed cells. This narrow distribution of emission wavelengths, which contrasts markedly with previous results at solid surfaces, allowed us to unambiguously identify single molecules of spectrally similar dyes. Crosstalk-free, multiplexed SRM was thus achieved for four dyes that were merely 10 nm apart in emission spectrum, with the three-dimensional SRM images of all four dyes being automatically aligned within one image channel. The ability to incorporate single-molecule fluorescence measurement with

  16. Thermophoretic forces on DNA measured with a single-molecule spring balance

    DEFF Research Database (Denmark)

    Pedersen, Jonas Nyvold; Lüscher, Christopher James; Marie, Rodolphe

    2014-01-01

    of the thermophoretic force in a static configuration finds forces up to 130 fN. This is eleven times stronger than the force experienced by the same molecule in the same thermal gradient in bulk, where the molecule shields itself. Our stronger forces stretch the middle of the molecule up to 80% of its contour length......We stretch a single DNA molecule with thermophoretic forces and measure these forces with a spring balance: the DNA molecule itself. It is an entropic spring which we calibrate, using as a benchmark its Brownian motion in the nanochannel that contains and prestretches it. This direct measurement...

  17. Single-molecule electronics: Cooling individual vibrational modes by the tunneling current.

    Science.gov (United States)

    Lykkebo, Jacob; Romano, Giuseppe; Gagliardi, Alessio; Pecchia, Alessandro; Solomon, Gemma C

    2016-03-21

    Electronic devices composed of single molecules constitute the ultimate limit in the continued downscaling of electronic components. A key challenge for single-molecule electronics is to control the temperature of these junctions. Controlling heating and cooling effects in individual vibrational modes can, in principle, be utilized to increase stability of single-molecule junctions under bias, to pump energy into particular vibrational modes to perform current-induced reactions, or to increase the resolution in inelastic electron tunneling spectroscopy by controlling the life-times of phonons in a molecule by suppressing absorption and external dissipation processes. Under bias the current and the molecule exchange energy, which typically results in heating of the molecule. However, the opposite process is also possible, where energy is extracted from the molecule by the tunneling current. Designing a molecular "heat sink" where a particular vibrational mode funnels heat out of the molecule and into the leads would be very desirable. It is even possible to imagine how the vibrational energy of the other vibrational modes could be funneled into the "cooling mode," given the right molecular design. Previous efforts to understand heating and cooling mechanisms in single molecule junctions have primarily been concerned with small models, where it is unclear which molecular systems they correspond to. In this paper, our focus is on suppressing heating and obtaining current-induced cooling in certain vibrational modes. Strategies for cooling vibrational modes in single-molecule junctions are presented, together with atomistic calculations based on those strategies. Cooling and reduced heating are observed for two different cooling schemes in calculations of atomistic single-molecule junctions.

  18. Small Molecules Facilitate Single Factor-Mediated Hepatic Reprogramming

    Directory of Open Access Journals (Sweden)

    Kyung Tae Lim

    2016-04-01

    Full Text Available Recent studies have shown that defined factors could lead to the direct conversion of fibroblasts into induced hepatocyte-like cells (iHeps. However, reported conversion efficiencies are very low, and the underlying mechanism of the direct hepatic reprogramming is largely unknown. Here, we report that direct conversion into iHeps is a stepwise transition involving the erasure of somatic memory, mesenchymal-to-epithelial transition, and induction of hepatic cell fate in a sequential manner. Through screening for additional factors that could potentially enhance the conversion kinetics, we have found that c-Myc and Klf4 (CK dramatically accelerate conversion kinetics, resulting in remarkably improved iHep generation. Furthermore, we identified small molecules that could lead to the robust generation of iHeps without CK. Finally, we show that Hnf1α supported by small molecules is sufficient to efficiently induce direct hepatic reprogramming. This approach might help to fully elucidate the direct conversion process and also facilitate the translation of iHep into the clinic.

  19. Small Molecules Facilitate Single Factor-Mediated Hepatic Reprogramming.

    Science.gov (United States)

    Lim, Kyung Tae; Lee, Seung Chan; Gao, Yimeng; Kim, Kee-Pyo; Song, Guangqi; An, Su Yeon; Adachi, Kenjiro; Jang, Yu Jin; Kim, Jonghun; Oh, Kyoung-Jin; Kwak, Tae Hwan; Hwang, Seon In; You, Jueng Soo; Ko, Kinarm; Koo, Seung-Hoi; Sharma, Amar Deep; Kim, Jong-Hoon; Hui, Lijian; Cantz, Tobias; Schöler, Hans R; Han, Dong Wook

    2016-04-26

    Recent studies have shown that defined factors could lead to the direct conversion of fibroblasts into induced hepatocyte-like cells (iHeps). However, reported conversion efficiencies are very low, and the underlying mechanism of the direct hepatic reprogramming is largely unknown. Here, we report that direct conversion into iHeps is a stepwise transition involving the erasure of somatic memory, mesenchymal-to-epithelial transition, and induction of hepatic cell fate in a sequential manner. Through screening for additional factors that could potentially enhance the conversion kinetics, we have found that c-Myc and Klf4 (CK) dramatically accelerate conversion kinetics, resulting in remarkably improved iHep generation. Furthermore, we identified small molecules that could lead to the robust generation of iHeps without CK. Finally, we show that Hnf1α supported by small molecules is sufficient to efficiently induce direct hepatic reprogramming. This approach might help to fully elucidate the direct conversion process and also facilitate the translation of iHep into the clinic. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    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

  1. Research Update: Molecular electronics: The single-molecule switch and transistor

    Directory of Open Access Journals (Sweden)

    Kai Sotthewes

    2014-01-01

    Full Text Available In order to design and realize single-molecule devices it is essential to have a good understanding of the properties of an individual molecule. For electronic applications, the most important property of a molecule is its conductance. Here we show how a single octanethiol molecule can be connected to macroscopic leads and how the transport properties of the molecule can be measured. Based on this knowledge we have realized two single-molecule devices: a molecular switch and a molecular transistor. The switch can be opened and closed at will by carefully adjusting the separation between the electrical contacts and the voltage drop across the contacts. This single-molecular switch operates in a broad temperature range from cryogenic temperatures all the way up to room temperature. Via mechanical gating, i.e., compressing or stretching of the octanethiol molecule, by varying the contact's interspace, we are able to systematically adjust the conductance of the electrode-octanethiol-electrode junction. This two-terminal single-molecule transistor is very robust, but the amplification factor is rather limited.

  2. Axial Colocalization of Single Molecules with Nanometer Accuracy Using Metal-Induced Energy Transfer.

    Science.gov (United States)

    Isbaner, Sebastian; Karedla, Narain; Kaminska, Izabela; Ruhlandt, Daja; Raab, Mario; Bohlen, Johann; Chizhik, Alexey; Gregor, Ingo; Tinnefeld, Philip; Enderlein, Jörg; Tsukanov, Roman

    2018-03-27

    Single-molecule localization based super-resolution microscopy has revolutionized optical microscopy and routinely allows for resolving structural details down to a few nanometers. However, there exists a rather large discrepancy between lateral and axial localization accuracy, the latter typically three to five times worse than the former. Here, we use single-molecule metal-induced energy transfer (smMIET) to localize single molecules along the optical axis, and to measure their axial distance with an accuracy of 5 nm. smMIET relies only on fluorescence lifetime measurements and does not require additional complex optical setups.

  3. Conformational analysis of single perfluoroalkyl chains by single-molecule real-time transmission electron microscopic imaging.

    Science.gov (United States)

    Harano, Koji; Takenaga, Shinya; Okada, Satoshi; Niimi, Yoshiko; Yoshikai, Naohiko; Isobe, Hiroyuki; Suenaga, Kazu; Kataura, Hiromichi; Koshino, Masanori; Nakamura, Eiichi

    2014-01-08

    Whereas a statistical average of molecular ensembles has been the conventional source of information on molecular structures, atomic resolution movies of single organic molecules obtained by single-molecule real-time transmission electron microscopy have recently emerged as a new tool to study the time evolution of the structures of individual molecules. The present work describes a proof-of-principle study of the determination of the conformation of each C-C bond in single perfluoroalkyl fullerene molecules encapsulated in a single-walled carbon nanotube (CNT) as well as those attached to the outer surface of a carbon nanohorn (CNH). Analysis of 82 individual molecules in CNTs under a 120 kV electron beam indicated that 6% of the CF2-CF2 bonds and about 20% of the CH2-CH2 bonds in the corresponding hydrocarbon analogue are in the gauche conformation. This comparison qualitatively matches the known conformational data based on time- and molecular-average as determined for ensembles. The transmission electron microscopy images also showed that the molecules entered the CNTs predominantly in one orientation. The molecules attached on a CNH surface moved more freely and exhibited more diverse conformation than those in a CNT, suggesting the potential applicability of this method for the determination of the dynamic shape of flexible molecules and of detailed conformations. We observed little sign of any decomposition of the specimen molecules, at least up to 10(7) e·nm(-2) (electrons/nm(2)) at 120 kV acceleration voltage. Decomposition of CNHs under irradiation with a 300 kV electron beam was suppressed by cooling to 77 K, suggesting that the decomposition is a chemical process. Several lines of evidence suggest that the graphitic substrate and the attached molecules are very cold.

  4. Effect of the HIV-1 nucleocapsid protein on reverse transcriptase pause sites revealed by single molecule microscopy

    Science.gov (United States)

    Jouonang, A.; Przybilla, F.; Godet, J.; Sharma, K. K.; Restlé, T.; de Rocquigny, H.; Darlix, J.-L.; Kenfack, C.; Didier, P.; Mély, Y.

    2013-02-01

    During reverse transcription, the HIV-1 RNA is converted by the reverse transcriptase (RT) into proviral DNA. RT is assisted by the HIV-1 nucleocapsid (NCp7) protein that notably increases the ability of RT to synthesize DNA through pause sites. Using single molecule FRET, we monitored the NCp7 effect on the binding of RT to nucleic acid sequences corresponding to two different pause sites. NCp7 was found to modify the distribution of RT orientations on the oligonucleotides and decrease the residence time of RT on one of the pause sites. These results give direct insight into the NCp7 molecular mechanism in reverse transcription.

  5. Flicker Noise as a Probe of Electronic Interaction at Metal-Single Molecule Interfaces.

    Science.gov (United States)

    Adak, Olgun; Rosenthal, Ethan; Meisner, Jeffery; Andrade, Erick F; Pasupathy, Abhay N; Nuckolls, Colin; Hybertsen, Mark S; Venkataraman, Latha

    2015-06-10

    Charge transport properties of metal-molecule interfaces depend strongly on the character of molecule-electrode interactions. Although through-bond coupled systems have attracted the most attention, through-space coupling is important in molecular systems when, for example, through-bond coupling is suppressed due to quantum interference effects. To date, a probe that clearly distinguishes these two types of coupling has not yet been demonstrated. Here, we investigate the origin of flicker noise in single molecule junctions and demonstrate how the character of the molecule-electrode coupling influences the flicker noise behavior of single molecule junctions. Importantly, we find that flicker noise shows a power law dependence on conductance in all junctions studied with an exponent that can distinguish through-space and through-bond coupling. Our results provide a new and powerful tool for probing and understanding coupling at the metal-molecule interface.

  6. Probing the local environment of a single OPE3 molecule using inelastic tunneling electron spectroscopy

    Directory of Open Access Journals (Sweden)

    Riccardo Frisenda

    2015-12-01

    Full Text Available We study single-molecule oligo(phenylene ethynylenedithiol junctions by means of inelastic electron tunneling spectroscopy (IETS. The molecule is contacted with gold nano-electrodes formed with the mechanically controllable break junction technique. We record the IETS spectrum of the molecule from direct current measurements, both as a function of time and electrode separation. We find that for fixed electrode separation the molecule switches between various configurations, which are characterized by different IETS spectra. Similar variations in the IETS signal are observed during atomic rearrangements upon stretching of the molecular junction. Using quantum chemistry calculations, we identity some of the vibrational modes which constitute a chemical fingerprint of the molecule. In addition, changes can be attributed to rearrangements of the local molecular environment, in particular at the molecule–electrode interface. This study shows the importance of taking into account the interaction with the electrodes when describing inelastic contributions to transport through single-molecule junctions.

  7. The elastic theory of a single DNA molecule

    Indian Academy of Sciences (India)

    We study the elastic responses of double- (ds) and single-stranded (ss) DNA at external force fields. A double-strand-polymer elastic model is constructed and solved by path integral methods and Monte Carlo simulations to understand the entropic elasticity, cooperative extensibility, and supercoiling property of dsDNA.

  8. The elastic theory of a single DNA molecule

    Indian Academy of Sciences (India)

    Abstract. We study the elastic responses of double- (ds) and single-stranded (ss) DNA at exter- nal force fields. A double-strand-polymer elastic model is constructed and solved by path integral methods and Monte Carlo simulations to understand the entropic elasticity, cooperative extensibil- ity, and supercoiling property of ...

  9. New tools to study biophysical properties of single molecules and single cells

    Directory of Open Access Journals (Sweden)

    Márcio S. Rocha

    2007-03-01

    Full Text Available We present a review on two new tools to study biophysical properties of single molecules and single cells. A laser incident through a high numerical aperture microscope objective can trap small dielectric particles near the focus. This arrangement is named optical tweezers. This technique has the advantage to permit manipulation of a single individual object. We use optical tweezers to measure the entropic elasticity of a single DNA molecule and its interaction with the drug Psoralen. Optical tweezers are also used to hold a kidney cell MDCK away from the substrate to allow precise volume measurements of this single cell during an osmotic shock. This procedure allows us to obtain information about membrane water permeability and regulatory volume increase. Defocusing microscopy is a recent technique invented in our laboratory, which allows the observation of transparent objects, by simply defocusing the microscope in a controlled way. Our physical model of a defocused microscope shows that the image contrast observed in this case is proportional to the defocus distance and to the curvature of the transparent object. Defocusing microscopy is very useful to study motility and mechanical properties of cells. We show here the application of defocusing microscopy to measurements of macrophage surface fluctuations and their influence on phagocytosis.Apresentamos uma revisão de duas novas técnicas para estudar propriedades biofísicas de moléculas únicas e células únicas. Um laser incidindo em uma objetiva de microscópio de grande abertura numérica é capaz de aprisionar pequenas partículas dielétricas na região próxima ao foco. Este aparato é chamado de pinça óptica. Esta técnica tem a grande vantagem de permitir a manipulação de um objeto individual. Usamos a pinça óptica para medir a elasticidade entrópica de uma molécula única de DNA em sua interação com o fármaco Psoralen. A pinça óptica também é usada para segurar

  10. Optical and Electrical Detection of Single Molecule Translocation through Carbon Nanotubes

    Science.gov (United States)

    Song, Weisi; Pang, Pei; He, Jin; Lindsay, Stuart

    2013-01-01

    Ion current through a single-walled carbon nanotube (SWCNT) was monitored at the same time as fluorescence was recorded from charged dye molecules translocating through the SWCNT. Fluorescence bursts generally follow ion-current peaks with a delay time consistent with diffusion from the end of the SWCNT to the fluorescence collection point. The fluorescence amplitude distribution of the bursts is consistent with single molecule signals. Thus each peak in the ion current flowing through the SWCNT is associated with the translocation of a single molecule. Ion current peaks (as opposed to blockades) were produced by both positively (Rhodamine 6G) and negatively (Alexa 546) charged molecules, showing that the charge filtering responsible for the current bursts is caused by the molecules themselves. PMID:23248975

  11. Estimating single molecule conductance from spontaneous evolution of a molecular contact

    Science.gov (United States)

    Gil, M.; Malinowski, T.; Iazykov, M.; Klein, H. R.

    2018-03-01

    We present an original method to estimate the conductivity of a single molecule anchored to nanometric-sized metallic electrodes, using a Mechanically Controlled Break Junction operated at room temperature in the liquid. We record the conductance through the metal/molecules/metal nanocontact while keeping the metallic electrodes at a fixed distance. Taking advantage of thermal diffusion and electromigration, we let the contact naturally explore the more stable configurations around a chosen conductance value. The conductance of a single molecule is estimated from a statistical analysis of raw conductance and conductance standard deviation data for molecular contacts containing up to 14 molecules. The single molecule conductance values are interpreted as time-averaged conductance of an ensemble of conformers at thermal equilibrium.

