Browse
Submit Data
Databases
API
Help

Dataset Information

18 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Elementary processes of DNA surface hybridization resolved by single-molecule kinetics: implication for macroscopic device performance.

ABSTRACT: Direct monitoring of single-molecule reactions has recently become a promising means of mechanistic investigation. However, the resolution of reaction pathways from single-molecule experiments remains elusive, primarily because of interference from extraneous processes such as bulk diffusion. Herein, we report a single-molecule kinetic investigation of DNA hybridization on a metal surface, as an example of a bimolecular association reaction. The tip of the scanning tunneling microscope (STM) was functionalized with single-stranded DNA (ssDNA), and hybridization with its complementary strand on an Au(111) surface was detected by the increase in the electrical conductance associated with the electron transport through the resulting DNA duplex. Kinetic analyses of the conductance changes successfully resolved the elementary processes, which involve not only the ssDNA strands and their duplex but also partially hybridized intermediate strands, and we found an increase in the hybridization efficiency with increasing the concentration of DNA in contrast to the knowledge obtained previously by conventional ensemble measurements. The rate constants derived from our single-molecule studies provide a rational explanation of these findings, such as the suppression of DNA melting on surfaces with higher DNA coverage. The present methodology, which relies on intermolecular conductance measurements, can be extended to a range of single-molecule reactions and to the exploration of novel chemical syntheses.

SUBMITTER: Harashima T

PROVIDER: S-EPMC8179252 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Measuring DNA Hybridization Kinetics in Live Cells Using a Time-Resolved 3D Single-Molecule Tracking Method.

Project description:Single-molecule detection enables direct characterization of annealing/melting kinetics of nucleic acids without the need for synchronization of molecular states, but the current experiments are not carried out in a native cellular context. Here we describe an integrated 3D single-molecule tracking and lifetime measurement method that can follow individual DNA molecules diffusing inside a mammalian cell and observe multiple annealing and melting events on the same molecules. By comparing the hybridization kinetics of the same DNA strand in vitro, we found the association constants can be 13- to 163-fold higher in the molecular crowding cellular environment.

| S-EPMC7179573 | biostudies-literature

Non-Langmuir Kinetics of DNA Surface Hybridization.

Project description:Hybridization of complementary single strands of DNA represents a very effective natural molecular recognition process widely exploited for diagnostic, biotechnology, and nanotechnology applications. A common approach relies on the immobilization on a surface of single-stranded DNA probes that bind complementary targets in solution. However, despite the deep knowledge on DNA interactions in bulk solution, the modeling of the same interactions on a surface are still challenging and perceived as strongly system dependent. Here, we show that a two-dimensional analysis of the kinetics of hybridization, performed at different target concentrations and probe surface densities by a label-free optical biosensor, reveals peculiar features inconsistent with an ideal Langmuir-like behavior. We propose a simple non-Langmuir kinetic model accounting for an enhanced electrostatic repulsion originating from the surface immobilization of nucleic acids and for steric hindrance close to full hybridization of the surface probes. The analysis of the kinetic data by the model enables quantifying the repulsive potential at the surface, as well as retrieving the kinetic parameters of isolated probes. We show that the strength and the kinetics of hybridization at large probe density can be improved by a three-dimensional immobilization strategy of probe strands with a double-stranded linker.

| S-EPMC7474173 | biostudies-literature

3D single-molecule tracking enables direct hybridization kinetics measurement in solution.

Project description:Single-molecule measurements of DNA hybridization kinetics are mostly performed on a surface or inside a trap. Here we demonstrate a time-resolved, 3D single-molecule tracking (3D-SMT) method that allows us to follow a freely diffusing ssDNA molecule in solution for hundreds of milliseconds or even seconds and observe multiple annealing and melting events taking place on the same molecule. This is achieved by combining confocal-feedback 3D-SMT with time-domain fluorescence lifetime measurement, where fluorescence lifetime serves as the indicator of hybridization. With sub-diffraction-limit spatial resolution in molecular tracking and 15 ms temporal resolution in monitoring the change of reporter's lifetime, we have demonstrated a full characterization of annealing rate (kon = 5.13 × 106 M-1 s-1), melting rate (koff = 9.55 s-1), and association constant (Ka = 0.54 μM-1) of an 8 bp duplex model system diffusing at 4.8 μm2 s-1. As our method completely eliminates the photobleaching artifacts and diffusion interference, our kon and koff results well represent the real kinetics in solution. Our binding kinetics measurement can be carried out in a low signal-to-noise ratio condition (SNR ≈ 1.4) where ∼130 recorded photons are sufficient for a lifetime estimation. Using a population-level analysis, we can characterize hybridization kinetics over a wide range (0.5-125 s-1), even beyond the reciprocals of the lifetime monitoring temporal resolution and the average track duration.

| S-EPMC5515391 | biostudies-literature

Label-free single-molecule detection of DNA-hybridization kinetics with a carbon nanotube field-effect transistor.

Project description:Single-molecule measurements of biomolecules can provide information about the molecular interactions and kinetics that are hidden in ensemble measurements. However, there is a requirement for techniques with improved sensitivity and time resolution for use in exploring biomolecular systems with fast dynamics. Here, we report the detection of DNA hybridization at the single-molecule level using a carbon nanotube field-effect transistor. By covalently attaching a single-stranded probe DNA sequence to a point defect in a carbon nanotube, we are able to measure two-level fluctuations in the conductance of the nanotube in the presence of a complementary DNA target. The kinetics of the system are studied as a function of temperature, allowing the measurement of rate constants, melting curves and activation energies for different sequences and target concentrations. The kinetics demonstrate non-Arrhenius behaviour, in agreement with DNA hybridization experiments using fluorescence correlation spectroscopy. This technique is label-free and could be used to probe single-molecule dynamics at microsecond timescales.

| S-EPMC3783941 | biostudies-literature

Elementary processes governing the evolution of road networks.

