DNA Unzipping Research Articles

High Nucleotide Skew Palindromic DNA Sequences Function as Potential Replication Origins due to their Unzipping Propensity.

Locations of DNA replication initiation in prokaryotes, called "origins of replication", are well-characterized. However, a mechanistic understanding of the sequence dependence of the local unzipping of double-stranded DNA, the first step towards replication initiation, is lacking. Here, utilizing a Markov chain model that was created to address the directional nature of DNA unzipping and replication, we model the sequence dependence of local melting of double-stranded linear DNA segments. We show that generalized palindromic sequences with high nucleotide skews have a low kinetic barrier for local melting near melting temperatures. This allows for such sequences to function as potential replication origins. We support our claim with evidence for high-skew palindromic sequences within the replication origins of mitochondrial DNA, bacteria, archaea and plasmids.

Numerical Investigation of Nucleotides’ Interaction Considering Changes Caused by Liquid Influences

This work is devoted to the interaction of nucleotides. The goal of this study is to learn or try to learn how the interaction between nucleotides with exposure to a liquid takes place. Will the interacting forces of the nucleotides be sufficient to approach the incision? A numerical imitation of the interaction is conducted using the discrete element method and a Gears predictor–corrector as part of the integrated scheme. In this work, the results reflect the dynamics of nucleotides: velocity, displacement, and force graphs are presented with and without the effect of the liquid. During changes caused by the influence of a liquid, the nucleotide interaction transforms and passes three stages: a full stop, one similar to viscous damping, and one similar to non-dissipative behaviors. The main contribution of this work is a better understanding of the behavior of infinitely small objects that would be difficult to observe in vivo. The changing influence of a liquid can transform into certain effects. As a result, a model is provided, which can be based on the results of well-known physical experiments (DNA unzipping) for modeling nucleotide interactions.

Engineering of Cas12a nuclease variants with enhanced genome-editing specificity.

The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a system is a powerful tool in gene editing; however, crRNA-DNA mismatches might induce unwanted cleavage events, especially at the distal end of the PAM. To minimize this limitation, we engineered a hyper fidelity AsCas12a variant carrying the mutations S186A/R301A/T315A/Q1014A/K414A (termed HyperFi-As) by modifying amino acid residues interacting with the target DNA and crRNA strand. HyperFi-As retains on-target activities comparable to wild-type AsCas12a (AsCas12aWT) in human cells. We demonstrated that HyperFi-As has dramatically reduced off-target effects in human cells, and HyperFi-As possessed notably a lower tolerance to mismatch at the position of the PAM-distal region compared with the wild type. Further, a modified single-molecule DNA unzipping assay at proper constant force was applied to evaluate the stability and transient stages of the CRISPR/Cas ribonucleoprotein (RNP) complex. Multiple states were sensitively detected during the disassembly of the DNA-Cas12a-crRNA complexes. On off-target DNA substrates, the HyperFi-As-crRNA was harder to maintain the R-loop complex state compared to the AsCas12aWT, which could explain exactly why the HyperFi-As has low off-targeting effects in human cells. Our findings provide a novel version of AsCas12a variant with low off-target effects, especially capable of dealing with the high off-targeting in the distal region from the PAM. An insight into how the AsCas12a variant behaves at off-target sites was also revealed at the single-molecule level and the unzipping assay to evaluate multiple states of CRISPR/Cas RNP complexes might be greatly helpful for a deep understanding of how CRISPR/Cas behaves and how to engineer it in future.

Communication between DNA and nucleotide binding sites facilitates stepping by the RecBCD helicase.