  12. Fluorescence spectroscopy of single molecules at room temperature and its applications

    Energy Technology Data Exchange (ETDEWEB)

    Ha, Taekjip [Univ. of California, Berkeley, CA (United States)

    1996-12-01

    We performed fluorescence spectroscopy of single and pairs of dye molecules on a surface at room temperature. Near field scanning optical microscope (NSOM) and far field scanning optical microscope with multi-color excitation/detection capability were built. The instrument is capable of optical imaging with 100nm resolution and has the sensitivity necessary for single molecule detection. A variety of dynamic events which cannot be observed from an ensemble of molecules is revealed when the molecules are probed one at a time. They include (1) spectral jumps correlated with dark states, (2) individually resolved quantum jumps to and from the meta-stable triplet state, (3) rotational jumps due to desorption/readsorption events of single molecules on the surface. For these studies, a computer controlled optical system which automatically and rapidly locates and performs spectroscopic measurements on single molecules was developed. We also studied the interaction between closely spaced pairs of molecules. In particular, fluorescence resonance energy transfer between a single resonant pair of donor and acceptor molecules was measured. Photodestruction dynamics of the donor or acceptor were used to determine the presence and efficiency of energy transfer Dual molecule spectroscopy was extended to a non-resonant pair of molecules to obtain high resolution differential distance information. By combining NSOM and dual color scheme, we studied the co-localization of parasite proteins and host proteins on a human red blood cell membrane infected with malaria. These dual-molecule techniques can be used to measure distances, relative orientations, and changes in distances/orientations of biological macromolecules with very good spatial, angular and temporal resolutions, hence opening new capabilities in the study of such systems.

  13. Dipolar molecules inside C-70: an electric field-driven room-temperature single-molecule switch

    Czech Academy of Sciences Publication Activity Database

    Foroutan-Nejad, C.; Andrushchenko, Valery; Straka, Michal

    2016-01-01

    Roč. 18, č. 48 (2016), s. 32673-32677 ISSN 1463-9076 R&D Projects: GA ČR(CZ) GA14-03564S Institutional support: RVO:61388963 Keywords : room-temperature single-molecule switch * electric field * endohedral fullerene * density functional calculations Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 4.123, year: 2016 http://pubs.rsc.org/en/content/articlepdf/2016/cp/c6cp06986j

  14. Detection of kinetic change points in piece-wise linear single molecule motion

    Science.gov (United States)

    Hill, Flynn R.; van Oijen, Antoine M.; Duderstadt, Karl E.

    2018-03-01

    Single-molecule approaches present a powerful way to obtain detailed kinetic information at the molecular level. However, the identification of small rate changes is often hindered by the considerable noise present in such single-molecule kinetic data. We present a general method to detect such kinetic change points in trajectories of motion of processive single molecules having Gaussian noise, with a minimum number of parameters and without the need of an assumed kinetic model beyond piece-wise linearity of motion. Kinetic change points are detected using a likelihood ratio test in which the probability of no change is compared to the probability of a change occurring, given the experimental noise. A predetermined confidence interval minimizes the occurrence of false detections. Applying the method recursively to all sub-regions of a single molecule trajectory ensures that all kinetic change points are located. The algorithm presented allows rigorous and quantitative determination of kinetic change points in noisy single molecule observations without the need for filtering or binning, which reduce temporal resolution and obscure dynamics. The statistical framework for the approach and implementation details are discussed. The detection power of the algorithm is assessed using simulations with both single kinetic changes and multiple kinetic changes that typically arise in observations of single-molecule DNA-replication reactions. Implementations of the algorithm are provided in ImageJ plugin format written in Java and in the Julia language for numeric computing, with accompanying Jupyter Notebooks to allow reproduction of the analysis presented here.

  15. Relaxation in Thin Polymer Films Mapped across the Film Thickness by Astigmatic Single-Molecule Imaging

    KAUST Repository

    Oba, Tatsuya

    2012-06-19

    We have studied relaxation processes in thin supported films of poly(methyl acrylate) at the temperature corresponding to 13 K above the glass transition by monitoring the reorientation of single perylenediimide molecules doped into the films. The axial position of the dye molecules across the thickness of the film was determined with a resolution of 12 nm by analyzing astigmatic fluorescence images. The average relaxation times of the rotating molecules do not depend on the overall thickness of the film between 20 and 110 nm. The relaxation times also do not show any dependence on the axial position within the films for the film thickness between 70 and 110 nm. In addition to the rotating molecules we observed a fraction of spatially diffusing molecules and completely immobile molecules. These molecules indicate the presence of thin (<5 nm) high-mobility surface layer and low-mobility layer at the interface with the substrate. (Figure presented) © 2012 American Chemical Society.

  16. Electron-vibron coupling effects on electron transport via a single-molecule magnet

    NARCIS (Netherlands)

    McCaskey, A.; Yamamoto, Y.; Warnock, M.; Burzuri, E.; Van der Zant, H.S.J.; Park, K.

    2015-01-01

    We investigate how the electron-vibron coupling influences electron transport via an anisotropic magnetic molecule, such as a single-molecule magnet (SMM) Fe4, by using a model Hamiltonian with parameter values obtained from density-functional theory (DFT). The magnetic anisotropy parameters,

  17. Fully Streched Single DNA Molecules in a Nanofluidic Chip Show Large-Scale Structural Variation

    DEFF Research Database (Denmark)

    Pedersen, Jonas Nyvold; Marie, Rodolphe; Bauer, D. L.

    2013-01-01

    When stretching and imaging DNA molecules in nanofluidic devices, it is important to know the relation between the physical length as measured in the lab and the distance along the contour of the DNA. Here a single DNA molecule longer than 1 Mbp is loaded into a nanofluidic device consisting of two...

  18. Integrated view of genome structure and sequence of a single DNA molecule in a nanofluidic device

    DEFF Research Database (Denmark)

    Marie, Rodolphe; Pedersen, Jonas Nyvold; L. V. Bauer, David

    2013-01-01

    We show how a bird’s-eye view of genomic structure can be obtained at ∼1-kb resolution from long (∼2 Mb) DNA molecules extracted from whole chromosomes in a nanofluidic laboratoryon-a-chip. We use an improved single-molecule denaturation mapping approach to detect repetitive elements and known...

  19. Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy

    Science.gov (United States)

    Neuman, Keir C.; Nagy, Attila

    2012-01-01

    Single-molecule force spectroscopy has emerged as a powerful tool to investigate the forces and motions associated with biological molecules and enzymatic activity. The most common force spectroscopy techniques are optical tweezers, magnetic tweezers and atomic force microscopy. These techniques are described and illustrated with examples highlighting current capabilities and limitations. PMID:18511917

  20. Single molecule charge transport : From a quantum mechanical to a classical description

    NARCIS (Netherlands)

    Kocherzhenko, A.A.; Grozema, F.C.; Siebbeles, L.D.A.

    2010-01-01

    This paper explores charge transport at the single molecule level. The conductive properties of both small organic molecules and conjugated polymers (molecular wires) are considered. In particular, the reasons for the transition from fully coherent to incoherent charge transport and the approaches

  1. A single molecule switch based on two Pd nanocrystals linked by a ...

    Indian Academy of Sciences (India)

    Tunneling spectroscopy measurements have been carried out on a single molecule device formed by two Pd nanocrystals (dia. ∼ 5 nm) electronically coupled by a conducting molecule, dimercaptodiphenylacetylene. The – data, obtained by positioning the tip over a nanocrystal electrode, exhibit negative differential ...

  2. Quantum interference effects at room temperature in OPV-based single-molecule junctions

    NARCIS (Netherlands)

    Arroyo Rodriguez, C.; Frisenda, R.; Moth-Poulsen, K.; Seldenthuis, J.S.; Bjornholm, T.; Van der Zant, H.S.

    2013-01-01

    Interference effects on charge transport through an individual molecule can lead to a notable modulation and suppression on its conductance. In this letter, we report the observation of quantum interference effects occurring at room temperature in single-molecule junctions based on

  3. Unified Model of Dynamic Forced Barrier Crossing in Single Molecules

    Energy Technology Data Exchange (ETDEWEB)

    Friddle, R W

    2007-06-21

    Thermally activated barrier crossing in the presence of an increasing load can reveal kinetic rate constants and energy barrier parameters when repeated over a range of loading rates. Here we derive a model of the mean escape force for all relevant loading rates--the complete force spectrum. Two well-known approximations emerge as limiting cases; one of which confirms predictions that single-barrier spectra should converge to a phenomenological description in the slow loading limit.

  4. Nanoscale and single-molecule interfacial electron transfer

    DEFF Research Database (Denmark)

    Hansen, Allan Glargaard; Wackerbarth, Hainer; Nielsen, Jens Ulrik

    2003-01-01

    Electrochemical science and technology in the 21st century have reached high levels of sophistication. A fundamental quantum mechanical theoretical frame for interfacial electrochemical electron transfer (ET) was introduced by Revaz Dogonadze. This frame has remained for four decades as a basis...... scanning tunneling microscopy (STM) and single-electron tunneling (SET, or Coulomb blockade) in electrochemical. systems directly in aqueous electrolyte solution and at room temperature. We illustrate the new theoretical formalism and its perspectives by recent cases of electrochemical SET, negative...

  5. Efficient use of single molecule time traces to resolve kinetic rates, models and uncertainties

    Science.gov (United States)

    Schmid, Sonja; Hugel, Thorsten

    2018-03-01

    Single molecule time traces reveal the time evolution of unsynchronized kinetic systems. Especially single molecule Förster resonance energy transfer (smFRET) provides access to enzymatically important time scales, combined with molecular distance resolution and minimal interference with the sample. Yet the kinetic analysis of smFRET time traces is complicated by experimental shortcomings—such as photo-bleaching and noise. Here we recapitulate the fundamental limits of single molecule fluorescence that render the classic, dwell-time based kinetic analysis unsuitable. In contrast, our Single Molecule Analysis of Complex Kinetic Sequences (SMACKS) considers every data point and combines the information of many short traces in one global kinetic rate model. We demonstrate the potential of SMACKS by resolving the small kinetic effects caused by different ionic strengths in the chaperone protein Hsp90. These results show an unexpected interrelation between conformational dynamics and ATPase activity in Hsp90.

  6. Targeting neurotransmitter receptors with nanoparticles in vivo allows single-molecule tracking in acute brain slices

    Science.gov (United States)

    Varela, Juan A.; Dupuis, Julien P.; Etchepare, Laetitia; Espana, Agnès; Cognet, Laurent; Groc, Laurent

    2016-03-01

    Single-molecule imaging has changed the way we understand many biological mechanisms, particularly in neurobiology, by shedding light on intricate molecular events down to the nanoscale. However, current single-molecule studies in neuroscience have been limited to cultured neurons or organotypic slices, leaving as an open question the existence of fast receptor diffusion in intact brain tissue. Here, for the first time, we targeted dopamine receptors in vivo with functionalized quantum dots and were able to perform single-molecule tracking in acute rat brain slices. We propose a novel delocalized and non-inflammatory way of delivering nanoparticles (NPs) in vivo to the brain, which allowed us to label and track genetically engineered surface dopamine receptors in neocortical neurons, revealing inherent behaviour and receptor activity regulations. We thus propose a NP-based platform for single-molecule studies in the living brain, opening new avenues of research in physiological and pathological animal models.

  7. 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.

  8. Break junction under electrochemical gating: testbed for single-molecule electronics.

    Science.gov (United States)

    Huang, Cancan; Rudnev, Alexander V; Hong, Wenjing; Wandlowski, Thomas

    2015-02-21

    Molecular electronics aims to construct functional molecular devices at the single-molecule scale. One of the major challenges is to construct a single-molecule junction and to further manipulate the charge transport through the molecular junction. Break junction techniques, including STM break junctions and mechanically controllable break junctions are considered as testbed to investigate and control the charge transport on a single-molecule scale. Moreover, additional electrochemical gating provides a unique opportunity to manipulate the energy alignment and molecular redox processes for a single-molecule junction. In this review, we start from the technical aspects of the break junction technique, then discuss the molecular structure-conductance correlation derived from break junction studies, and, finally, emphasize electrochemical gating as a promising method for the functional molecular devices.

  9. SISGR: Room Temperature Single-Molecule Detection and Imaging by Stimulated Emission Microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Xiaoliang Sunney [Harvard Univ., Cambridge, MA (United States). Dept. of Chemistry and Chemical Biology

    2017-03-13

    Single-molecule spectroscopy has made considerable impact on many disciplines including chemistry, physics, and biology. To date, most single-molecule spectroscopy work is accomplished by detecting fluorescence. On the other hand, many naturally occurring chromophores, such as retinal, hemoglobin and cytochromes, do not have detectable fluorescence. There is an emerging need for single-molecule spectroscopy techniques that do not require fluorescence. In the last proposal period, we have successfully demonstrated stimulated emission microscopy, single molecule absorption, and stimulated Raman microscopy based on a high-frequency modulation transfer technique. These first-of-a- kind new spectroscopy/microscopy methods tremendously improved our ability to observe molecules that fluorescence weakly, even to the limit of single molecule detection for absorption measurement. All of these methods employ two laser beams: one (pump beam) excites a single molecule to a real or virtual excited state, and the other (probe beam) monitors the absorption/emission property of the single. We extract the intensity change of the probe beam with high sensitivity by implementing a high-frequency phase-sensitive detection scheme, which offers orders of magnitude improvement in detection sensitivity over direct absorption/emission measurement. However, single molecule detection based on fluorescence or absorption is fundamentally limited due to their broad spectral response. It is important to explore other avenues in single molecule detection and imaging which provides higher molecular specificity for studying a wide variety of heterogeneous chemical and biological systems. This proposal aimed to achieve single-molecule detection sensitivity with near resonance stimulated Raman scattering (SRS) microscopy. SRS microscopy was developed in our lab as a powerful technique for imaging heterogeneous samples based on their intrinsic vibrational contrasts, which provides much higher molecular

  10. DNA origami as biocompatible surface to match single-molecule and ensemble experiments

    Science.gov (United States)

    Gietl, Andreas; Holzmeister, Phil; Grohmann, Dina; Tinnefeld, Philip

    2012-01-01

    Single-molecule experiments on immobilized molecules allow unique insights into the dynamics of molecular machines and enzymes as well as their interactions. The immobilization, however, can invoke perturbation to the activity of biomolecules causing incongruities between single molecule and ensemble measurements. Here we introduce the recently developed DNA origami as a platform to transfer ensemble assays to the immobilized single molecule level without changing the nano-environment of the biomolecules. The idea is a stepwise transfer of common functional assays first to the surface of a DNA origami, which can be checked at the ensemble level, and then to the microscope glass slide for single-molecule inquiry using the DNA origami as a transfer platform. We studied the structural flexibility of a DNA Holliday junction and the TATA-binding protein (TBP)-induced bending of DNA both on freely diffusing molecules and attached to the origami structure by fluorescence resonance energy transfer. This resulted in highly congruent data sets demonstrating that the DNA origami does not influence the functionality of the biomolecule. Single-molecule data collected from surface-immobilized biomolecule-loaded DNA origami are in very good agreement with data from solution measurements supporting the fact that the DNA origami can be used as biocompatible surface in many fluorescence-based measurements. PMID:22523083

  11. Multicolour single molecule imaging in cells with near infra-red dyes.

    Directory of Open Access Journals (Sweden)

    Christopher J Tynan

    Full Text Available The autofluorescence background of biological samples impedes the detection of single molecules when imaging. The most common method of reducing the background is to use evanescent field excitation, which is incompatible with imaging beyond the surface of biological samples. An alternative would be to use probes that can be excited in the near infra-red region of the spectrum, where autofluorescence is low. Such probes could also increase the number of labels that can be imaged in multicolour single molecule microscopes. Despite being widely used in ensemble imaging, there is a currently a shortage of information available for selecting appropriate commercial near infra-red dyes for single molecule work. It is therefore important to characterise available near infra-red dyes relevant to multicolour single molecule imaging.A range of commercially available near infra-red dyes compatible with multi-colour imaging was screened to find the brightest and most photostable candidates. Image series of immobilised samples of the brightest dyes (Alexa 700, IRDye 700DX, Alexa 790 and IRDye 800CW were analysed to obtain the mean intensity of single dye molecules, their photobleaching rates and long period blinking kinetics. Using the optimum dye pair, we have demonstrated for the first time widefield, multi-colour, near infra-red single molecule imaging using a supercontinuum light source in MCF-7 cells.We have demonstrated that near infra-red dyes can be used to avoid autofluorescence background in samples where restricting the illumination volume of visible light fails or is inappropriate. We have also shown that supercontinuum sources are suited to single molecule multicolour imaging throughout the 470-1000 nm range. Our measurements of near infra-red dye properties will enable others to select optimal dyes for single molecule imaging.

