Browse
Submit Data
Databases
API
Help

Dataset Information

23 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Single-Molecule Monitoring of the Structural Switching Dynamics of Nucleic Acids through Controlling Fluorescence Blinking.

ABSTRACT: Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful tool to investigate the dynamics of biomolecular events in real time. However, it requires two fluorophores and can be applied only to dynamics that accompany large changes in distance between the molecules. Herein, we introduce a method for kinetic analysis based on control of fluorescence blinking (KACB), a general approach to investigate the dynamics of biomolecules by using a single fluorophore. By controlling the kinetics of the redox reaction the blinking kinetics or pattern can be controlled to be affected by microenvironmental changes around a fluorophore (rKACB), thereby enabling real-time single-molecule measurement of the structure-changing dynamics of nucleic acids.

SUBMITTER: Kawai K

PROVIDER: S-EPMC5725658 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Single-Molecule Monitoring of the Structural Switching Dynamics of Nucleic Acids through Controlling Fluorescence Blinking.

Kawai Kiyohiko K Miyata Takafumi T Shimada Naohiko N Ito Syoji S Miyasaka Hiroshi H Maruyama Atsushi A

Angewandte Chemie (International ed. in English) 20171024 48

Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful tool to investigate the dynamics of biomolecular events in real time. However, it requires two fluorophores and can be applied only to dynamics that accompany large changes in distance between the molecules. Herein, we introduce a method for kinetic analysis based on control of fluorescence blinking (KACB), a general approach to investigate the dynamics of biomolecules by using a single fluorophore. By controlling the ...[more]

PMID: 28990725

Similar Datasets

Controlling the fluorescence of ordinary oxazine dyes for single-molecule switching and superresolution microscopy.

Project description:Fluorescent molecular switches have widespread potential for use as sensors, material applications in electro-optical data storages and displays, and superresolution fluorescence microscopy. We demonstrate that adjustment of fluorophore properties and environmental conditions allows the use of ordinary fluorescent dyes as efficient single-molecule switches that report sensitively on their local redox condition. Adding or removing reductant or oxidant, switches the fluorescence of oxazine dyes between stable fluorescent and nonfluorescent states. At low oxygen concentrations, the off-state that we ascribe to a radical anion is thermally stable with a lifetime in the minutes range. The molecular switches show a remarkable reliability with intriguing fatigue resistance at the single-molecule level: Depending on the switching rate, between 400 and 3,000 switching cycles are observed before irreversible photodestruction occurs. A detailed picture of the underlying photoinduced and redox reactions is elaborated. In the presence of both reductant and oxidant, continuous switching is manifested by "blinking" with independently controllable on- and off-state lifetimes in both deoxygenated and oxygenated environments. This "continuous switching mode" is advantageously used for imaging actin filament and actin filament bundles in fixed cells with subdiffraction-limited resolution.

| S-EPMC2688868 | biostudies-other

Design and In Vitro Evaluation of Splice-Switching Oligonucleotides Bearing Locked Nucleic Acids, Amido-Bridged Nucleic Acids, and Guanidine-Bridged Nucleic Acids.

Project description:Our group previously developed a series of bridged nucleic acids (BNAs), including locked nucleic acids (LNAs), amido-bridged nucleic acids (AmNAs), and guanidine-bridged nucleic acids (GuNAs), to impart specific characteristics to oligonucleotides such as high-affinity binding and enhanced enzymatic resistance. In this study, we designed a series of LNA-, AmNA-, and GuNA-modified splice-switching oligonucleotides (SSOs) with different lengths and content modifications. We measured the melting temperature (Tm) of each designed SSO to investigate its binding affinity for RNA strands. We also investigated whether the single-stranded SSOs formed secondary structures using UV melting analysis without complementary RNA. As a result, the AmNA-modified SSOs showed almost the same Tm values as the LNA-modified SSOs, with decreased secondary structure formation in the former. In contrast, the GuNA-modified SSOs showed slightly lower Tm values than the LNA-modified SSOs, with no inhibition of secondary structures. We also evaluated the exon skipping activities of the BNAs in vitro at both the mRNA and protein expression levels. We found that both AmNA-modified SSOs and GuNA-modified SSOs showed higher exon skipping activities than LNA-modified SSOs but each class must be appropriately designed in terms of length and modification content.

| S-EPMC8037388 | biostudies-literature

Single molecule mass photometry of nucleic acids.