Double-strand DNA breaks are the severest type of genomic damage, requiring rapid response to ensure survival. RecBCD helicase in prokaryotes initiates processive and rapid DNA unzipping, essential for break repair. The energetics of RecBCD during translocation along the DNA track are quantitatively not defined. Specifically, it's essential to understand the mechanism by which RecBCD switches between its binding states to enable its translocation. Here, we determine, by systematic affinity measurements, the degree of coupling between DNA and nucleotide binding to RecBCD. In the presence of ADP, RecBCD binds weakly to DNA that harbors a double overhang mimicking an unwinding intermediate. Consistently, RecBCD binds weakly to ADP in the presence of the same DNA. We did not observe coupling between DNA and nucleotide binding for DNA molecules having only a single overhang, suggesting that RecBCD subunits must both bind DNA to 'sense' the nucleotide state. On the contrary, AMPpNp shows weak coupling as RecBCD remains strongly bound to DNA in its presence. Detailed thermodynamic analysis of the RecBCD reaction mechanism suggests an 'energetic compensation' between RecB and RecD, which may be essential for rapid unwinding. Our findings provide the basis for a plausible stepping mechanism' during the processive translocation of RecBCD.

Force-induced unzipping of DNA in the presence of solvent molecules

The melting of double-stranded DNA (dsDNA) in the presence of solvent molecules is a fundamental process with significant implications for understanding the thermal and mechanical behavior of DNA and its interactions with the surrounding environment. The solvents play an essential role in the structural transformation of DNA subjected to a pulling force. In this study, we simulate the thermal and force induced denaturation of dsDNA and elucidate the solvent dependent melting behavior, identifying key factors that influence the stability of DNA melting in presence of solvent molecules. Using a statistical model, we first find the melting profile of short heterogeneous DNA molecules in the presence of solvent molecules in Force ensemble. We also investigate the effect of solvent's strengths on the melting profile of DNA. In the force ensemble, we consider two homogeneous DNA chains and apply the force on different locations along the chain in the presence of solvent molecules. Different pathways manifest the melting of the molecule in both ensembles, and we found several interesting features of melting DNA in a constant force ensemble, such as lower critical force when the chain is pulled from the base pair close to a solvent molecule. The results provide new insights into the force-induced unzipping of DNA and could be used to develop new methods for controlling the unzipping process. By providing a better understanding of melting and unzipping of dsDNA in the presence of solvent molecules, this study provides valuable guidelines for predicting DNA thermodynamic quantities and for designing DNA nanostructures.

Stochastic resonance in a model of a periodically driven DNA: Multiple transitions, scaling, and sequence dependence.

We numerically study stochastic resonance in the unzipping of a model double-stranded DNA by a periodic force. We observe multiple peaks in stochastic resonance in the output signal as the driving force frequency is varied for different force amplitudes, temperature, chain length, and chain heterogeneity. Multiple peaks point to the existence of multiple stable and metastable states, which correspond to dynamical states of partially zipped and unzipped conformations and transitions between them. We quantify such transitions by looking at the time evolution of the fraction of bound base pairs. We obtain phase diagrams in the force amplitude-temperature plane both in the resonance frequency of the primary peak and the output signal at the peak value. We further obtain an excellent scaling behavior of the output signal for changing lengths of the DNA. Resonance behavior is also affected by chain heterogeneity as it depends strongly on which base pair the periodic forcing is applied.

Experimental Test of Sinai's Model in DNA Unzipping.

The experimental measurement of correlation functions and critical exponents in disordered systems is key to testing renormalization group (RG) predictions. We mechanically unzip single DNA hairpins with optical tweezers, an experimental realization of the diffusive motion of a particle in a one-dimensional random force field, known as the Sinai model. We measure the unzipping forces F_{w} as a function of the trap position w in equilibrium and calculate the force-force correlator Δ_{m}(w), its amplitude, and correlation length, finding agreement with theoretical predictions. We study the universal scaling properties since the effective trap stiffness m^{2} decreases upon unzipping. Fluctuations of the position of the base pair at the unzipping junction u scales as u∼m^{-ζ}, with a roughness exponent ζ=1.34±0.06, in agreement with the analytical prediction ζ=4/3. Our study provides a single-molecule test of the functional RG approach for disordered elastic systems in equilibrium.

Unpicking DNA translocation in nanopores with simultaneous single-molecule fluorescence and optical single channel recording.