  12. Multicolour Single Molecule Imaging in Cells with Near Infra-Red Dyes

    Science.gov (United States)

    Tynan, Christopher J.; Clarke, David T.; Coles, Benjamin C.; Rolfe, Daniel J.; Martin-Fernandez, Marisa L.; Webb, Stephen E. D.

    2012-01-01

    Background The autofluorescence background of biological samples impedes the detection of single molecules when imaging. The most common method of reducing the background is to use evanescent field excitation, which is incompatible with imaging beyond the surface of biological samples. An alternative would be to use probes that can be excited in the near infra-red region of the spectrum, where autofluorescence is low. Such probes could also increase the number of labels that can be imaged in multicolour single molecule microscopes. Despite being widely used in ensemble imaging, there is a currently a shortage of information available for selecting appropriate commercial near infra-red dyes for single molecule work. It is therefore important to characterise available near infra-red dyes relevant to multicolour single molecule imaging. Methodology/Principal Findings A range of commercially available near infra-red dyes compatible with multi-colour imaging was screened to find the brightest and most photostable candidates. Image series of immobilised samples of the brightest dyes (Alexa 700, IRDye 700DX, Alexa 790 and IRDye 800CW) were analysed to obtain the mean intensity of single dye molecules, their photobleaching rates and long period blinking kinetics. Using the optimum dye pair, we have demonstrated for the first time widefield, multi-colour, near infra-red single molecule imaging using a supercontinuum light source in MCF-7 cells. Conclusions/Significance We have demonstrated that near infra-red dyes can be used to avoid autofluorescence background in samples where restricting the illumination volume of visible light fails or is inappropriate. We have also shown that supercontinuum sources are suited to single molecule multicolour imaging throughout the 470–1000 nm range. Our measurements of near infra-red dye properties will enable others to select optimal dyes for single molecule imaging. PMID:22558412

  13. Stretching, twisting and supercoiling in short, single DNA molecules

    Science.gov (United States)

    Lam, Pui-Man; Zhen, Yi

    2018-02-01

    We had combined the Neukirch-Marko model that describes the extension, torque and supercoiling in single, stretched and twisted DNA of infinite contour length, with a form of the free energy suggested by Sinha and Samuels to describe short DNA, with contour length only a few times the persistence length. We find that the free energy of the stretched but untwisted DNA, is significantly modified from its infinitely length value and this in turn modifies significantly the torque and supercoiling. We show that this is consistent with short DNA being more flexible than infinitely long DNA. We hope our results will stimulate experimental investigation of torque and supercoiling in short DNA.

  14. Magnetization relaxation of single molecule magnets after field cooling

    Science.gov (United States)

    Fernandez, Julio F.; Alonso, Juan J.

    2004-03-01

    Magnetic clusters, such as Fe8 and Mn_12, behave at low temperatures as large single spins S. In crystals, anisotropy energies U allow magnetic relaxation only through tunneling at k_BTstackrelspins with dipolar interactions. To mimic tunneling effects, a spin on a lattice site where h is within some tunnel window -h_w

  15. Single-molecule fluorescence microscopy review: shedding new light on old problems.

    Science.gov (United States)

    Shashkova, Sviatlana; Leake, Mark C

    2017-08-31

    Fluorescence microscopy is an invaluable tool in the biosciences, a genuine workhorse technique offering exceptional contrast in conjunction with high specificity of labelling with relatively minimal perturbation to biological samples compared with many competing biophysical techniques. Improvements in detector and dye technologies coupled to advances in image analysis methods have fuelled recent development towards single-molecule fluorescence microscopy, which can utilize light microscopy tools to enable the faithful detection and analysis of single fluorescent molecules used as reporter tags in biological samples. For example, the discovery of GFP, initiating the so-called 'green revolution', has pushed experimental tools in the biosciences to a completely new level of functional imaging of living samples, culminating in single fluorescent protein molecule detection. Today, fluorescence microscopy is an indispensable tool in single-molecule investigations, providing a high signal-to-noise ratio for visualization while still retaining the key features in the physiological context of native biological systems. In this review, we discuss some of the recent discoveries in the life sciences which have been enabled using single-molecule fluorescence microscopy, paying particular attention to the so-called 'super-resolution' fluorescence microscopy techniques in live cells, which are at the cutting-edge of these methods. In particular, how these tools can reveal new insights into long-standing puzzles in biology: old problems, which have been impossible to tackle using other more traditional tools until the emergence of new single-molecule fluorescence microscopy techniques. © 2017 The Author(s).

  16. Extracting physics of life at the molecular level: A review of single-molecule data analyses.

    Science.gov (United States)

    Colomb, Warren; Sarkar, Susanta K

    2015-06-01

    Studying individual biomolecules at the single-molecule level has proved very insightful recently. Single-molecule experiments allow us to probe both the equilibrium and nonequilibrium properties as well as make quantitative connections with ensemble experiments and equilibrium thermodynamics. However, it is important to be careful about the analysis of single-molecule data because of the noise present and the lack of theoretical framework for processes far away from equilibrium. Biomolecular motion, whether it is free in solution, on a substrate, or under force, involves thermal fluctuations in varying degrees, which makes the motion noisy. In addition, the noise from the experimental setup makes it even more complex. The details of biologically relevant interactions, conformational dynamics, and activities are hidden in the noisy single-molecule data. As such, extracting biological insights from noisy data is still an active area of research. In this review, we will focus on analyzing both fluorescence-based and force-based single-molecule experiments and gaining biological insights at the single-molecule level. Inherently nonequilibrium nature of biological processes will be highlighted. Simulated trajectories of biomolecular diffusion will be used to compare and validate various analysis techniques. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. A Single Molecule Study of Two Bacteriophage Epigenetic Switches

    Science.gov (United States)

    Wang, Haowei

    Epigenetic switches allow organisms to evolve into different states by activating/repressing different sets of genes without mutations of the underlying DNA sequence. The study of epigenetic switches is very important to understand the mechanism of human development, the origin of cancer, mental illness and fundamental processes such as gene regulation. The coliphage lambda epigenetic switch, which allows switching from lysogeny to lysis, has been studied for more than 50 years as a paradigm, and has recently received renewed attention. Atomic force microscopy (AFM) was used here to show that the lambda repressor oligomerizes on DNA, primarily as a dodecamer, to secure a DNA loop, which is the basis of the lambda switch. This study also provides support for the idea that specifically bound repressor stabilizes adjacent, non-specifically bound repressor molecules, which confers robustness to the switch. 186 is a member of a different coliphage family. One of the major differences between the two coliphage families is that lambda phages can be induced to switch from the lysogenic to the lytic state by UV radiation, but most coliphages of P2 family, to which 186 belongs, cannot. Interaction between coliphage 186 repressor and DNA is characterized by AFM and tethered particle motion (TPM). To expedite analysis of the AFM data, MatLab codes were written to automate the laborious, manual tracing procedures. The programs automatically recognize DNA segments and protein particles in an image, in order to measure the DNA length and position of bound particles as well as their height, diameter and volume. Application of these algorithms greatly improved the efficiency of AFM analysis. It was showed that 186 CI dimers form heptameric wheels, which induce DNA wrapping and different kinds of DNA looping producing various conformations of nucleoprotein complexes. Information about the dynamics of DNA wrapping and looping on 186 CI particles was also obtained by TPM.

  18. Gene length and detection bias in single cell RNA sequencing protocols [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Belinda Phipson

    2017-04-01

    Full Text Available Background: Single cell RNA sequencing (scRNA-seq has rapidly gained popularity for profiling transcriptomes of hundreds to thousands of single cells. This technology has led to the discovery of novel cell types and revealed insights into the development of complex tissues. However, many technical challenges need to be overcome during data generation. Due to minute amounts of starting material, samples undergo extensive amplification, increasing technical variability. A solution for mitigating amplification biases is to include unique molecular identifiers (UMIs, which tag individual molecules. Transcript abundances are then estimated from the number of unique UMIs aligning to a specific gene, with PCR duplicates resulting in copies of the UMI not included in expression estimates. Methods: Here we investigate the effect of gene length bias in scRNA-Seq across a variety of datasets that differ in terms of capture technology, library preparation, cell types and species. Results: We find that scRNA-seq datasets that have been sequenced using a full-length transcript protocol exhibit gene length bias akin to bulk RNA-seq data. Specifically, shorter genes tend to have lower counts and a higher rate of dropout. In contrast, protocols that include UMIs do not exhibit gene length bias, with a mostly uniform rate of dropout across genes of varying length. Across four different scRNA-Seq datasets profiling mouse embryonic stem cells (mESCs, we found the subset of genes that are only detected in the UMI datasets tended to be shorter, while the subset of genes detected only in the full-length datasets tended to be longer. Conclusions: We find that the choice of scRNA-seq protocol influences the detection rate of genes, and that full-length datasets exhibit gene-length bias. In addition, despite clear differences between UMI and full-length transcript data, we illustrate that full-length and UMI data can be combined to reveal the underlying biology

  19. Accurate quantification of microRNA via single strand displacement reaction on DNA origami motif.

    Directory of Open Access Journals (Sweden)

    Jie Zhu

    Full Text Available DNA origami is an emerging technology that assembles hundreds of staple strands and one single-strand DNA into certain nanopattern. It has been widely used in various fields including detection of biological molecules such as DNA, RNA and proteins. MicroRNAs (miRNAs play important roles in post-transcriptional gene repression as well as many other biological processes such as cell growth and differentiation. Alterations of miRNAs' expression contribute to many human diseases. However, it is still a challenge to quantitatively detect miRNAs by origami technology. In this study, we developed a novel approach based on streptavidin and quantum dots binding complex (STV-QDs labeled single strand displacement reaction on DNA origami to quantitatively detect the concentration of miRNAs. We illustrated a linear relationship between the concentration of an exemplary miRNA as miRNA-133 and the STV-QDs hybridization efficiency; the results demonstrated that it is an accurate nano-scale miRNA quantifier motif. In addition, both symmetrical rectangular motif and asymmetrical China-map motif were tested. With significant linearity in both motifs, our experiments suggested that DNA Origami motif with arbitrary shape can be utilized in this method. Since this DNA origami-based method we developed owns the unique advantages of simple, time-and-material-saving, potentially multi-targets testing in one motif and relatively accurate for certain impurity samples as counted directly by atomic force microscopy rather than fluorescence signal detection, it may be widely used in quantification of miRNAs.

  20. Accurate Quantification of microRNA via Single Strand Displacement Reaction on DNA Origami Motif

    Science.gov (United States)

    Lou, Jingyu; Li, Weidong; Li, Sheng; Zhu, Hongxin; Yang, Lun; Zhang, Aiping; He, Lin; Li, Can

    2013-01-01

    DNA origami is an emerging technology that assembles hundreds of staple strands and one single-strand DNA into certain nanopattern. It has been widely used in various fields including detection of biological molecules such as DNA, RNA and proteins. MicroRNAs (miRNAs) play important roles in post-transcriptional gene repression as well as many other biological processes such as cell growth and differentiation. Alterations of miRNAs' expression contribute to many human diseases. However, it is still a challenge to quantitatively detect miRNAs by origami technology. In this study, we developed a novel approach based on streptavidin and quantum dots binding complex (STV-QDs) labeled single strand displacement reaction on DNA origami to quantitatively detect the concentration of miRNAs. We illustrated a linear relationship between the concentration of an exemplary miRNA as miRNA-133 and the STV-QDs hybridization efficiency; the results demonstrated that it is an accurate nano-scale miRNA quantifier motif. In addition, both symmetrical rectangular motif and asymmetrical China-map motif were tested. With significant linearity in both motifs, our experiments suggested that DNA Origami motif with arbitrary shape can be utilized in this method. Since this DNA origami-based method we developed owns the unique advantages of simple, time-and-material-saving, potentially multi-targets testing in one motif and relatively accurate for certain impurity samples as counted directly by atomic force microscopy rather than fluorescence signal detection, it may be widely used in quantification of miRNAs. PMID:23990889

  1. Evaluation of the Electronic Structure of Single-Molecule Junctions Based on Current-Voltage and Thermopower Measurements: Application to C60 Single-Molecule Junction.

    Science.gov (United States)

    Komoto, Yuki; Isshiki, Yuji; Fujii, Shintaro; Nishino, Tomoaki; Kiguchi, Manabu

    2017-02-16

    The electronic structure of molecular junctions has a significant impact on their transport properties. Despite the decisive role of the electronic structure, a complete characterization of the electronic structure remains a challenge. This is because there is no straightforward way of measuring electron spectroscopy for an individual molecule trapped in a nanoscale gap between two metal electrodes. Herein, a comprehensive approach to obtain a detailed description of the electronic structure in single-molecule junctions based on the analysis of current-voltage (I-V) and thermoelectric characteristics is described. It is shown that the electronic structure of the prototypical C 60 single-molecule junction can be resolved by analyzing complementary results of the I-V and thermoelectric measurement. This combined approach confirmed that the C 60 single-molecule junction was highly conductive with molecular electronic conductances of 0.033 and 0.003 G 0 and a molecular Seebeck coefficient of -12 μV K -1 . In addition, we revealed that charge transport was mediated by a LUMO whose energy level was located 0.5≈0.6 eV above the Fermi level of the Au electrode. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Spin transport properties of single metallocene molecules attached to single-walled carbon nanotubes via nickel adatoms

    Science.gov (United States)

    Wei, Peng; Sun, Lili; Benassi, Enrico; Shen, Ziyong; Sanvito, Stefano; Hou, Shimin

    2011-06-01

    The spin-dependent transport properties of single ferrocene, cobaltocene, and nickelocene molecules attached to the sidewall of a (4,4) armchair single-walled carbon nanotube via a Ni adatom are investigated by using a self-consistent ab initio approach that combines the non-equilibrium Green's function formalism with the spin density functional theory. Our calculations show that the Ni adatom not only binds strongly to the sidewall of the nanotube, but also maintains the spin degeneracy and affects little the transmission around the Fermi level. When the Ni adatom further binds to a metallocene molecule, its density of states is modulated by that of the molecule and electron scattering takes place in the nanotube. In particular, we find that for both cobaltocene and nickelocene the transport across the nanotube becomes spin-polarized. This demonstrates that metallocene molecules and carbon nanotubes can become a promising materials platform for applications in molecular spintronics.

  3. Suppression of mRNA Nanoparticle Transfection in Human Fibroblasts by Selected Interferon Inhibiting Small Molecule Compounds.

    Science.gov (United States)

    Liu, Yang; Krishnan, Manoj N; Phua, Kyle K L

    2017-07-31

    In vitro transcribed (IVT) mRNA is increasingly applied in lieu of DNA to deliver reprogramming genes to fibroblasts for stem cell derivation. However, IVT mRNA induces interferon (IFN) responses from mammalian cells that reduces transfection efficiency. It has been previously suggested that small molecule inhibitors of IFN are a viable strategy to enhance mRNA transfection efficiency. Herein, we screen a list of commercially available small molecules, including published IFN inhibitors, for their potential to enhance mRNA transfection in BJ fibroblasts. Transfection enhancement is quantified by relative mean fluorescence intensity of translated green fluorescent protein (GFP) in treated cells compared to dimethyl sulfoxide treated controls. Within toxicological constrains, all tested small molecules did not enhance mRNA transfection in BJ fibroblasts while a third of the tested compounds unexpectedly inhibited GFP expression even though IFN-β production is inhibited. Based on the results of our study, we conclude that small molecule inhibitors, including IFN inhibitors, tested in this study do not enhance in vitro mRNA transfection efficiency in human fibroblasts.

  4. Chemical Principles and Interference in the Electrical Conductance of Single Molecules

    DEFF Research Database (Denmark)

    Borges, Anders Christian

    The electrical conductance of single molecules are routinely reported in the scientific literature and off-resonant coherent tunneling is believed to be the mechanism for transport in some of these experiments. In these experiments it is observed that, in spite of similar molecular structures......-Tunneling Microscope Break-Junction experiments (STM-BJ). It is demonstrated that these links can be used to design molecules exhibiting surprising interference effects and to interpret and predict the trends in the characteristic conductance of single molecules without resorting to numerical computational methods...