Project description:Urbanisation is a fundamental phenomenon whose quantitative characterisation is still inadequate. We report here the empirical analysis of a unique data set regarding almost 200 years of evolution of the road network in a large area located north of Milan (Italy). We find that urbanisation is characterised by the homogenisation of cell shapes, and by the stability throughout time of high-centrality roads which constitute the backbone of the urban structure, confirming the importance of historical paths. We show quantitatively that the growth of the network is governed by two elementary processes: (i) 'densification', corresponding to an increase in the local density of roads around existing urban centres and (ii) 'exploration', whereby new roads trigger the spatial evolution of the urbanisation front. The empirical identification of such simple elementary mechanisms suggests the existence of general, simple properties of urbanisation and opens new directions for its modelling and quantitative description.

| S-EPMC3291042 | biostudies-literature

Secondary structure effects on DNA hybridization kinetics: a solution versus surface comparison.

Project description:The hybridization kinetics for a series of designed 25mer probe-target pairs having varying degrees of secondary structure have been measured by UV absorbance and surface plasmon resonance (SPR) spectroscopy in solution and on the surface, respectively. Kinetic rate constants derived from the resultant data decrease with increasing probe and target secondary structure similarly in both solution and surface environments. Specifically, addition of three intramolecular base pairs in the probe and target structure slow hybridization by a factor of two. For individual strands containing four or more intramolecular base pairs, hybridization cannot be described by a traditional two-state model in solution-phase nor on the surface. Surface hybridization rates are also 20- to 40-fold slower than solution-phase rates for identical sequences and conditions. These quantitative findings may have implications for the design of better biosensors, particularly those using probes with deliberate secondary structure.

| S-EPMC1488884 | biostudies-other

Optimal estimation of ion-channel kinetics from macroscopic currents.

Project description:Markov modeling provides an effective approach for modeling ion channel kinetics. There are several search algorithms for global fitting of macroscopic or single-channel currents across different experimental conditions. Here we present a particle swarm optimization(PSO)-based approach which, when used in combination with golden section search (GSS), can fit macroscopic voltage responses with a high degree of accuracy (errors within 1%) and reasonable amount of calculation time (less than 10 hours for 20 free parameters) on a desktop computer. We also describe a method for initial value estimation of the model parameters, which appears to favor identification of global optimum and can further reduce the computational cost. The PSO-GSS algorithm is applicable for kinetic models of arbitrary topology and size and compatible with common stimulation protocols, which provides a convenient approach for establishing kinetic models at the macroscopic level.

| S-EPMC3335051 | biostudies-literature

Macroscopic volume change of dynamic hydrogels induced by reversible DNA hybridization.

Project description:Molecular recognition is fundamental to the specific interactions between molecules, of which the best known examples are antibody-antigen binding and cDNA hybridization. Reversible manipulation of the molecular recognition events is still a very challenging topic, and such studies are often performed at the molecular level. An important consideration is the collection of changes at the molecular level to provide macroscopic observables. This research makes use of photoresponsive molecular recognition for the fabrication of novel photoregulated dynamic materials. Specifically, a dynamic hydrogel was prepared by grafting azobenzene-tethered ssDNA and its cDNA to the hydrogel network. The macroscopic volume of the hydrogel can be manipulated through the photoreversible DNA hybridization controlled by alternate irradiation of UV and visible light. The effects of synthetic parameters including the concentration of DNA, polymer monomer, and permanent cross-linker are also discussed.

| S-EPMC3407576 | biostudies-literature

Uncovering the role of elementary processes in network evolution.

Project description:The growth and evolution of networks has elicited considerable interest from the scientific community and a number of mechanistic models have been proposed to explain their observed degree distributions. Various microscopic processes have been incorporated in these models, among them, node and edge addition, vertex fitness and the deletion of nodes and edges. The existing models, however, focus on specific combinations of these processes and parameterize them in a way that makes it difficult to elucidate the role of the individual elementary mechanisms. We therefore formulated and solved a model that incorporates the minimal processes governing network evolution. Some contribute to growth such as the formation of connections between existing pair of vertices, while others capture deletion; the removal of a node with its corresponding edges, or the removal of an edge between a pair of vertices. We distinguish between these elementary mechanisms, identifying their specific role on network evolution.

| S-EPMC3794373 | biostudies-literature

Hybridization kinetics is different inside cells.

Project description:It is generally expected that the kinetics of reactions inside living cells differs from the situation in bulk solutions. Macromolecular crowding and specific binding interactions could change the diffusion properties and the availability of free molecules. Their impact on reaction kinetics in the relevant context of living cells is still elusive, mainly because the difficulty of capturing fast kinetics in vivo. This article shows spatially resolved measurements of DNA hybridization kinetics in single living cells. HeLa cells were transfected with a FRET-labeled dsDNA probe by lipofection. We characterized the hybridization reaction kinetics with a kinetic range of 10 micros to 1 s by a combination of laser-driven temperature oscillations and stroboscopic fluorescence imaging. The time constant of the hybridization depended on DNA concentration within individual cells and between cells. A quantitative analysis of the concentration dependence revealed several-fold accelerated kinetics as compared with free solution for a 16-bp probe and decelerated kinetics for a 12-bp probe. We did not find significant effects of crowding agents on the hybridization kinetics in vitro. Our results suggest that the reaction rates in vivo are specifically modulated by binding interactions for the two probes, possibly triggered by their different lengths. In general, the presented imaging modality of temperature oscillation optical lock-in microscopy allows to probe biomolecular interactions in different cell compartments in living cells for systems biology.

| S-EPMC2793311 | 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