Nanopore-based DNA sequencing offers high throughput and long reads by measuring blockades in current as a strand is translocated through the pore, providing a single-molecule readout with high time resolution. However, electrical measurements alone offer only information regarding the current block itself; to improve nanopore sensing, a molecular picture of the steps of analyte binding, capture and translocation is required. Optical single channel recording of calcium flux through the nanopore enables parallel single-molecule capture events to be distinguished. We combine optical single channel recording with a single molecule fluorescence readout of DNA unzipping to correlate the steps of individual translocations and image the location of the system components. By annealing short oligomers bearing fluorophore-quencher pairs to the translocating DNA strand, we map the process of nanopore capture, threading and unzipping.

Nonequilibrium Thermodynamics of DNA Nanopore Unzipping.

Using theory and simulations, we carried out a first systematic characterization of DNA unzipping via nanopore translocation. Starting from partially unzipped states, we found three dynamical regimes depending on the applied force f: (i)heterogeneous DNA retraction and rezipping (f<17 pN), (ii)normal (17 pN<f<60 pN), and (iii)anomalous (f>60 pN) drift-diffusive behavior. We show that the normal drift-diffusion regime can be effectively modeled as a one-dimensional stochastic process in a tilted periodic potential. We use the theory of stochastic processes to recover the potential from nonequilibrium unzipping trajectories and show that it corresponds to the free-energy landscape for single-base-pair unzipping. Applying this general approach to other single-molecule systems with periodic potentials ought to yield detailed free-energy landscapes from out-of-equilibrium trajectories.

5-Methyl-cytosine stabilizes DNA but hinders DNA hybridization revealed by magnetic tweezers and simulations.

5-Methyl-cytosine (5mC) is one of the most important DNA modifications and plays versatile biological roles. It is well known that 5mC stabilizes DNA duplexes. However, it remains unclear how 5mC affects the kinetics of DNA melting and hybridization. Here, we studied the kinetics of unzipping and rezipping using a 502-bp DNA hairpin by single-molecule magnetic tweezers. Under constant loading rates, 5mC increases the unzipping force but counterintuitively decreases the rezipping force at various salt and temperature conditions. Under constant forces, the non-methylated DNA hops between metastable states during unzipping and rezipping, which implies low energy barriers. Surprisingly, the 5mC DNA can't rezip after fully unzipping unless much lower forces are applied, where it rezips stochastically in a one-step manner, which implies 5mC kinetically hinders DNA hybridization and high energy barriers in DNA hybridization. All-atom molecular dynamics simulations reveal that the 5mC kinetically hinders DNA hybridization due to steric effects rather than electrostatic effects caused by the additional methyl groups of cytosines. Considering the possible high speed of DNA unzipping and zipping during replication and transcription, our findings provide new insights into the biological roles of 5mC.

Simultaneous Dual-Site Identification of 5mC/8oG in DNA Triplex Using a Nanopore Sensor.

DNA triplex participates in delivering site-specific epigenetic modifications critical for the regulation of gene expression. Among these marks, 5mC with 8oG functions comprehensively on gene expression. Recently, few research studies have emphasized the necessity of incorporation detection of 5mC with 8oG using one DNA triplex at the same time. Herein, DNA triplex structure was designed and tailored for the site-specific identification of 5mC with 8oG by means of nanopore electroanalysis. The identification was associated with the distinguishable current modulation types caused by DNA unzipping through the nanopore in an electrical field. Results demonstrated that the epigenetic modification proximity to the latch zone or constriction area of the nanopore enables differentiation of modification series at single nucleotide resolution in one DNA triplex, at both physiological and mildly acidic environment. In addition, our nanopore method enables the kinetic and thermodynamic studies to calculate the free energy of modified DNA triplex with applied potentials. Gibbs' energy provided the direct evidence that the DNA triplex with these epigenetic modifications is more stable in acidic environment. Considering modified DNA functions significantly in gene expression, the presented method may provide future opportunities to understand incorporating epigenetic mechanisms of many dysregulated biological processes on the basis of accurate detection.

Role of Hoogsteen interaction in the stability of different phases of triplex DNA.

A simple coarse-grained model of DNA which includes both Watson-Crick and Hoogsteen base pairing has been used to study the melting and unzipping of triplex DNA. Using Langevin dynamics simulations, we reproduce the qualitative features of one-step and two-step thermal melting of triplex as seen in experiments. The thermal melting phase diagram shows the existence of a stable interchain three-strand complex (bubble-bound state). Our studies based on the mechanical unzipping of a triplex revealed that it is mechanically more stable compared to an isolated duplex-DNA.