  5. Electric-Field Control of Interfering Transport Pathways in a Single-Molecule Anthraquinone Transistor

    Science.gov (United States)

    Koole, Max; Thijssen, Jos M.; Valkenier, Hennie; Hummelen, Jan C.; Zant, Herre S. J. van der

    2015-08-01

    It is understood that molecular conjugation plays an important role in charge transport through single-molecule junctions. Here, we investigate electron transport through an anthraquinone based single-molecule three-terminal device. With the use of an electric-field induced by a gate electrode, the molecule is reduced resulting into a ten-fold increase in the off-resonant differential conductance. Theoretical calculations link the change in differential conductance to a reduction-induced change in conjugation, thereby lifting destructive interference of transport pathways.

  6. Full counting statistics of a single-molecule quantum dot

    Science.gov (United States)

    Dong, Bing; Ding, G. H.; Lei, X. L.

    2013-08-01

    We investigate the full counting statistics of a single quantum dot strongly coupled to a local phonon and weakly tunnel connected to two metallic electrodes. By employing the generalized nonequilibrium Green-function method and the Lang-Firsov transformation, we derive an explicit analytical formula for the cumulant generating function, which makes one able to identify distinctly the elastic and inelastic contributions to the current and zero-frequency shot noise. We find that at zero temperature, the inelastic effect causes upward steps in the current and downward jumps in the noise at the bias voltages corresponding to the opening of the inelastic channels, which are ascribed to the vibration-induced complex dependencies of electronic self-energies on the energy and bias voltage. More interestingly, the Fano factor exhibits oscillatory behavior with increasing bias voltage and its minimum value is observed to be smaller than one-half.

  7. Surface single-molecule dynamics controlled by entropy at low temperatures

    Science.gov (United States)

    Gehrig, J. C.; Penedo, M.; Parschau, M.; Schwenk, J.; Marioni, M. A.; Hudson, E. W.; Hug, H. J.

    2017-02-01

    Configuration transitions of individual molecules and atoms on surfaces are traditionally described using an Arrhenius equation with energy barrier and pre-exponential factor (attempt rate) parameters. Characteristic parameters can vary even for identical systems, and pre-exponential factors sometimes differ by orders of magnitude. Using low-temperature scanning tunnelling microscopy (STM) to measure an individual dibutyl sulfide molecule on Au(111), we show that the differences arise when the relative position of tip apex and molecule changes by a fraction of the molecule size. Altering the tip position on that scale modifies the transition's barrier and attempt rate in a highly correlated fashion, which results in a single-molecular enthalpy-entropy compensation. Conversely, appropriately positioning the STM tip allows selecting the operating point on the compensation line and modifying the transition rates. The results highlight the need to consider entropy in transition rates of single molecules, even at low temperatures.

  8. Analyzing abundance of mRNA molecules with a near-infrared fluorescence technique.

    Science.gov (United States)

    Chen, Ying; Pan, Yan; Zhang, Beibei; Wang, Jinke

    2014-01-01

    This study describes a simple method for analyzing the abundance of mRNA molecules in a total DNA sample. Due to the dependence on the near-infrared fluorescence technique, this method is named near-infrared fluorescence gene expression detection (NIRF-GED). The procedure has three steps: (1) isolating total RNA from detected samples and reverse-transcription into cDNA with a biotin-labeled oligo dT; (2) hybridizing cDNA to oligonucleotide probes coupled to a 96-well microplate; and (3) detecting biotins with NIRF-labeled streptavidin. The method was evaluated by performing proof-in-concept detections of absolute and relative expressions of housekeeping and NF-κB target genes in HeLa cells. As a result, the absolute expression of three genes, Ccl20, Cxcl2, and Gapdh, in TNF-α-uninduced HeLa cells was determined with a standard curve constructed on the same microplate, and the relative expression of five genes, Ccl20, Cxcl2, Il-6, STAT5A, and Gapdh, in TNF-α-induced and -uninduced HeLa cells was measured by using NIRF-GED. The results were verified by quantitative PCR (qPCR) and DNA microarray detections. The biggest advantage of NIRF-GED over the current techniques lies in its independence of exponential or linear amplification of nucleic acids. Moreover, NIRF-GED also has several other benefits, including high sensitivity as low as several fmols, absolute quantification in the range of 9 to 147 fmols, low cDNA consumption similar to qPCR template, and the current medium throughput in 96-well microplate format and future high throughput in DNA microarray format. NIRF-GED thus provides a new tool for analyzing gene transcripts and other nucleic acid molecules.

  9. A Single-Molecule Barcoding System using Nanoslits for DNA Analysis

    Science.gov (United States)

    Jo, Kyubong; Schramm, Timothy M.; Schwartz, David C.

    Single DNA molecule approaches are playing an increasingly central role in the analytical genomic sciences because single molecule techniques intrinsically provide individualized measurements of selected molecules, free from the constraints of bulk techniques, which blindly average noise and mask the presence of minor analyte components. Accordingly, a principal challenge that must be addressed by all single molecule approaches aimed at genome analysis is how to immobilize and manipulate DNA molecules for measurements that foster construction of large, biologically relevant data sets. For meeting this challenge, this chapter discusses an integrated approach for microfabricated and nanofabricated devices for the manipulation of elongated DNA molecules within nanoscale geometries. Ideally, large DNA coils stretch via nanoconfinement when channel dimensions are within tens of nanometers. Importantly, stretched, often immobilized, DNA molecules spanning hundreds of kilobase pairs are required by all analytical platforms working with large genomic substrates because imaging techniques acquire sequence information from molecules that normally exist in free solution as unrevealing random coils resembling floppy balls of yarn. However, nanoscale devices fabricated with sufficiently small dimensions fostering molecular stretching make these devices impractical because of the requirement of exotic fabrication technologies, costly materials, and poor operational efficiencies. In this chapter, such problems are addressed by discussion of a new approach to DNA presentation and analysis that establishes scaleable nanoconfinement conditions through reduction of ionic strength; stiffening DNA molecules thus enabling their arraying for analysis using easily fabricated devices that can also be mass produced. This new approach to DNA nanoconfinement is complemented by the development of a novel labeling scheme for reliable marking of individual molecules with fluorochrome labels

  10. Quantifying and optimizing single-molecule switching nanoscopy at high speeds.

    Directory of Open Access Journals (Sweden)

    Yu Lin

    Full Text Available Single-molecule switching nanoscopy overcomes the diffraction limit of light by stochastically switching single fluorescent molecules on and off, and then localizing their positions individually. Recent advances in this technique have greatly accelerated the data acquisition speed and improved the temporal resolution of super-resolution imaging. However, it has not been quantified whether this speed increase comes at the cost of compromised image quality. The spatial and temporal resolution depends on many factors, among which laser intensity and camera speed are the two most critical parameters. Here we quantitatively compare the image quality achieved when imaging Alexa Fluor 647-immunolabeled microtubules over an extended range of laser intensities and camera speeds using three criteria - localization precision, density of localized molecules, and resolution of reconstructed images based on Fourier Ring Correlation. We found that, with optimized parameters, single-molecule switching nanoscopy at high speeds can achieve the same image quality as imaging at conventional speeds in a 5-25 times shorter time period. Furthermore, we measured the photoswitching kinetics of Alexa Fluor 647 from single-molecule experiments, and, based on this kinetic data, we developed algorithms to simulate single-molecule switching nanoscopy images. We used this software tool to demonstrate how laser intensity and camera speed affect the density of active fluorophores and influence the achievable resolution. Our study provides guidelines for choosing appropriate laser intensities for imaging Alexa Fluor 647 at different speeds and a quantification protocol for future evaluations of other probes and imaging parameters.

  11. 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.

  12. Preparation of zinc sulfide nanocrystallites from single-molecule precursors

    Science.gov (United States)

    Palve, Anil M.; Garje, Shivram S.

    2011-07-01

    Zinc sulfide nanocrystallites were prepared using Zinc(II) thiosemicarbazone complexes of the types Zn(L) 2 and ZnCl 2(LH) 2 (where, LH=thiosemicarbazones of cinnamaldehyde, 4-chlorobenzaldehyde, indol-3-carboxaldehyde and thiophene-2-carboxaldehyde) as single source precursors by solvothermal decomposition in ethylene glycol and ethylene diamine in few cases. The materials were characterized by powder X-ray diffraction (XRD), transmission electron microscopy (TEM), selected area electron diffraction, energy dispersive X-ray analysis and UV-vis and IR spectroscopy. Solvothermal decomposition in ethylene glycol resulted in the formation of hexagonal ZnS (JCPDS: 36-1450) as evident from the XRD patterns. However, XRD shows formation of hybrid material, ZnS 0.5EN in case of solvothermal decomposition in ethylenediamine. Infrared spectra authenticate the capping of ethylene glycol and ethylenediamine on ZnS and ZnS 0.5EN, respectively. TEM images showed formation of spherical nanoparticles for the materials obtained from ethylene glycol, whereas plate-like morphology is observed in case of materials obtained from ethylene diamine. The blue shift of absorption bands compared to bands of bulk materials in the UV-vis spectra supports the formation of smaller particles.

  13. Nanofluidic single-molecule sorting of DNA: a new concept in separation and analysis of biomolecules towards ultimate level performance

    International Nuclear Information System (INIS)

    Yamamoto, Takatoki; Fujii, Teruo

    2010-01-01

    Separation and separation-based analysis of biomolecules are fundamentally important techniques in the field of biotechnology. These techniques, however, depend on stochastic processes that intrinsically involve uncertainty, and thus it is not possible to achieve 100% separation accuracy. Theoretically, the ultimate resolution and sensitivity should be realized in a single-molecule system because of the deterministic nature of single-molecule manipulation. Here, we have proposed and experimentally demonstrated the concept of a 'single-molecule sorter' that detects and correctly identifies individual single molecules, realizing the ultimate level of resolution and sensitivity for any separation-based technology. The single-molecule sorter was created using a nanofluidic network consisting of a single inlet channel that branches off into multiple outlet channels. It includes two major functional elements, namely a single-molecule detection and identification element and a flow path switching element to accurately separate single molecules. With this system we have successfully demonstrated the world's first single-molecule sorting using DNA as a sample molecule. In the future, we hope to expand the application of such devices to comprehensive sorting of single-proteins from a single cell. We also believe that in addition to the single-molecule sorting method reported here, other types of single-molecule based processes will emerge and find use in a wide variety of applications.

  14. A single-stranded architecture for cotranscriptional folding of RNA nanostructures

    DEFF Research Database (Denmark)

    Geary, Cody; Rothemund, Paul; Andersen, Ebbe Sloth

    2014-01-01

    . We introduce an architecture for designing artificial RNA structures that fold from a single strand, in which arrays of antiparallel RNA helices are precisely organized by RNA tertiary motifs and a new type of crossover pattern. We constructed RNA tiles that assemble into hexagonal lattices......Artificial DNA and RNA structures have been used as scaffolds for a variety of nanoscale devices. In comparison to DNA structures, RNA structures have been limited in size, but they also have advantages: RNA can fold during transcription and thus can be genetically encoded and expressed in cells...

  15. Development of new photon-counting detectors for single-molecule fluorescence microscopy

    Science.gov (United States)

    Michalet, X.; Colyer, R. A.; Scalia, G.; Ingargiola, A.; Lin, R.; Millaud, J. E.; Weiss, S.; Siegmund, Oswald H. W.; Tremsin, Anton S.; Vallerga, John V.; Cheng, A.; Levi, M.; Aharoni, D.; Arisaka, K.; Villa, F.; Guerrieri, F.; Panzeri, F.; Rech, I.; Gulinatti, A.; Zappa, F.; Ghioni, M.; Cova, S.

    2013-01-01

    Two optical configurations are commonly used in single-molecule fluorescence microscopy: point-like excitation and detection to study freely diffusing molecules, and wide field illumination and detection to study surface immobilized or slowly diffusing molecules. Both approaches have common features, but also differ in significant aspects. In particular, they use different detectors, which share some requirements but also have major technical differences. Currently, two types of detectors best fulfil the needs of each approach: single-photon-counting avalanche diodes (SPADs) for point-like detection, and electron-multiplying charge-coupled devices (EMCCDs) for wide field detection. However, there is room for improvements in both cases. The first configuration suffers from low throughput owing to the analysis of data from a single location. The second, on the other hand, is limited to relatively low frame rates and loses the benefit of single-photon-counting approaches. During the past few years, new developments in point-like and wide field detectors have started addressing some of these issues. Here, we describe our recent progresses towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. We also discuss our development of large area photon-counting cameras achieving subnanosecond resolution for fluorescence lifetime imaging applications at the single-molecule level. PMID:23267185

  16. Blinking effect and the use of quantum dots in single molecule spectroscopy

    International Nuclear Information System (INIS)

    Rombach-Riegraf, Verena; Oswald, Peter; Bienert, Roland; Petersen, Jan; Domingo, M.P.; Pardo, Julian; Gräber, P.; Galvez, E.M.

    2013-01-01

    Highlights: ► It is possible to eliminate the blinking effect of a water-soluble QD. ► We provide a direct method to study protein function and dynamics at the single level. ► QD, potent tool for single molecule studies of biochemical and biological processes. -- Abstract: Luminescent semiconductor nanocrystals (quantum dots, QD) have unique photo-physical properties: high photostability, brightness and narrow size-tunable fluorescence spectra. Due to their unique properties, QD-based single molecule studies have become increasingly more popular during the last years. However QDs show a strong blinking effect (random and intermittent light emission), which may limit their use in single molecule fluorescence studies. QD blinking has been widely studied and some hypotheses have been done to explain this effect. Here we summarise what is known about the blinking effect in QDs, how this phenomenon may affect single molecule studies and, on the other hand, how the “on”/“off” states can be exploited in diverse experimental settings. In addition, we present results showing that site-directed binding of QD to cysteine residues of proteins reduces the blinking effect. This option opens a new possibility of using QDs to study protein–protein interactions and dynamics by single molecule fluorescence without modifying the chemical composition of the solution or the QD surface.

  17. Supramolecular Systems and Chemical Reactions in Single-Molecule Break Junctions.

    Science.gov (United States)

    Li, Xiaohui; Hu, Duan; Tan, Zhibing; Bai, Jie; Xiao, Zongyuan; Yang, Yang; Shi, Jia; Hong, Wenjing

    2017-04-01

    The major challenges of molecular electronics are the understanding and manipulation of the electron transport through the single-molecule junction. With the single-molecule break junction techniques, including scanning tunneling microscope break junction technique and mechanically controllable break junction technique, the charge transport through various single-molecule and supramolecular junctions has been studied during the dynamic fabrication and continuous characterization of molecular junctions. This review starts from the charge transport characterization of supramolecular junctions through a variety of noncovalent interactions, such as hydrogen bond, π-π interaction, and electrostatic force. We further review the recent progress in constructing highly conductive molecular junctions via chemical reactions, the response of molecular junctions to external stimuli, as well as the application of break junction techniques in controlling and monitoring chemical reactions in situ. We suggest that beyond the measurement of single molecular conductance, the single-molecule break junction techniques provide a promising access to study molecular assembly and chemical reactions at the single-molecule scale.