Project description:Mass photometry is a recently developed methodology capable of measuring the mass of individual proteins under solution conditions. Here, we show that this approach is equally applicable to nucleic acids, enabling their facile, rapid and accurate detection and quantification using sub-picomoles of sample. The ability to count individual molecules directly measures relative concentrations in complex mixtures without need for separation. Using a dsDNA ladder, we find a linear relationship between the number of bases per molecule and the associated imaging contrast for up to 1200 bp, enabling us to quantify dsDNA length with up to 2 bp accuracy. These results introduce mass photometry as an accurate, rapid and label-free single molecule method complementary to existing DNA characterization techniques.

| S-EPMC7515692 | biostudies-literature

Measuring single-molecule nucleic acid dynamics in solution by two-color filtered ratiometric fluorescence correlation spectroscopy.

Project description:This work presents a general method for determining single-molecule intramolecular dynamics in biomolecules by using a reporter fluorophore, whose fluorescence is quenched or partially quenched as a result of intramolecular motion, and a remote observer fluorophore. These fluorophores were excited independently with two different lasers, and the ratio of the two fluorophores' fluorescence was calculated. The time-varying ratio was then filtered to reduce contributions from molecules outside the overlapped laser volume and then correlated. The rates of opening and closing of a DNA hairpin were measured by using both fluorescence correlation spectroscopy and this method for comparison. We found at 50 pM, where molecules were studied one by one as they diffused through the probe volume, we obtained accurate opening and closing rates and could also measure dynamic heterogeneity. To demonstrate applicability to a more complex biological molecule we then probed intramolecular motions in the dimer of a human telomerase RNA fragment (hTR(380-444)), in the presence of an excess of monomer. The motion was found to occur on the time scale of 180-750 micros and slowed with increasing magnesium ion concentration. Blocking experiments using complementary oligonucleotides suggested that the motion involves substantial changes in dimer tertiary structure. This method appears to be a general method for selectively studying intramolecular motion in large biomolecules or complexes.

| S-EPMC521954 | biostudies-literature

Predicting secondary structural folding kinetics for nucleic acids.

Project description:We report a new computational approach to the prediction of RNA secondary structure folding kinetics. In this approach, each elementary kinetic step is represented as the transformation between two secondary structures that differ by a helix. Based on the free energy landscape analysis, we identify three types of dominant pathways and the rate constants for the kinetic steps: 1), formation; 2), disruption of a helix stem; and 3), helix formation with concomitant partial melting of a competing (incompatible) helix. The third pathway, termed the tunneling pathway, is the low-barrier dominant pathway for the conversion between two incompatible helices. Comparisons with experimental data indicate that this new method is quite reliable in predicting the kinetics for RNA secondary structural folding and structural rearrangements. The approach presented here may provide a robust first step for further systematic development of a predictive theory for the folding kinetics for large RNAs.

| S-EPMC2856163 | biostudies-literature

Dioxaphosphorinane-constrained nucleic Acid dinucleotides as tools for structural tuning of nucleic acids.

Project description:We describe a rational approach devoted to modulate the sugar-phosphate backbone geometry of nucleic acids. Constraints were generated by connecting one oxygen of the phosphate group to a carbon of the sugar moiety. The so-called dioxaphosphorinane rings were introduced at key positions along the sugar-phosphate backbone allowing the control of the six-torsion angles ? to ? defining the polymer structure. The syntheses of all the members of the D-CNA family are described, and we emphasize the effect on secondary structure stabilization of a couple of diastereoisomers of ?,?-D-CNA exhibiting wether B-type canonical values or not.

| S-EPMC3488415 | biostudies-literature

Structured Waters Mediate Small Molecule Binding to G-Quadruplex Nucleic Acids.