Hysteresis loop area scaling exponents in DNA unzipping by a periodic force: A Langevin dynamics simulation study.

Using Langevin dynamics simulations, we study the hysteresis in unzipping of longer double-stranded DNA chains whose ends are subjected to a time-dependent periodic force with frequency ω and amplitude G keeping the other end fixed. We find that the area of the hysteresis loop, A_{loop}, scales as 1/ω at higher frequencies, whereas it scales as (G-G_{c})^{α}ω^{β} with exponents α=1 and β=1.25 in the low-frequency regime. These values are same as the exponents obtained in Monte Carlo simulation studies of a directed self-avoiding walk model of a homopolymer DNA [R. Kapri, Phys. Rev. E 90, 062719 (2014)10.1103/PhysRevE.90.062719], and the block copolymer DNA [R.K. Yadav and R. Kapri, Phys. Rev. E 103, 012413 (2021)2470-004510.1103/PhysRevE.103.012413] on a square lattice, and differs from the values reported earlier using Langevin dynamics simulation studies on a much shorter DNA hairpins.

Extended and dynamic linker histone-DNA Interactions control chromatosome compaction

Chromatosomes play a fundamental role in chromatin regulation, but a detailed understanding of their structure is lacking, partially due to their complex dynamics. Using single-molecule DNA unzipping with optical tweezers, we reveal that linker histone interactions with DNA are remarkably extended, with the C-terminal domain binding both DNA linkers as far as approximately±140bp from the dyad. In addition to a symmetrical compaction of the nucleosome core governed by globular domain contacts at the dyad, the C-terminal domain compacts the nucleosome's entry and exit. These interactions are dynamic, exhibit rapid binding and dissociation, are sensitive to phosphorylation of a specific residue, and are crucial to determining the symmetry of the chromatosome's core. Extensive unzipping of the linker DNA, which mimics its invasion by motor proteins, shifts H1 into an asymmetric, off-dyad configuration and triggers nucleosome decompaction, highlighting the plasticity of the chromatosome structure and its potential regulatory role.

Curaxin-Induced DNA Topology Alterations Trigger the Distinct Binding Response of CTCF and FACT at the Single-Molecule Level

The candidate anticancer drug curaxins can insert into DNA base pairs and efficiently inhibit the growth of various cancers. However, how curaxins alter the genomic DNA structure and affect the DNA binding property of key proteins remains to be clarified. Here, we first showed that curaxin CBL0137 strongly stabilizes the interaction between the double strands of DNA and reduces DNA bending and twist rigidity simultaneously, by single-molecule magnetic tweezers. More importantly, we found that CBL0137 greatly impairs the binding of CTCF but facilitates trapping FACT on DNA. We revealed that CBL0137 clamps the DNA double helix that may induce a huge barrier for DNA unzipping during replication and transcription and causes the distinct binding response of CTCF and FACT on DNA. Our work provides a novel mechanical insight into CBL0137's anticancer mechanisms at the nucleic acid level.

Unzipping of a double-stranded block copolymer DNA by a periodic force.

Using Monte Carlo simulations, we study the hysteresis in unzipping of a double-stranded block copolymer DNA with -A_{n}B_{n}- repeat units. Here A and B represent two different types of base pairs having two and three bonds, respectively, and 2n represents the number of such base pairs in a unit. The end of the DNA are subjected to a time-dependent periodic force with frequency (ω) and amplitude (g_{0}) keeping the other end fixed. We find that the equilibrium force-temperature phase diagram for the static force is independent of the DNA sequence. For a periodic force case, the results are found to be dependent on the block copolymer DNA sequence and on the base pair type on which the periodic force is acting. We observe hysteresis loops of various shapes and sizes and obtain the scaling of loop area both at low- and high-frequency regimes.

An Exactly Solvable Stochastic Kinetic Theory of Single-Molecule Force Experiments.