  18. Blinking effect and the use of quantum dots in single molecule spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Rombach-Riegraf, Verena; Oswald, Peter; Bienert, Roland; Petersen, Jan [Albert-Ludwigs-Universitaet Freiburg, Institut fuer Physikalische Chemie, Albertstrasse 23a, 79104 Freiburg (Germany); Domingo, M.P. [Instituto de Carboquimica (CSIC), Miguel Luesma 4, 50018 Zaragoza (Spain); Pardo, Julian [Grupo Apoptosis, Inmunidad y Cancer, Departamento Bioquimica y Biologia Molecular y Celular, Fac. Ciencias, Universidad de Zaragoza, Zaragoza (Spain); Fundacion Aragon I-D (ARAID), Gobierno de Aragon, Zaragoza (Spain); Immune Effector Cells Group, Aragon Health Research Institute (IIS Aragon), Biomedical Research Centre of Aragon (CIBA) Fundacion Aragon I-D - ARAID, Gobierno de Aragon, Zaragoza (Spain); Graeber, P. [Albert-Ludwigs-Universitaet Freiburg, Institut fuer Physikalische Chemie, Albertstrasse 23a, 79104 Freiburg (Germany); Galvez, E.M., E-mail: eva@icb.csic.es [Instituto de Carboquimica (CSIC), Miguel Luesma 4, 50018 Zaragoza (Spain); Immune Effector Cells Group, Aragon Health Research Institute (IIS Aragon), Biomedical Research Centre of Aragon (CIBA) Fundacion Aragon I-D - ARAID, Gobierno de Aragon, Zaragoza (Spain)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer It is possible to eliminate the blinking effect of a water-soluble QD. Black-Right-Pointing-Pointer We provide a direct method to study protein function and dynamics at the single level. Black-Right-Pointing-Pointer QD, potent tool for single molecule studies of biochemical and biological processes. -- Abstract: Luminescent semiconductor nanocrystals (quantum dots, QD) have unique photo-physical properties: high photostability, brightness and narrow size-tunable fluorescence spectra. Due to their unique properties, QD-based single molecule studies have become increasingly more popular during the last years. However QDs show a strong blinking effect (random and intermittent light emission), which may limit their use in single molecule fluorescence studies. QD blinking has been widely studied and some hypotheses have been done to explain this effect. Here we summarise what is known about the blinking effect in QDs, how this phenomenon may affect single molecule studies and, on the other hand, how the 'on'/'off' states can be exploited in diverse experimental settings. In addition, we present results showing that site-directed binding of QD to cysteine residues of proteins reduces the blinking effect. This option opens a new possibility of using QDs to study protein-protein interactions and dynamics by single molecule fluorescence without modifying the chemical composition of the solution or the QD surface.

  19. Ninth international conference on hole burning, single molecule and related spectroscopies: science and applications (HBSM 2006)

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2006-07-01

    This conference was organized around 9 sessions: -) single molecule, -) quantum optics, -) hole-burning materials and mechanisms, -) single nano-particle spectroscopy, -) dephasing and spectral diffusion, -) microwave photonics, -) biological systems, -) rare earth doped materials, -) novel laser sources. This document gathers only the slides of the presentations.

  20. Ninth international conference on hole burning, single molecule and related spectroscopies: science and applications (HBSM 2006)

    International Nuclear Information System (INIS)

    2006-01-01

    This conference was organized around 9 sessions: -) single molecule, -) quantum optics, -) hole-burning materials and mechanisms, -) single nano-particle spectroscopy, -) dephasing and spectral diffusion, -) microwave photonics, -) biological systems, -) rare earth doped materials, -) novel laser sources. This document gathers only the slides of the presentations

  1. 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.

  2. Radiation- and phonon-bottleneck--induced tunneling in the Fe8 single-molecule magnet

    Science.gov (United States)

    Bal, M.; Friedman, Jonathan R.; Chen, W.; Tuominen, M. T.; Beedle, C. C.; Rumberger, E. M.; Hendrickson, D. N.

    2008-04-01

    We measure magnetization changes in a single crystal of the single-molecule magnet Fe8 when exposed to intense, short (spin dynamics, allowing observation of thermally assisted resonant tunneling between spin states at the 100 ns time scale. Detailed numerical simulations quantitatively reproduce the data and yield a spin-phonon relaxation time T1~40 ns.

  3. Spectral multitude and spectral dynamics reflect changing conjugation length in single molecules of oligophenylenevinylenes

    KAUST Repository

    Kobayashi, Hiroyuki

    2012-01-01

    Single-molecule study of phenylenevinylene oligomers revealed distinct spectral forms due to different conjugation lengths which are determined by torsional defects. Large spectral jumps between different spectral forms were ascribed to torsional flips of a single phenylene ring. These spectral changes reflect the dynamic nature of electron delocalization in oligophenylenevinylenes and enable estimation of the phenylene torsional barriers. © 2012 The Owner Societies.

  4. Metal-Controlled Magnetoresistance at Room Temperature in Single-Molecule Devices.

    Science.gov (United States)

    Aragonès, Albert C; Aravena, Daniel; Valverde-Muñoz, Francisco J; Real, José Antonio; Sanz, Fausto; Díez-Pérez, Ismael; Ruiz, Eliseo

    2017-04-26

    The appropriate choice of the transition metal complex and metal surface electronic structure opens the possibility to control the spin of the charge carriers through the resulting hybrid molecule/metal spinterface in a single-molecule electrical contact at room temperature. The single-molecule conductance of a Au/molecule/Ni junction can be switched by flipping the magnetization direction of the ferromagnetic electrode. The requirements of the molecule include not just the presence of unpaired electrons: the electronic configuration of the metal center has to provide occupied or empty orbitals that strongly interact with the junction metal electrodes and that are close in energy to their Fermi levels for one of the electronic spins only. The key ingredient for the metal surface is to provide an efficient spin texture induced by the spin-orbit coupling in the topological surface states that results in an efficient spin-dependent interaction with the orbitals of the molecule. The strong magnetoresistance effect found in this kind of single-molecule wire opens a new approach for the design of room-temperature nanoscale devices based on spin-polarized currents controlled at molecular level.

  5. Drift correction for single-molecule imaging by molecular constraint field, a distance minimum metric

    International Nuclear Information System (INIS)

    Han, Renmin; Wang, Liansan; Xu, Fan; Zhang, Yongdeng; Zhang, Mingshu; Liu, Zhiyong; Ren, Fei; Zhang, Fa

    2015-01-01

    The recent developments of far-field optical microscopy (single molecule imaging techniques) have overcome the diffraction barrier of light and improve image resolution by a factor of ten compared with conventional light microscopy. These techniques utilize the stochastic switching of probe molecules to overcome the diffraction limit and determine the precise localizations of molecules, which often requires a long image acquisition time. However, long acquisition times increase the risk of sample drift. In the case of high resolution microscopy, sample drift would decrease the image resolution. In this paper, we propose a novel metric based on the distance between molecules to solve the drift correction. The proposed metric directly uses the position information of molecules to estimate the frame drift. We also designed an algorithm to implement the metric for the general application of drift correction. There are two advantages of our method: First, because our method does not require space binning of positions of molecules but directly operates on the positions, it is more natural for single molecule imaging techniques. Second, our method can estimate drift with a small number of positions in each temporal bin, which may extend its potential application. The effectiveness of our method has been demonstrated by both simulated data and experiments on single molecular images

  6. Assembly and diploid architecture of an individual human genome via single-molecule technologies.

    Science.gov (United States)

    Pendleton, Matthew; Sebra, Robert; Pang, Andy Wing Chun; Ummat, Ajay; Franzen, Oscar; Rausch, Tobias; Stütz, Adrian M; Stedman, William; Anantharaman, Thomas; Hastie, Alex; Dai, Heng; Fritz, Markus Hsi-Yang; Cao, Han; Cohain, Ariella; Deikus, Gintaras; Durrett, Russell E; Blanchard, Scott C; Altman, Roger; Chin, Chen-Shan; Guo, Yan; Paxinos, Ellen E; Korbel, Jan O; Darnell, Robert B; McCombie, W Richard; Kwok, Pui-Yan; Mason, Christopher E; Schadt, Eric E; Bashir, Ali

    2015-08-01

    We present the first comprehensive analysis of a diploid human genome that combines single-molecule sequencing with single-molecule genome maps. Our hybrid assembly markedly improves upon the contiguity observed from traditional shotgun sequencing approaches, with scaffold N50 values approaching 30 Mb, and we identified complex structural variants (SVs) missed by other high-throughput approaches. Furthermore, by combining Illumina short-read data with long reads, we phased both single-nucleotide variants and SVs, generating haplotypes with over 99% consistency with previous trio-based studies. Our work shows that it is now possible to integrate single-molecule and high-throughput sequence data to generate de novo assembled genomes that approach reference quality.

  7. Insects' RNA Profiling Reveals Absence of "Hidden Break" in 28S Ribosomal RNA Molecule of Onion Thrips, Thrips tabaci.

    Science.gov (United States)

    Macharia, Rosaline Wanjiru; Ombura, Fidelis Levi; Aroko, Erick Onyango

    2015-01-01

    With an exception of aphids, insects' 28S rRNA is thought to harbor a "hidden break" which cleaves under denaturing conditions to comigrate with 18S rRNA band to exhibit a degraded appearance on native agarose gels. The degraded appearance confounds determination of RNA integrity in laboratories that rely on gel electrophoresis. To provide guidelines for RNA profiles, RNA from five major insect orders, namely, Diptera, Hemiptera, Thysanoptera, Hymenoptera, and Lepidoptera, was compared under denaturing and nondenaturing conditions. This study confirmed that although present in most of insect's RNA, the "hidden break" is absent in the 28S rRNA of onion thrips, Thrips tabaci. On the other hand, presence of "hidden break" was depicted in whiteflies' 28S rRNA despite their evolutionary grouping under same order with aphids. Divergence of 28S rRNA sequences confirms variation of both size and composition of gap region among insect species. However, phylogeny reconstruction does not support speciation as a possible source of the hidden break in insect's 28S rRNA. In conclusion, we show that RNA from a given insect order does not conform to a particular banding profile and therefore this approach cannot be reliably used to characterize newly discovered species.

  8. Nonequilibrium Chemical Effects in Single-Molecule SERS Revealed by Ab Initio Molecular Dynamics Simulations

    Energy Technology Data Exchange (ETDEWEB)

    Fischer, Sean A.; Apra, Edoardo; Govind, Niranjan; Hess, Wayne P.; El-Khoury, Patrick Z.

    2017-02-03

    Recent developments in nanophotonics have paved the way for achieving significant advances in the realm of single molecule chemical detection, imaging, and dynamics. In particular, surface-enhanced Raman scattering (SERS) is a powerful analytical technique that is now routinely used to identify the chemical identity of single molecules. Understanding how nanoscale physical and chemical processes affect single molecule SERS spectra and selection rules is a challenging task, and is still actively debated. Herein, we explore underappreciated chemical phenomena in ultrasensitive SERS. We observe a fluctuating excited electronic state manifold, governed by the conformational dynamics of a molecule (4,4’-dimercaptostilbene, DMS) interacting with a metallic cluster (Ag20). This affects our simulated single molecule SERS spectra; the time trajectories of a molecule interacting with its unique local environment dictates the relative intensities of the observable Raman-active vibrational states. Ab initio molecular dynamics of a model Ag20-DMS system are used to illustrate both concepts in light of recent experimental results.

  9. Single-molecule chemical reaction reveals molecular reaction kinetics and dynamics.

    Science.gov (United States)

    Zhang, Yuwei; Song, Ping; Fu, Qiang; Ruan, Mingbo; Xu, Weilin

    2014-06-25

    Understanding the microscopic elementary process of chemical reactions, especially in condensed phase, is highly desirable for improvement of efficiencies in industrial chemical processes. Here we show an approach to gaining new insights into elementary reactions in condensed phase by combining quantum chemical calculations with a single-molecule analysis. Elementary chemical reactions in liquid-phase, revealed from quantum chemical calculations, are studied by tracking the fluorescence of single dye molecules undergoing a reversible redox process. Statistical analyses of single-molecule trajectories reveal molecular reaction kinetics and dynamics of elementary reactions. The reactivity dynamic fluctuations of single molecules are evidenced and probably arise from either or both of the low-frequency approach of the molecule to the internal surface of the SiO2 nanosphere or the molecule diffusion-induced memory effect. This new approach could be applied to other chemical reactions in liquid phase to gain more insight into their molecular reaction kinetics and the dynamics of elementary steps.

  10. Electronic Transport in Single Molecule Junctions: Control of the Molecule-Electrode Coupling Through Intramolecular Tunneling Barriers

    DEFF Research Database (Denmark)

    Danilov, Andrey; Kubatkin, Sergey; Kafanov, Sergey

    2008-01-01

    We report on single molecule electron transport measurements of two oligophenylenevinylene (OPV3) derivatives placed in a nanogap between gold (Au) or lead (Pb) electrodes in a field effect transistor device. Both derivatives contain thiol end groups that allow chemical binding to the electrodes....... One derivative has additional methylene groups separating the thiols from the delocalized -electron system. The insertion of methylene groups changes the open state conductance by 3-4 orders of magnitude and changes the transport mechanism from a coherent regime with finite zero-bias conductance...

  11. Studying σ 54-dependent transcription at the single-molecule level using alternating-laser excitation (ALEX) spectroscopy

    Science.gov (United States)

    Heilemann, M.; Lymperopoulos, K.; Wigneshweraraj, S. R.; Buck, M.; Kapanidis, A. N.

    2007-07-01

    We present single-molecule fluorescence studies of σ 54-dependent gene-transcription complexes using singlemolecule fluorescence resonance energy transfer (smFRET) and alternating-laser excitation (ALEX) spectroscopy. The ability to study one biomolecule at the time allowed us to resolve and analyze sample heterogeneities and extract structural information on subpopulations and transient intermediates of transcription; such information is hidden in bulk experiments. Using site-specifically labeled σ 54 derivatives and site-specifically labeled promoter-DNA fragments, we demonstrate that we can observe single diffusing σ 54-DNA and transcription-initiation RNA polymerase-σ 54- DNA complexes, and that we can measure distances within such complexes; the identity of the complexes has been confirmed using electrophoretic-mobility-shift assays. Our studies pave the way for understanding the mechanism of abortive initiation and promoter escape in σ 54-dependent transcription.

  12. Single-molecule imaging and manipulation of biomolecular machines and systems.

    Science.gov (United States)

    Iino, Ryota; Iida, Tatsuya; Nakamura, Akihiko; Saita, Ei-Ichiro; You, Huijuan; Sako, Yasushi

    2018-02-01

    Biological molecular machines support various activities and behaviors of cells, such as energy production, signal transduction, growth, differentiation, and migration. We provide an overview of single-molecule imaging methods involving both small and large probes used to monitor the dynamic motions of molecular machines in vitro (purified proteins) and in living cells, and single-molecule manipulation methods used to measure the forces, mechanical properties and responses of biomolecules. We also introduce several examples of single-molecule analysis, focusing primarily on motor proteins and signal transduction systems. Single-molecule analysis is a powerful approach to unveil the operational mechanisms both of individual molecular machines and of systems consisting of many molecular machines. Quantitative, high-resolution single-molecule analyses of biomolecular systems at the various hierarchies of life will help to answer our fundamental question: "What is life?" This article is part of a Special Issue entitled "Biophysical Exploration of Dynamical Ordering of Biomolecular Systems" edited by Dr. Koichi Kato. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. 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.

  14. Novel exons and splice variants in the human antibody heavy chain identified by single cell and single molecule sequencing.

    Directory of Open Access Journals (Sweden)

    Christopher Vollmers

    Full Text Available Antibody heavy chains contain a variable and a constant region. The constant region of the antibody heavy chain is encoded by multiple groups of exons which define the isotype and therefore many functional characteristics of the antibody. We performed both single B cell RNAseq and long read single molecule sequencing of antibody heavy chain transcripts and were able to identify novel exons for IGHA1 and IGHA2 as well as novel isoforms for IGHM antibody heavy chain.

  15. madSTORM: a superresolution technique for large-scale multiplexing at single-molecule accuracy

    Science.gov (United States)

    Yi, Jason; Manna, Asit; Barr, Valarie A.; Hong, Jennifer; Neuman, Keir C.; Samelson, Lawrence E.

    2016-01-01

    Investigation of heterogeneous cellular structures using single-molecule localization microscopy has been limited by poorly defined localization accuracy and inadequate multiplexing capacity. Using fluorescent nanodiamonds as fiducial markers, we define and achieve localization precision required for single-molecule accuracy in dSTORM images. Coupled with this advance, our new multiplexing strategy, madSTORM, allows accurate targeting of multiple molecules using sequential binding and elution of fluorescent antibodies. madSTORM is used on an activated T-cell to localize 25 epitopes, 14 of which are on components of the same multimolecular T-cell receptor complex. We obtain an average localization precision of 2.6 nm, alignment error of 2.0 nm, and molecules within structures. Probing the molecular topology of complex signaling cascades and other heterogeneous networks is feasible with madSTORM. PMID:27708141

  16. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.

    Science.gov (United States)

    Chomczynski, P; Sacchi, N

    1987-04-01

    A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.

  17. Integrated Transmission Electron and Single-Molecule Fluorescence Microscopy Correlates Reactivity with Ultrastructure in a Single Catalyst Particle

    OpenAIRE

    Hendriks, Frank C.; Mohammadian, Sajjad; Ristanovic, Zoran; Kalirai, Samanbir; Meirer, Florian; Vogt, Eelco T. C.; Bruijnincx, Pieter C. A.; Gerritsen, Hans; Weckhuysen, Bert M.