Project description:The role of G-quadruplexes in human cancers is increasingly well-defined. Accordingly, G-quadruplexes can be suitable drug targets and many small molecules have been identified to date as G-quadruplex binders, some using computer-based design methods and co-crystal structures. The role of bound water molecules in the crystal structures of G-quadruplex-small molecule complexes has been analyzed in this study, focusing on the water arrangements in several G-quadruplex ligand complexes. One is the complex between the tetrasubstituted naphthalene diimide compound MM41 and a human intramolecular telomeric DNA G-quadruplex, and the others are in substituted acridine bimolecular G-quadruplex complexes. Bridging water molecules form most of the hydrogen-bond contacts between ligands and DNA in the parallel G-quadruplex structures examined here. Clusters of structured water molecules play essential roles in mediating between ligand side chain groups/chromophore core and G-quadruplex. These clusters tend to be conserved between complex and native G-quadruplex structures, suggesting that they more generally serve as platforms for ligand binding, and should be taken into account in docking and in silico studies.

| S-EPMC8781208 | biostudies-literature

Recognition of single-stranded nucleic acids by small-molecule splicing modulators.

Project description:Risdiplam is the first approved small-molecule splicing modulator for the treatment of spinal muscular atrophy (SMA). Previous studies demonstrated that risdiplam analogues have two separate binding sites in exon 7 of the SMN2 pre-mRNA: (i) the 5'-splice site and (ii) an upstream purine (GA)-rich binding site. Importantly, the sequence of this GA-rich binding site significantly enhanced the potency of risdiplam analogues. In this report, we unambiguously determined that a known risdiplam analogue, SMN-C2, binds to single-stranded GA-rich RNA in a sequence-specific manner. The minimum required binding sequence for SMN-C2 was identified as GAAGGAAGG. We performed all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method, which captured spontaneous binding of a risdiplam analogue to the target nucleic acids. We uncovered, for the first time, a ligand-binding pocket formed by two sequential GAAG loop-like structures. The simulation findings were highly consistent with experimental data obtained from saturation transfer difference (STD) NMR and structure-affinity-relationship studies of the risdiplam analogues. Together, these studies illuminate us to understand the molecular basis of single-stranded purine-rich RNA recognition by small-molecule splicing modulators with an unprecedented binding mode.

| S-EPMC8373063 | biostudies-literature

Fluorescence Blinking as an Output Signal for Biosensing.

Project description:We demonstrate the first biosensing strategy that relies on quantum dot (QD) fluorescence blinking to report the presence of a target molecule. Unlike other biosensors that utilize QDs, our method does not require the analyte to induce any fluorescence intensity or color changes, making it readily applicable to a wide range of target species. Instead, our approach relies on the understanding that blinking, a single particle phenomenon, is obscured when several QDs lie within the detection volume of a confocal microscope. If QDs are engineered to aggregate when they encounter a particular target molecule, the observation of quasi-continuous emission should indicate its presence. As proof of concept, we programmed DNAs to drive rapid isothermal assembly of QDs in the presence of a target strand (oncogene K-ras). The assemblies, confirmed by various gel techniques, contained multiple QDs and were readily distinguished from free QDs by the absence of blinking.

| S-EPMC6054459 | biostudies-literature

Single-molecule fluorescence measurements of ribosomal translocation dynamics.

Project description:We employ single-molecule fluorescence resonance energy transfer (smFRET) to study structural dynamics over the first two elongation cycles of protein synthesis, using ribosomes containing either Cy3-labeled ribosomal protein L11 and A- or P-site Cy5-labeled tRNA or Cy3- and Cy5-labeled tRNAs. Pretranslocation (PRE) complexes demonstrate fluctuations between classical and hybrid forms, with concerted motions of tRNAs away from L11 and from each other when classical complex converts to hybrid complex. EF-G?GTP binding to both hybrid and classical PRE complexes halts these fluctuations prior to catalyzing translocation to form the posttranslocation (POST) complex. EF-G dependent translocation from the classical PRE complex proceeds via transient formation of a short-lived hybrid intermediate. A-site binding of either EF-G to the PRE complex or of aminoacyl-tRNA?EF-Tu ternary complex to the POST complex markedly suppresses ribosome conformational lability.

| S-EPMC3090999 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data