The unfolding of a protein in single-molecule pulling experiments subjected to a constant force (force-clamp) and constant velocity (force-ramp) is analyzed by introducing an exactly solvable two-state kinetic model framed based on the general stochastic approach of discrete state and continuous time formulation. The effect of perturbation is interpreted in the presence of dynamic disorder, resulting from intrinsic conformational fluctuations, by deriving an exact analytical expression for the unfolding time distribution, which in turn allowed us to calculate the expressions for the quantities of experimental interest explicitly. In particular, the novelty of our method lies in the fact that it reduced the need for a lengthy calculation, contrary to the previous dynamic disorder studies, and provides a fairly concise but sufficient mathematical analysis, which becomes much easier to implement quantitatively. We tested our results against the measured data from a number of force unfolding experiments on various proteins, ubiquitin, titin, and filamin, and the force unzipping of DNA and observed excellent agreement in each case. This asserts the reliability of the present technique, which suggests a plausible extension of the stochastic kinetic theory in single-molecule force experiments beyond its present-day widespread implications.

An in vitro DNA Sensor-based Assay to Measure Receptor-specific Adhesion Forces of Eukaryotic Cells and Pathogens.

Motility of eukaryotic cells or pathogens within tissues is mediated by the turnover of specific interactions with other cells or with the extracellular matrix. Biophysical characterization of these ligand-receptor adhesions helps to unravel the molecular mechanisms driving migration. Traction force microscopy or optical tweezers are typically used to measure the cellular forces exerted by cells on a substrate. However, the spatial resolution of traction force microscopy is limited to ~2 µm and performing experiments with optical traps is very time-consuming. Here we present the production of biomimetic surfaces that enable specific cell adhesion via synthetic ligands and at the same time monitor the transmitted forces by using molecular tension sensors. The ligands were coupled to double-stranded DNA probes with defined force thresholds for DNA unzipping. Receptor-mediated forces in the pN range are thereby semi-quantitatively converted into fluorescence signals, which can be detected by standard fluorescence microscopy at the resolution limit (~0.2 µm). The modular design of the assay allows to vary the presented ligands and the mechanical strength of the DNA probes, which provides a number of possibilities to probe the adhesion of different eukaryotic cell types and pathogens and is exemplified here with osteosarcoma cells and Plasmodium berghei Sporozoites.

The Possibility of Blocking the Process of DNA Base Pairs Opening by Hydrogen Peroxide

One of the most progressive methods of cancer treatment is the ion beam therapy. Simulations of the water radiolysis show that the most long-living species in the cell medium are hydrogen peroxide (H2O2) molecules. But up to the present time, the role of H2O2 molecules in the deactivation of cancer cells has not been determined yet. To understand the possible role of H2O2 in the ion beam therapy, the competitive interaction of H2O and H2O2 molecules with nucleic bases in a pair on the different stages of genetic information transfer is studied in the present work. The method of atom-atomic potential functions is used in the calculations. It is shown that some configurations of A·T, and G·C complementary pairs are stabilized muchbetter by an H2O2 molecule as compared to a water molecule. The formation of such interaction complexes can terminate the processes of DNA unzipping by enzymes and consequently block the genetic information transfer processes in cancer cells during the ion beam treatment. An experimental method of verification of the interaction of hydrogen peroxide with nucleic base pairs is proposed.

Sequence-Dependent Unzipping Dynamics of DNA Hairpins in a Nanopore.

By applying an electric field to an insulating membrane, movement of charged particles through a nanopore can be induced. The measured ionic current reports on biomolecules passing through the nanopore. In this paper, we explore the sequence-dependent dynamics of DNA unzipping using our recently developed coarse-grained model. We estimated three molecular profiles (the potential of mean force, position-dependent diffusion coefficient, and position-dependent effective charge) for the DNA unzipping of four hairpins with different sequences. We found that the molecular profiles are correlated with the ionic current and molecular events. We also explored the unzipping kinetics using Brownian dynamics. We found that the effect of hairpin structure on the unzipping/translocation times is not only energetic (weaker hairpins unzip more quickly) but also kinetic (different unzipping and translocation pathways play an important role).