    2018-01-01

    Establishing structure–activity relationships in complex, hierarchically structured nanomaterials, such as fluid catalytic cracking (FCC) catalysts, requires characterization with complementary, correlated analysis techniques. An integrated setup has been developed to perform transmission electron microscopy (TEM) and single-molecule fluorescence (SMF) microscopy on such nanostructured samples. Correlated structure–reactivity information was obtained for 100 nm thin, microtomed sections of a ...

  18. Minimizing pulling geometry errors in atomic force microscope single molecule force spectroscopy.

    Science.gov (United States)

    Rivera, Monica; Lee, Whasil; Ke, Changhong; Marszalek, Piotr E; Cole, Daniel G; Clark, Robert L

    2008-10-01

    In atomic force microscopy-based single molecule force spectroscopy (AFM-SMFS), it is assumed that the pulling angle is negligible and that the force applied to the molecule is equivalent to the force measured by the instrument. Recent studies, however, have indicated that the pulling geometry errors can drastically alter the measured force-extension relationship of molecules. Here we describe a software-based alignment method that repositions the cantilever such that it is located directly above the molecule's substrate attachment site. By aligning the applied force with the measurement axis, the molecule is no longer undergoing combined loading, and the full force can be measured by the cantilever. Simulations and experimental results verify the ability of the alignment program to minimize pulling geometry errors in AFM-SMFS studies.

  19. Sequence variation of the human immunodeficiency virus primer-binding site suggests the use of an alternative tRNA(Lys) molecule in reverse transcription

    NARCIS (Netherlands)

    Das, A. T.; Klaver, B.; Berkhout, B.

    1997-01-01

    Retroviruses use a cellular tRNA molecule as primer for reverse transcription. The complementarity between the 3' end of this tRNA and a sequence near the 5' end of the viral RNA, the primer-binding site (PBS), allows the primer to anneal onto the viral RNA. During reverse transcription 18

  20. Manifestation of Spin Selection Rules on the Quantum Tunneling of Magnetization in a Single Molecule Magnet

    OpenAIRE

    Henderson, J. J.; Koo, C.; Feng, P. L.; del Barco, E.; Hill, S.; Tupitsyn, I. S.; Stamp, P. C. E.; Hendrickson, D. N.

    2009-01-01

    We present low temperature magnetometry measurements on a new Mn3 single-molecule magnet (SMM) in which the quantum tunneling of magnetization (QTM) displays clear evidence for quantum mechanical selection rules. A QTM resonance appearing only at elevated temperatures demonstrates tunneling between excited states with spin projections differing by a multiple of three: this is dictated by the C3 symmetry of the molecule, which forbids pure tunneling from the lowest metastable state. Resonances...

  1. Aberration-accounting calibration for 3D single-molecule localization microscopy

    Science.gov (United States)

    Cabriel, Clément; Bourg, Nicolas; Dupuis, Guillaume; Lévêque-Fort, Sandrine

    2018-01-01

    We propose a straightforward sample-based technique to calibrate the axial detection in 3D single molecule localization microscopy (SMLM). Using microspheres coated with fluorescent molecules, the calibration curves of PSF-shaping- or intensity-based measurements can be obtained for any required depth range from a few hundreds of nanometers to several tens of microns. This experimental method takes into account the effect of the spherical aberration without requiring computational correction.

  2. Effect of ligand substitution on the exchange interactions in {Mn12}-type single-molecule magnets

    OpenAIRE

    Boukhvalov, D. W.; Dobrovitski, V. V.; Kögerler, P.; Al-Saqer, M.; Katsnelson, M. I.; Lichtenstein, A. I.; Harmon, B. N.

    2010-01-01

    We investigate how the ligand substitution affects the intra-molecular spin exchange interactions, studying a prototypal family of single-molecule magnets comprising dodecanuclear cluster molecules [Mn12O12(COOR)16]. We identify a simple scheme based on accumulated Pauling electronegativity numbers (a.e.n.) of the carboxylate ligand groups (R). The redistribution of the electron density, controlled by a.e.n. of a ligand, changes the degree of hybridization between 3d electrons of manganese an...

  3. Super-Resolution Imaging of Molecular Emission Spectra and Single Molecule Spectral Fluctuations.

    Science.gov (United States)

    Mlodzianoski, Michael J; Curthoys, Nikki M; Gunewardene, Mudalige S; Carter, Sean; Hess, Samuel T

    2016-01-01

    Localization microscopy can image nanoscale cellular details. To address biological questions, the ability to distinguish multiple molecular species simultaneously is invaluable. Here, we present a new version of fluorescence photoactivation localization microscopy (FPALM) which detects the emission spectrum of each localized molecule, and can quantify changes in emission spectrum of individual molecules over time. This information can allow for a dramatic increase in the number of different species simultaneously imaged in a sample, and can create super-resolution maps showing how single molecule emission spectra vary with position and time in a sample.

  4. Single-Molecule Rotational Switch on a Dangling Bond Dimer Bearing.

    Science.gov (United States)

    Godlewski, Szymon; Kawai, Hiroyo; Kolmer, Marek; Zuzak, Rafał; Echavarren, Antonio M; Joachim, Christian; Szymonski, Marek; Saeys, Mark

    2016-09-27

    One of the key challenges in the construction of atomic-scale circuits and molecular machines is to design molecular rotors and switches by controlling the linear or rotational movement of a molecule while preserving its intrinsic electronic properties. Here, we demonstrate both the continuous rotational switching and the controlled step-by-step single switching of a trinaphthylene molecule adsorbed on a dangling bond dimer created on a hydrogen-passivated Ge(001):H surface. The molecular switch is on-surface assembled when the covalent bonds between the molecule and the dangling bond dimer are controllably broken, and the molecule is attached to the dimer by long-range van der Waals interactions. In this configuration, the molecule retains its intrinsic electronic properties, as confirmed by combined scanning tunneling microscopy/spectroscopy (STM/STS) measurements, density functional theory calculations, and advanced STM image calculations. Continuous switching of the molecule is initiated by vibronic excitations when the electrons are tunneling through the lowest unoccupied molecular orbital state of the molecule. The switching path is a combination of a sliding and rotation motion over the dangling bond dimer pivot. By carefully selecting the STM conditions, control over discrete single switching events is also achieved. Combined with the ability to create dangling bond dimers with atomic precision, the controlled rotational molecular switch is expected to be a crucial building block for more complex surface atomic-scale devices.

  5. Single-molecule detection of dihydroazulene photo-thermal reaction using break junction technique

    Science.gov (United States)

    Huang, Cancan; Jevric, Martyn; Borges, Anders; Olsen, Stine T.; Hamill, Joseph M.; Zheng, Jue-Ting; Yang, Yang; Rudnev, Alexander; Baghernejad, Masoud; Broekmann, Peter; Petersen, Anne Ugleholdt; Wandlowski, Thomas; Mikkelsen, Kurt V.; Solomon, Gemma C.; Brøndsted Nielsen, Mogens; Hong, Wenjing

    2017-05-01

    Charge transport by tunnelling is one of the most ubiquitous elementary processes in nature. Small structural changes in a molecular junction can lead to significant difference in the single-molecule electronic properties, offering a tremendous opportunity to examine a reaction on the single-molecule scale by monitoring the conductance changes. Here, we explore the potential of the single-molecule break junction technique in the detection of photo-thermal reaction processes of a photochromic dihydroazulene/vinylheptafulvene system. Statistical analysis of the break junction experiments provides a quantitative approach for probing the reaction kinetics and reversibility, including the occurrence of isomerization during the reaction. The product ratios observed when switching the system in the junction does not follow those observed in solution studies (both experiment and theory), suggesting that the junction environment was perturbing the process significantly. This study opens the possibility of using nano-structured environments like molecular junctions to tailor product ratios in chemical reactions.

  6. Single-molecule, structural and functional studies of Listeria monocytogenes Ca2+-ATPase

    DEFF Research Database (Denmark)

    Dyla, Mateusz

    -ion transport (e.g. H+ for Ca2+-ATPases). P-type ATPases undergo major conformational changes during their functional cycle, as has been learned from a wealth of atomic-resolution X-ray crystallographic structures (4). In this work, single-molecule, structural and functional studies were employed to investigate...... the dynamics and mechanism of the transport cycle of P-type ATPase at a single molecule level. A representative P-type ATPase, the Listeria monocytogenes Ca2+-ATPase (LMCA1) was engineered and characterized to facilitate smFRET studies. Pairs of cysteines were introduced and reacted with maleimide derivatives...... transitions to the E2 state triggered by ATP binding and phosphorylation were very brief, and could only be characterized in a dephosphorylation-deficient LMCA1 mutant. Owing to a spontaneous dephosphorylation of this mutant, full transport cycles at a single-molecule resolution were observed for the first...

  7. Single-molecule three-color FRET with both negligible spectral overlap and long observation time.

    Directory of Open Access Journals (Sweden)

    Sanghwa Lee

    Full Text Available Full understanding of complex biological interactions frequently requires multi-color detection capability in doing single-molecule fluorescence resonance energy transfer (FRET experiments. Existing single-molecule three-color FRET techniques, however, suffer from severe photobleaching of Alexa 488, or its alternative dyes, and have been limitedly used for kinetics studies. In this work, we developed a single-molecule three-color FRET technique based on the Cy3-Cy5-Cy7 dye trio, thus providing enhanced observation time and improved data quality. Because the absorption spectra of three fluorophores are well separated, real-time monitoring of three FRET efficiencies was possible by incorporating the alternating laser excitation (ALEX technique both in confocal microscopy and in total-internal-reflection fluorescence (TIRF microscopy.

  8. PhotoGate microscopy: tracking single molecules in a cytoplasm (Conference Presentation)

    Science.gov (United States)

    Yildiz, Ahmet

    2016-02-01

    Tracking single molecules inside cells reveals the dynamics of biological processes, including receptor trafficking, signaling and cargo transport. However, individual molecules often cannot be resolved inside cells due to their high density in the cellular environment. We developed a photobleaching gate assay, which controls the number of fluorescent particles in a region of interest by repeatedly photobleaching its boundary. Using this method, we tracked single particles at surface densities two orders of magnitude higher than the single-molecule detection limit. We observed ligand-induced dimerization of epidermal growth factor receptors (EGFR) on a live cell membrane. In addition, we tracked individual intraflagellar transport (IFT) trains along the length of a cilium and observed their remodeling at the ciliary tip.

  9. Surface-enhanced Raman spectroscopy of dyes: from single molecules to the artists' canvas.

    Science.gov (United States)

    Wustholz, Kristin L; Brosseau, Christa L; Casadio, Francesca; Van Duyne, Richard P

    2009-09-14

    This perspective presents recent surface-enhanced Raman spectroscopy (SERS) studies of dyes, with applications to the fields of single-molecule spectroscopy and art conservation. First we describe the development and outlook of single-molecule SERS (SMSERS). Rather than providing an exhaustive review of the literature, SMSERS experiments that we consider essential for its future development are emphasized. Shifting from single-molecule to ensemble-averaged experiments, we describe recent efforts toward SERS analysis of colorants in precious artworks. Our intention is to illustrate through these examples that the forward development of SERS is dependent upon both fundamental (e.g., SMSERS) and applied (e.g., on-the-specimen SERS of historical art objects) investigations and that the future of SERS is very bright indeed.

  10. Influence of quantum dot labels on single molecule movement in the plasma membrane

    DEFF Research Database (Denmark)

    Clausen, Mathias P.; Lagerholm, B. Christoffer

    2011-01-01

    Single particle tracking results are very dependent on the probe that is used. In this study we have investigated the influence that functionalized quantum dots (QDs) have on the recorded movement in single molecule tracking experiments of plasma membrane species in live cells. Potential issues...... in labeling single molecules with QDs (and other particles e.g. gold particles) are induction of cross-linking of the target molecules, which can cause activation of signaling pathways or reduced mobility, and steric hindrance as a result of the probe size. Cross-linking can be a result of the multivalent...... functionalization tag (e.g. streptavidin (sAv)) or the presence of multiple mono- or multivalent functionalization tags per QD. In this work, we have compared commercially available sAv-QDs of different sizes with custom prepared Co enzyme A (CoA)-QDs both targeting a GPI-anchored protein modified with either...

  11. Mapping Nanoscale Hotspots with Single-Molecule Emitters Assembled into Plasmonic Nanocavities Using DNA Origami

    Science.gov (United States)

    Chikkaraddy, Rohit; Turek, V. A.; Kongsuwan, Nuttawut; Benz, Felix; Carnegie, Cloudy; van de Goor, Tim; de Nijs, Bart; Demetriadou, Angela; Hess, Ortwin; Keyser, Ulrich F.; Baumberg, Jeremy J.

    2018-01-01

    Fabricating nanocavities in which optically-active single quantum emitters are precisely positioned, is crucial for building nanophotonic devices. Here we show that self-assembly based on robust DNA-origami constructs can precisely position single molecules laterally within sub-5nm gaps between plasmonic substrates that support intense optical confinement. By placing single-molecules at the center of a nanocavity, we show modification of the plasmon cavity resonance before and after bleaching the chromophore, and obtain enhancements of $\\geq4\\times10^3$ with high quantum yield ($\\geq50$%). By varying the lateral position of the molecule in the gap, we directly map the spatial profile of the local density of optical states with a resolution of $\\pm1.5$ nm. Our approach introduces a straightforward non-invasive way to measure and quantify confined optical modes on the nanoscale.

  12. Single-molecule detection of dihydroazulene photo-thermal reaction using break junction technique

    DEFF Research Database (Denmark)

    Huang, Cancan; Jevric, Martyn; Borges, Anders Christian

    2017-01-01

    a quantitative approach for probing the reaction kinetics and reversibility, including the occurrence of isomerization during the reaction. The product ratios observed when switching the system in the junction does not follow those observed in solution studies (both experiment and theory), suggesting......Charge transport by tunnelling is one of the most ubiquitous elementary processes in nature. Small structural changes in a molecular junction can lead to significant difference in the single-molecule electronic properties, offering a tremendous opportunity to examine a reaction on the single......-molecule scale by monitoring the conductance changes. Here, we explore the potential of the single-molecule break junction technique in the detection of photo-thermal reaction processes of a photochromic dihydroazulene/vinylheptafulvene system. Statistical analysis of the break junction experiments provides...

  13. Three-Dimensional Localization of Single Molecules for Super-Resolution Imaging and Single-Particle Tracking.

    Science.gov (United States)

    von Diezmann, Alex; Shechtman, Yoav; Moerner, W E

    2017-06-14

    Single-molecule super-resolution fluorescence microscopy and single-particle tracking are two imaging modalities that illuminate the properties of cells and materials on spatial scales down to tens of nanometers or with dynamical information about nanoscale particle motion in the millisecond range, respectively. These methods generally use wide-field microscopes and two-dimensional camera detectors to localize molecules to much higher precision than the diffraction limit. Given the limited total photons available from each single-molecule label, both modalities require careful mathematical analysis and image processing. Much more information can be obtained about the system under study by extending to three-dimensional (3D) single-molecule localization: without this capability, visualization of structures or motions extending in the axial direction can easily be missed or confused, compromising scientific understanding. A variety of methods for obtaining both 3D super-resolution images and 3D tracking information have been devised, each with their own strengths and weaknesses. These include imaging of multiple focal planes, point-spread-function engineering, and interferometric detection. These methods may be compared based on their ability to provide accurate and precise position information on single-molecule emitters with limited photons. To successfully apply and further develop these methods, it is essential to consider many practical concerns, including the effects of optical aberrations, field dependence in the imaging system, fluorophore labeling density, and registration between different color channels. Selected examples of 3D super-resolution imaging and tracking are described for illustration from a variety of biological contexts and with a variety of methods, demonstrating the power of 3D localization for understanding complex systems.

  14. Enhancement of single guide RNA transcription for efficient CRISPR/Cas-based genomic engineering.

    Science.gov (United States)

    Ui-Tei, Kumiko; Maruyama, Shohei; Nakano, Yuko

    2017-06-01

    Genomic engineering using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein is a promising approach for targeting the genomic DNA of virtually any organism in a sequence-specific manner. Recent remarkable advances in CRISPR/Cas technology have made it a feasible system for use in therapeutic applications and biotechnology. In the CRISPR/Cas system, a guide RNA (gRNA), interacting with the Cas protein, recognizes a genomic region with sequence complementarity, and the double-stranded DNA at the target site is cleaved by the Cas protein. A widely used gRNA is an RNA polymerase III (pol III)-driven single gRNA (sgRNA), which is produced by artificial fusion of CRISPR RNA (crRNA) and trans-activation crRNA (tracrRNA). However, we identified a TTTT stretch, known as a termination signal of RNA pol III, in the scaffold region of the sgRNA. Here, we revealed that sgRNA carrying a TTTT stretch reduces the efficiency of sgRNA transcription due to premature transcriptional termination, and decreases the efficiency of genome editing. Unexpectedly, it was also shown that the premature terminated sgRNA may have an adverse effect of inducing RNA interference. Such disadvantageous effects were avoided by substituting one base in the TTTT stretch.

  15. Folding and ligand recognition of the TPP riboswitch aptamer at single-molecule resolution.

    Science.gov (United States)

    Haller, Andrea; Altman, Roger B; Soulière, Marie F; Blanchard, Scott C; Micura, Ronald

    2013-03-12

    Thiamine pyrophosphate (TPP)-sensitive mRNA domains are the most prevalent riboswitches known. Despite intensive investigation, the complex ligand recognition and concomitant folding processes in the TPP riboswitch that culminate in the regulation of gene expression remain elusive. Here, we used single-molecule fluorescence resonance energy transfer imaging to probe the folding landscape of the TPP aptamer domain in the absence and presence of magnesium and TPP. To do so, distinct labeling patterns were used to sense the dynamics of the switch helix (P1) and the two sensor arms (P2/P3 and P4/P5) of the aptamer domain. The latter structural elements make interdomain tertiary contacts (L5/P3) that span a region immediately adjacent to the ligand-binding site. In each instance, conformational dynamics of the TPP riboswitch were influenced by ligand binding. The P1 switch helix, formed by the 5' and 3' ends of the aptamer domain, adopts a predominantly folded structure in the presence of Mg(2+) alone. However, even at saturating concentrations of Mg(2+) and TPP, the P1 helix, as well as distal regions surrounding the TPP-binding site, exhibit an unexpected degree of residual dynamics and disperse kinetic behaviors. Such plasticity results in a persistent exchange of the P3/P5 forearms between open and closed configurations that is likely to facilitate entry and exit of the TPP ligand. Correspondingly, we posit that such features of the TPP aptamer domain contribute directly to the mechanism of riboswitch-mediated translational regulation.

  16. Identification of Putative Coffee Rust Mycoparasites via Single-Molecule DNA Sequencing of Infected Pustules.

    Science.gov (United States)

    James, Timothy Y; Marino, John A; Perfecto, Ivette; Vandermeer, John

    2016-01-15

    The interaction of crop pests with their natural enemies is a fundament to their control. Natural enemies of fungal pathogens of crops are poorly known relative to those of insect pests, despite the diversity of fungal pathogens and their economic importance. Currently, many regions across Latin America are experiencing unprecedented epidemics of coffee rust (Hemileia vastatrix). Identification of natural enemies of coffee rust could aid in developing management strategies or in pinpointing species that could be used for biocontrol. In the present study, we characterized fungal communities associated with coffee rust lesions by single-molecule DNA sequencing of fungal rRNA gene bar codes from leaf discs (≈28 mm(2)) containing rust lesions and control discs with no rust lesions. The leaf disc communities were hyperdiverse in terms of fungi, with up to 69 operational taxonomic units (putative species) per control disc, and the diversity was only slightly reduced in rust-infected discs, with up to 63 putative species. However, geography had a greater influence on the fungal community than whether the disc was infected by coffee rust. Through comparisons between control and rust-infected leaf discs, as well as taxonomic criteria, we identified 15 putative mycoparasitic fungi. These fungi are concentrated in the fungal family Cordycipitaceae and the order Tremellales. These data emphasize the complexity of diverse fungi of unknown ecological function within a leaf that might influence plant disease epidemics or lead to the development of species for biocontrol of fungal disease. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  17. Single-Molecule Fluorescence Microscopy Reveals Local Diffusion Coefficients in the Pore Network of an Individual Catalyst Particle

    NARCIS (Netherlands)

    Hendriks, Frank|info:eu-repo/dai/nl/412642697; Meirer, Florian; Kubarev, Alexey V.; Ristanovic, Zoran|info:eu-repo/dai/nl/328233005; Roeffaers, Maarten B J; Vogt, Eelco T. C.|info:eu-repo/dai/nl/073717398; Bruijnincx, Pieter C. A.|info:eu-repo/dai/nl/33799529X; Weckhuysen, Bert M.|info:eu-repo/dai/nl/285484397

    2017-01-01

    We used single-molecule fluorescence microscopy to study self-diffusion of a feedstock-like probe molecule with nanometer accuracy in the macropores of a micrometer-sized, real-life fluid catalytic cracking (FCC) particle. Movies of single fluorescent molecules allowed their movement through the

  18. Dynamic encapsulation of corannulene molecules into a single-walled carbon nanotube.

    Science.gov (United States)

    Joko, Y; Sasaki, R; Shintani, K

    2017-10-18

    The morphology of corannulene molecules encapsulated in a single-walled carbon nanotube (SWCNT) is addressed using atomistic simulations. Firstly, dynamic simulation (DS) of encapsulation of corannulene molecules into a SWCNT is performed using a molecular dynamics (MD) method. It is revealed that corannulene molecules encapsulated in a SWCNT tend to form concave-concave (CC) dimers, and these dimers make stacks tilting against the SWCNT axis or take an arrangement such that their convex surfaces face the inner wall of the SWCNT. This tendency arises from the fact that the van der Waals interactions between the convex surfaces of the corannulene molecules and the inner wall of the SWCNT dominate in their dynamic encapsulation into the SWCNT, and CC dimers are favored based on the energetics. Next, conjugate gradient (CG) energy minimizations starting from two kinds of initial arrangement of corannulene molecules in a SWCNT, concave-convex (CV) and CC/convex-convex (VV) arrangements, are performed. The CG energy minimizations confirm the result of DS that CC dimers are the structural motif of corannulene molecules in a SWCNT. From the final configurations of both the simulations, the tilt angles and intermolecular distances of the stacked molecules are calculated. With increasing the SWCNT diameter, the tilt angles decrease while the intermolecular distances remain almost constant. The tilt angles of the stacked corannulene molecules are approximately expressed by a semi-analytical formula which is derived on the basis of a geometrical constraint condition.

  19. Demonstration of Single-Barium-Ion Sensitivity for Neutrinoless Double-Beta Decay Using Single-Molecule Fluorescence Imaging

    Science.gov (United States)

    McDonald, A. D.; Jones, B. J. P.; Nygren, D. R.; Adams, C.; Álvarez, V.; Azevedo, C. D. R.; Benlloch-Rodríguez, J. M.; Borges, F. I. G. M.; Botas, A.; Cárcel, S.; Carrión, J. V.; Cebrián, S.; Conde, C. A. N.; Díaz, J.; Diesburg, M.; Escada, J.; Esteve, R.; Felkai, R.; Fernandes, L. M. P.; Ferrario, P.; Ferreira, A. L.; Freitas, E. D. C.; Goldschmidt, A.; Gómez-Cadenas, J. J.; González-Díaz, D.; Gutiérrez, R. M.; Guenette, R.; Hafidi, K.; Hauptman, J.; Henriques, C. A. O.; Hernandez, A. I.; Hernando Morata, J. A.; Herrero, V.; Johnston, S.; Labarga, L.; Laing, A.; Lebrun, P.; Liubarsky, I.; López-March, N.; Losada, M.; Martín-Albo, J.; Martínez-Lema, G.; Martínez, A.; Monrabal, F.; Monteiro, C. M. B.; Mora, F. J.; Moutinho, L. M.; Muñoz Vidal, J.; Musti, M.; Nebot-Guinot, M.; Novella, P.; Palmeiro, B.; Para, A.; Pérez, J.; Querol, M.; Repond, J.; Renner, J.; Riordan, S.; Ripoll, L.; Rodríguez, J.; Rogers, L.; Santos, F. P.; dos Santos, J. M. F.; Simón, A.; Sofka, C.; Sorel, M.; Stiegler, T.; Toledo, J. F.; Torrent, J.; Tsamalaidze, Z.; Veloso, J. F. C. A.; Webb, R.; White, J. T.; Yahlali, N.; NEXT Collaboration

    2018-03-01

    A new method to tag the barium daughter in the double-beta decay of Xe 136 is reported. Using the technique of single molecule fluorescent imaging (SMFI), individual barium dication (Ba++ ) resolution at a transparent scanning surface is demonstrated. A single-step photobleach confirms the single ion interpretation. Individual ions are localized with superresolution (˜2 nm ), and detected with a statistical significance of 12.9 σ over backgrounds. This lays the foundation for a new and potentially background-free neutrinoless double-beta decay technology, based on SMFI coupled to high pressure xenon gas time projection chambers.

  20. Demonstration of Single-Barium-Ion Sensitivity for Neutrinoless Double-Beta Decay Using Single-Molecule Fluorescence Imaging

    Energy Technology Data Exchange (ETDEWEB)

    McDonald, A. D.; Jones, B. J. P.; Nygren, D. R.; Adams, C.; Álvarez, V.; Azevedo, C. D. R.; Benlloch-Rodríguez, J. M.; Borges, F. I. G. M.; Botas, A.; Cárcel, S.; Carrión, J. V.; Cebrián, S.; Conde, C. A. N.; Díaz, J.; Diesburg, M.; Escada, J.; Esteve, R.; Felkai, R.; Fernandes, L. M. P.; Ferrario, P.; Ferreira, A. L.; Freitas, E. D. C.; Goldschmidt, A.; Gómez-Cadenas, J. J.; González-Díaz, D.; Gutiérrez, R. M.; Guenette, R.; Hafidi, K.; Hauptman, J.; Henriques, C. A. O.; Hernandez, A. I.; Hernando Morata, J. A.; Herrero, V.; Johnston, S.; Labarga, L.; Laing, A.; Lebrun, P.; Liubarsky, I.; López-March, N.; Losada, M.; Martín-Albo, J.; Martínez-Lema, G.; Martínez, A.; Monrabal, F.; Monteiro, C. M. B.; Mora, F. J.; Moutinho, L. M.; Muñoz Vidal, J.; Musti, M.; Nebot-Guinot, M.; Novella, P.; Palmeiro, B.; Para, A.; Pérez, J.; Querol, M.; Repond, J.; Renner, J.; Riordan, S.; Ripoll, L.; Rodríguez, J.; Rogers, L.; Santos, F. P.; dos Santos, J. M. F.; Simón, A.; Sofka, C.; Sorel, M.; Stiegler, T.; Toledo, J. F.; Torrent, J.; Tsamalaidze, Z.; Veloso, J. F. C. A.; Webb, R.; White, J. T.; Yahlali, N.

    2018-03-01

    A new method to tag the barium daughter in the double beta decay of $^{136}$Xe is reported. Using the technique of single molecule fluorescent imaging (SMFI), individual barium dication (Ba$^{++}$) resolution at a transparent scanning surface has been demonstrated. A single-step photo-bleach confirms the single ion interpretation. Individual ions are localized with super-resolution ($\\sim$2~nm), and detected with a statistical significance of 12.9~$\\sigma$ over backgrounds. This lays the foundation for a new and potentially background-free neutrinoless double beta decay technology, based on SMFI coupled to high pressure xenon gas time projection chambers.

  1. Shedding light on protein folding, structural and functional dynamics by single molecule studies

    DEFF Research Database (Denmark)

    Bavishi, Krutika; Hatzakis, Nikos

    2014-01-01

    The advent of advanced single molecule measurements unveiled a great wealth of dynamic information revolutionizing our understanding of protein dynamics and behavior in ways unattainable by conventional bulk assays. Equipped with the ability to record distribution of behaviors rather than the mean...... property of a population, single molecule measurements offer observation and quantification of the abundance, lifetime and function of multiple protein states. They also permit the direct observation of the transient and rarely populated intermediates in the energy landscape that are typically averaged out...

  2. Single Molecule Atomic Force Microscopy Studies of Photosensitized Singlet Oxygen Behavior on a DNA Origami Template

    DEFF Research Database (Denmark)

    Helmig, Sarah Wendelboe; Rotaru, Alexandru; Arian, Dumitru

    2010-01-01

    DNA origami, the folding of a long single-stranded DNA sequence (scaffold strand) by hundreds of short synthetic oligonucleotides (staple strands) into parallel aligned helices, is a highly efficient method to form advanced self-assembled DNA-architectures. Since molecules and various materials can...... be conjugated to each of the short staple strands, the origami method offers a unique possibility of arranging molecules and materials in well-defined positions on a structured surface. Here we combine the action of light with AFM and DNA nanostructures to study the production of singlet oxygen from a single...

  3. Polymerase specific error rates and profiles identified by single molecule sequencing.

    Science.gov (United States)

    Hestand, Matthew S; Van Houdt, Jeroen; Cristofoli, Francesca; Vermeesch, Joris R

    2016-01-01

    DNA polymerases have an innate error rate which is polymerase and DNA context specific. Historically the mutational rate and profiles have been measured using a variety of methods, each with their own technical limitations. Here we used the unique properties of single molecule sequencing to evaluate the mutational rate and profiles of six DNA polymerases at the sequence level. In addition to accurately determining mutations in double strands, single molecule sequencing also captures direction specific transversions and transitions through the analysis of heteroduplexes. Not only did the error rates vary, but also the direction specific transitions differed among polymerases. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Comparative single-molecule and ensemble myosin enzymology: sulfoindocyanine ATP and ADP derivatives.

    OpenAIRE

    Oiwa, K; Eccleston, J F; Anson, M; Kikumoto, M; Davis, C T; Reid, G P; Ferenczi, M A; Corrie, J E; Yamada, A; Nakayama, H; Trentham, D R

    2000-01-01

    Single-molecule and macroscopic reactions of fluorescent nucleotides with myosin have been compared. The single-molecule studies serve as paradigms for enzyme-catalyzed reactions and ligand-receptor interactions analyzed as individual stochastic processes. Fluorescent nucleotides, called Cy3-EDA-ATP and Cy5-EDA-ATP, were derived by coupling the dyes Cy3.29.OH and Cy5.29.OH (compounds XI and XIV, respectively, in, Bioconjug. Chem. 4:105-111)) with 2'(3')-O-[N-(2-aminoethyl)carbamoyl]ATP (EDA-A...

  5. Manipulation and Motion of Organelles and Single Molecules in Living Cells

    DEFF Research Database (Denmark)

    Norregaard, Kamilla; Metzler, Ralf; Ritter, Christine M.

    2017-01-01

    driving many cellular processes. The forces on a molecular scale are exactly in the range that can be manipulated and probed with single molecule force spectroscopy. The natural environment of a biomolecule is inside a living cell, hence, this is the most relevant environment for probing their function....... In vivo studies are, however, challenged by the complexity of the cell. In this review, we start with presenting relevant theoretical tools for analyzing single molecule data obtained in intracellular environments followed by a description of state-of-the art visualization techniques. The most commonly...

  6. Preparation and single molecule structure of electroactive polysilane end-grafted on a crystalline silicon surface

    Science.gov (United States)

    Furukawa, Kazuaki; Ebata, Keisuke

    2000-12-01

    Electrically active polysilanes of poly(methylphenylsilane) (PMPS) and poly[bis(p-n-butylphenyl)silane] (PBPS), which are, respectively, known as a good hole transporting material and a near-ultraviolet electroluminescent material, are end-grafted directly on a crystalline silicon surface. The single polysilane molecules are clearly distinguished one from the other on the surface by means of atomic force microscopy observations. End-grafted single molecules of PMPS are observed as dots while end-grafted PBPS appear as worms extending for more than 100 nm on the crystalline silicon surface.

  7. Controlled switching of single-molecule junctions by mechanical motion of a phenyl ring

    Directory of Open Access Journals (Sweden)

    Yuya Kitaguchi

    2015-10-01

    Full Text Available Mechanical methods for single-molecule control have potential for wide application in nanodevices and machines. Here we demonstrate the operation of a single-molecule switch made functional by the motion of a phenyl ring, analogous to the lever in a conventional toggle switch. The switch can be actuated by dual triggers, either by a voltage pulse or by displacement of the electrode, and electronic manipulation of the ring by chemical substitution enables rational control of the on-state conductance. Owing to its simple mechanics, structural robustness, and chemical accessibility, we propose that phenyl rings are promising components in mechanical molecular devices.

  8. Photon-by-Photon Hidden Markov Model Analysis for Microsecond Single-Molecule FRET Kinetics.

    Science.gov (United States)

    Pirchi, Menahem; Tsukanov, Roman; Khamis, Rashid; Tomov, Toma E; Berger, Yaron; Khara, Dinesh C; Volkov, Hadas; Haran, Gilad; Nir, Eyal

    2016-12-29

    The function of biological macromolecules involves large-scale conformational dynamics spanning multiple time scales, from microseconds to seconds. Such conformational motions, which may involve whole domains or subunits of a protein, play a key role in allosteric regulation. There is an urgent need for experimental methods to probe the fastest of these motions. Single-molecule fluorescence experiments can in principle be used for observing such dynamics, but there is a lack of analysis methods that can extract the maximum amount of information from the data, down to the microsecond time scale. To address this issue, we introduce H 2 MM, a maximum likelihood estimation algorithm for photon-by-photon analysis of single-molecule fluorescence resonance energy transfer (FRET) experiments. H 2 MM is based on analytical estimators for model parameters, derived using the Baum-Welch algorithm. An efficient and effective method for the calculation of these estimators is introduced. H 2 MM is shown to accurately retrieve the reaction times from ∼1 s to ∼10 μs and even faster when applied to simulations of freely diffusing molecules. We further apply this algorithm to single-molecule FRET data collected from Holliday junction molecules and show that at low magnesium concentrations their kinetics are as fast as ∼10 4 s -1 . The new algorithm is particularly suitable for experiments on freely diffusing individual molecules and is readily incorporated into existing analysis packages. It paves the way for the broad application of single-molecule fluorescence to study ultrafast functional dynamics of biomolecules.

  9. Towards 3D structure prediction of large RNA molecules: an integer programming framework to insert local 3D motifs in RNA secondary structure.

    Science.gov (United States)

    Reinharz, Vladimir; Major, François; Waldispühl, Jérôme

    2012-06-15

    The prediction of RNA 3D structures from its sequence only is a milestone to RNA function analysis and prediction. In recent years, many methods addressed this challenge, ranging from cycle decomposition and fragment assembly to molecular dynamics simulations. However, their predictions remain fragile and limited to small RNAs. To expand the range and accuracy of these techniques, we need to develop algorithms that will enable to use all the structural information available. In particular, the energetic contribution of secondary structure interactions is now well documented, but the quantification of non-canonical interactions-those shaping the tertiary structure-is poorly understood. Nonetheless, even if a complete RNA tertiary structure energy model is currently unavailable, we now have catalogues of local 3D structural motifs including non-canonical base pairings. A practical objective is thus to develop techniques enabling us to use this knowledge for robust RNA tertiary structure predictors. In this work, we introduce RNA-MoIP, a program that benefits from the progresses made over the last 30 years in the field of RNA secondary structure prediction and expands these methods to incorporate the novel local motif information available in databases. Using an integer programming framework, our method refines predicted secondary structures (i.e. removes incorrect canonical base pairs) to accommodate the insertion of RNA 3D motifs (i.e. hairpins, internal loops and k-way junctions). Then, we use predictions as templates to generate complete 3D structures with the MC-Sym program. We benchmarked RNA-MoIP on a set of 9 RNAs with sizes varying from 53 to 128 nucleotides. We show that our approach (i) improves the accuracy of canonical base pair predictions; (ii) identifies the best secondary structures in a pool of suboptimal structures; and (iii) predicts accurate 3D structures of large RNA molecules. RNA-MoIP is publicly available at: http://csb.cs.mcgill.ca/RNAMoIP.

  10. DNA Origami Directed Au Nanostar Dimers for Single-Molecule Surface-Enhanced Raman Scattering.

    Science.gov (United States)

    Tanwar, Swati; Haldar, Krishna Kanta; Sen, Tapasi

    2017-12-06

    We demonstrate the synthesis of Au nanostar dimers with tunable interparticle gap and controlled stoichiometry assembled on DNA origami. Au nanostars with uniform and sharp tips were immobilized on rectangular DNA origami dimerized structures to create nanoantennas containing monomeric and dimeric Au nanostars. Single Texas red (TR) dye was specifically attached in the junction of the dimerized origami to act as a Raman reporter molecule. The SERS enhancement factors of single TR dye molecules located in the conjunction region in dimer structures having interparticle gaps of 7 and 13 nm are 2 × 10 10 and 8 × 10 9 , respectively, which are strong enough for single analyte detection. The highly enhanced electromagnetic field generated by the plasmon coupling between sharp tips and cores of two Au nanostars in the wide conjunction region allows the accommodation and specific detection of large biomolecules. Such DNA-directed assembled nanoantennas with controlled interparticle separation distance and stoichiometry, and well-defined geometry, can be used as excellent substrates in single-molecule SERS spectroscopy and will have potential applications as a reproducible platform in single-molecule sensing.

  11. Electrostatic melting in a single-molecule field-effect transistor with applications in genomic identification

    Science.gov (United States)

    Vernick, Sefi; Trocchia, Scott M.; Warren, Steven B.; Young, Erik F.; Bouilly, Delphine; Gonzalez, Ruben L.; Nuckolls, Colin; Shepard, Kenneth L.

    2017-05-01

    The study of biomolecular interactions at the single-molecule level holds great potential for both basic science and biotechnology applications. Single-molecule studies often rely on fluorescence-based reporting, with signal levels limited by photon emission from single optical reporters. The point-functionalized carbon nanotube transistor, known as the single-molecule field-effect transistor, is a bioelectronics alternative based on intrinsic molecular charge that offers significantly higher signal levels for detection. Such devices are effective for characterizing DNA hybridization kinetics and thermodynamics and enabling emerging applications in genomic identification. In this work, we show that hybridization kinetics can be directly controlled by electrostatic bias applied between the device and the surrounding electrolyte. We perform the first single-molecule experiments demonstrating the use of electrostatics to control molecular binding. Using bias as a proxy for temperature, we demonstrate the feasibility of detecting various concentrations of 20-nt target sequences from the Ebolavirus nucleoprotein gene in a constant-temperature environment.

  12. 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 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...... and for genome searches. A simple and powerful probability model for ranking RNA matches is proposed. We demonstrate viability of the entire setup by identifying the DNA template of a series of RNAs of biological and of in vitro transcriptional origin in complete microbial genomes and by identifying authentic 16...

  13. Switching behavior of double-decker single molecule magnets on a metal surface

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Yingshuang; Schwoebel, Joerg; Hoffmann, Germar; Brede, Jens; Wiesendanger, Roland [University of Hamburg, Hamburg (Germany); Dillulo, Andrew [Ohio University, Athens (United States); Klyatskaya, Svetlana [Karlsruhe Institute of Technology, Karlsruhe (Germany); Ruben, Mario [Karlsruhe Institute of Technology, Karlsruhe (Germany); Universite de Strasbourg, Strasbourg (France)

    2011-07-01

    Single molecule magnets (SMM) are most promising materials for spin based molecular electronics. Due to their large magnetic anisotropy stabilized by inside chemical bonds, SMM can potentially be used for information storage at the single molecule level. For applications, it is of importance to adsorb the SMM onto surfaces and to study their subsequent conformational, electronic and magnetic properties. We have investigated the adsorption behavior of Tb and Dy based double-decker SMM on an Ir(111) surface with low temperature scanning tunneling microscopy and spectroscopy. It is found that Tb double-decker molecules bind tightly to the Ir(111) surface. By resonantly injecting tunneling electrons into its LUMO or HOMO state, the Tb double-decker molecule can be switched from a four-lobed structure to an eight-lobed structure. After switching, energy positions of the HOMO and LUMO states both shift closer to the Fermi level. Dy double-decker molecules also exhibit the same switching properties on the Ir(111) surface. The switching behavior of the molecules is tentatively attributed to a conformational change of the double-decker molecular frame.

  14. A general approach to break the concentration barrier in single-molecule imaging

    KAUST Repository

    Loveland, Anna B.

    2012-09-09

    Single-molecule fluorescence imaging is often incompatible with physiological protein concentrations, as fluorescence background overwhelms an individual molecule\\'s signal. We solve this problem with a new imaging approach called PhADE (PhotoActivation, Diffusion and Excitation). A protein of interest is fused to a photoactivatable protein (mKikGR) and introduced to its surface-immobilized substrate. After photoactivation of mKikGR near the surface, rapid diffusion of the unbound mKikGR fusion out of the detection volume eliminates background fluorescence, whereupon the bound molecules are imaged. We labeled the eukaryotic DNA replication protein flap endonuclease 1 with mKikGR and added it to replication-competent Xenopus laevis egg extracts. PhADE imaging of high concentrations of the fusion construct revealed its dynamics and micrometer-scale movements on individual, replicating DNA molecules. Because PhADE imaging is in principle compatible with any photoactivatable fluorophore, it should have broad applicability in revealing single-molecule dynamics and stoichiometry of macromolecular protein complexes at previously inaccessible fluorophore concentrations. © 2012 Nature America, Inc. All rights reserved.

  15. Radio frequency scanning tunneling spectroscopy for single-molecule spin resonance.

    Science.gov (United States)

    Müllegger, Stefan; Tebi, Stefano; Das, Amal K; Schöfberger, Wolfgang; Faschinger, Felix; Koch, Reinhold

    2014-09-26

    We probe nuclear and electron spins in a single molecule even beyond the electromagnetic dipole selection rules, at readily accessible magnetic fields (few mT) and temperatures (5 K) by resonant radio-frequency current from a scanning tunneling microscope. We achieve subnanometer spatial resolution combined with single-spin sensitivity, representing a 10 orders of magnitude improvement compared to existing magnetic resonance techniques. We demonstrate the successful resonant spectroscopy of the complete manifold of nuclear and electronic magnetic transitions of up to ΔI(z)=±3 and ΔJ(z)=±12 of single quantum spins in a single molecule. Our method of resonant radio-frequency scanning tunneling spectroscopy offers, atom-by-atom, unprecedented analytical power and spin control with an impact on diverse fields of nanoscience and nanotechnology.

  16. Nanomechanical DNA origami 'single-molecule beacons' directly imaged by atomic force microscopy

    Science.gov (United States)

    Kuzuya, Akinori; Sakai, Yusuke; Yamazaki, Takahiro; Xu, Yan; Komiyama, Makoto

    2011-01-01

    DNA origami involves the folding of long single-stranded DNA into designed structures with the aid of short staple strands; such structures may enable the development of useful nanomechanical DNA devices. Here we develop versatile sensing systems for a variety of chemical and biological targets at molecular resolution. We have designed functional nanomechanical DNA origami devices that can be used as 'single-molecule beacons', and function as pinching devices. Using 'DNA origami pliers' and 'DNA origami forceps', which consist of two levers ~170 nm long connected at a fulcrum, various single-molecule inorganic and organic targets ranging from metal ions to proteins can be visually detected using atomic force microscopy by a shape transition of the origami devices. Any detection mechanism suitable for the target of interest, pinching, zipping or unzipping, can be chosen and used orthogonally with differently shaped origami devices in the same mixture using a single platform. PMID:21863016

  17. Kinetics of CrPV and HCV IRES-mediated eukaryotic translation using single-molecule fluorescence microscopy.

    Science.gov (United States)

    Bugaud, Olivier; Barbier, Nathalie; Chommy, Hélène; Fiszman, Nicolas; Le Gall, Antoine; Dulin, David; Saguy, Matthieu; Westbrook, Nathalie; Perronet, Karen; Namy, Olivier

    2017-11-01

    Protein synthesis is a complex multistep process involving many factors that need to interact in a coordinated manner to properly translate the messenger RNA. As translating ribosomes cannot be synchronized over many elongation cycles, single-molecule studies have been introduced to bring a deeper understanding of prokaryotic translation dynamics. Extending this approach to eukaryotic translation is very appealing, but initiation and specific labeling of the ribosomes are much more complicated. Here, we use a noncanonical translation initiation based on internal ribosome entry sites (IRES), and we monitor the passage of individual, unmodified mammalian ribosomes at specific fluorescent milestones along mRNA. We explore initiation by two types of IRES, the intergenic IRES of cricket paralysis virus (CrPV) and the hepatitis C (HCV) IRES, and show that they both strongly limit the rate of the first elongation steps compared to the following ones, suggesting that those first elongation cycles do not correspond to a canonical elongation. This new system opens the possibility of studying both IRES-mediated initiation and elongation kinetics of eukaryotic translation and will undoubtedly be a valuable tool to investigate the role of translation machinery modifications in human diseases. © 2017 Bugaud et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  18. Fabrication of Low Noise Borosilicate Glass Nanopores for Single Molecule Sensing.

    Directory of Open Access Journals (Sweden)

    Jayesh A Bafna

    Full Text Available We show low-cost fabrication and characterization of borosilicate glass nanopores for single molecule sensing. Nanopores with diameters of ~100 nm were fabricated in borosilicate glass capillaries using laser assisted glass puller. We further achieve controlled reduction and nanometer-size control in pore diameter by sculpting them under constant electron beam exposure. We successfully fabricate pore diameters down to 6 nm. We next show electrical characterization and low-noise behavior of these borosilicate nanopores and compare their taper geometries. We show, for the first time, a comprehensive characterization of glass nanopore conductance across six-orders of magnitude (1M-1μM of salt conditions, highlighting the role of buffer conditions. Finally, we demonstrate single molecule sensing capabilities of these devices with real-time translocation experiments of individual λ-DNA molecules. We observe distinct current blockage signatures of linear as well as folded DNA molecules as they undergo voltage-driven translocation through the glass nanopores. We find increased signal to noise for single molecule detection for higher trans-nanopore driving voltages. We propose these nanopores will expand the realm of applications for nanopore platform.

  19. Detection of gas molecules on single Mn adatom adsorbed graphyne: a DFT-D study

    Science.gov (United States)

    Lu, Zhansheng; Lv, Peng; Ma, Dongwei; Yang, Xinwei; Li, Shuo; Yang, Zongxian

    2018-02-01

    As one of the prominent applications in intelligent systems, gas sensing technology has attracted great interest in both industry and academia. In the current study, the pristine graphyne (GY) without and with a single Mn atom is investigated to detect the gas molecules (CO, CH4, CO2, NH3, NO and O2). The pristine GY is promising to detect O2 molecules because of its chemical adsorption on GY with large electron transfer. The great stability of the Mn/GY is found, and the Mn atom prefers to anchor at the alkyne ring as a single atom. Upon single Mn atom anchoring, the sensitivity and selectivity of GY based gas sensors is significantly improved for various molecules, except CH4. The recovery time of the Mn/GY after detecting the gas molecules may help to appraise the detection efficiency for the Mn/GY. The current study will help to understand the mechanism of detecting the gas molecules, and extend the potentially fascinating applications of GY-based materials.

  20. Structure-spectrophotometric selectivity relationship in interactions of quercetin related flavonoids with double stranded and single stranded RNA

    Science.gov (United States)

    Piantanida, Ivo; Mašić, Lozika; Rusak, Gordana

    2009-04-01

    Interactions of five flavonoids with dsRNA and single stranded ssRNA were studied by UV/vis titrations. The results obtained supported the intercalative binding mode as a dominant interaction of studied flavonoids with dsRNA as well as major interaction with ssRNA. Furthermore, changes of the UV/vis spectra of flavonoids induced by addition of poly G or poly C, respectively, are significantly stronger than changes induced by double stranded poly G-poly C, pointing to essential role of the free poly G or poly C sequence (not hydrogen bonded in double helix). Exclusively poly G caused significant batochromic shift of the UV/vis maxima of all studied flavonoids, whereby the intensity of batochromic shift is nicely correlated to the number of OH groups of flavonoid. Unlikely to poly G, addition of poly A and poly U induced measurable changes only in the UV/vis spectra of flavonoids characterised by no OH (galangin) or three OH groups (myricetin) on the phenyl part of the molecule. Consequently, flavonoids with one- or two-OH groups on the phenyl part of the molecule (luteolin, fisetin, kaempferol) specifically differentiate between poly A, poly U (negligible changes in the UV/Vis spectra) and poly G (strong changes in the UV/Vis spectra) as well as poly C (moderate changes in the UV/Vis spectra).