Melting Of Double-stranded DNA Research Articles

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.

Strong and Specific Recognition of CAG/CTG Repeat DNA (5'-dWGCWGCW-3') by a Cyclic Pyrrole-Imidazole Polyamide.

Abnormally expanded CAG/CTG repeat DNA sequences lead to a variety of neurological diseases, such as Huntington's disease. Here, we synthesized a cyclic pyrrole‐imidazole polyamide (cPIP), which can bind to the minor groove of the CAG/CTG DNA sequence. The double‐stranded DNA melting temperature (T m) and surface plasmon resonance assays revealed the high binding affinity of the cPIP. In addition, next‐generation sequencing showed that the cPIP had high specificity for its target DNA sequence.

Fuel-Driven Transient DNA Strand Displacement Circuitry with Self-Resetting Function.

Toehold-mediated DNA strand displacement (DSD) is a powerful strategy to engineer dynamics in DNA-based devices and for molecular computing. However, facile strategies for autonomously self-resettable DSD cascades with programmable lifetimes are still missing. Here, we concatenate an ATP-powered ligation/restriction network with toehold-mediated DSD reactions realizing ATP-driven transient DSD with self-resetting behavior. The ATP-fueled ligation biases strand displacement reactions by increasing the toehold length and increasing the local concentration by covalent fixation, while the concurrent endonuclease restriction eliminates this bias, allowing to reset the system. The lifetimes and adaptive dynamic steady states for the DSD are engineered by the ATP-fueled uphill-driven nonequilibrium ligation/restriction system. By programming the toeholds for downstream DSD reaction networks, we realize ATP-fueled transient DSD cascades. A higher level of fuel-driven automaton is achieved by combining two subsystems for the transient DSD, where the expelled strands are each other's input strands and the restriction-induced double-stranded DNA melting resets the systems.

How global DNA unwinding causes non-uniform stress distribution and melting of DNA.

DNA unwinding is an important process that controls binding of proteins, gene expression and melting of double-stranded DNA. In a series of all-atom MD simulations on two DNA molecules containing a transcription start TATA-box sequence we demonstrate that application of a global restraint on the DNA twisting dramatically changes the coupling between helical parameters and the distribution of deformation energy along the sequence. Whereas only short range nearest-neighbor coupling is observed in the relaxed case, long-range coupling is induced in the globally restrained case. With increased overall unwinding the elastic deformation energy is strongly non-uniformly distributed resulting ultimately in a local melting transition of only the TATA box segment during the simulations. The deformation energy tends to be stored more in cytidine/guanine rich regions associated with a change in conformational substate distribution. Upon TATA box melting the deformation energy is largely absorbed by the melting bubble with the rest of the sequences relaxing back to near B-form. The simulations allow us to characterize the structural changes and the propagation of the elastic energy but also to calculate the associated free energy change upon DNA unwinding up to DNA melting. Finally, we design an Ising model for predicting the local melting transition based on empirical parameters. The direct comparison with the atomistic MD simulations indicates a remarkably good agreement for the predicted necessary torsional stress to induce a melting transition, for the position and length of the melted region and for the calculated associated free energy change between both approaches.

Electrochemical Melting of DNA: Optimization and Application

A biosensor is an analytical device that can detect, quantitate, and/or characterize biologically-relevant molecules, often utilizing biologically-derived recognition elements, such as DNA oligomers or enzymes. Applications in point-of-care diagnostics, drug development, and forensic toxicology require portable, rapid, reliable, and easy-to-use devices. Electrochemistry provides a promising platform for such devices due to its potential for parallel measurements, its sensitivity, and its ease-of-miniaturization. In this work, gold electrodes modified with self-assembled monolayers of DNA are utilized to study the electric field assisted melting of double-stranded DNA (i.e. “e-melting”). We have previously demonstrated the ability to detect single mismatches in short DNA oligomers using this approach (J. Electrochem. Soc. 2019 volume 166, issue 4, B236-B242). Broadly, this project aims to (1) develop a better understanding of the mechanism of e-melting and (2) correlate e-melting behavior (kinetics, dependence on applied potential, ionic strength, etc.) to DNA structure and stability. Such analysis has the potential to provide fast and portable screening of biological samples for specific sequences, presence of mismatches, and other mutations. In addition, this method could provide a new platform for fundamental biophysical studies aimed at understanding the behavior of DNA in high electric fields. We will present several key improvements to the procedure for formation of the DNA monolayers using a potential pulse routine during electrode modification. This procedure has now replaced the “passive” method used in previous work and has decreased the amounts of reagents, decreased the reaction time, and increased reproducibility of the DNA surface coverage. This has made possible a correlation between the electrochemical signal (peak height obtained from voltammetric measurements) and the DNA surface coverage (DNA molecules per cm2). Additionally, improvements to the electrochemical melting routine itself will also be presented.These improvements in the methodology allows more controlled and reproducible studies, decreasing the signal-to-noise, and increasing sensitivity to small changes in DNA stability or structure. Results from the application of this method towards the study of interactions between DNA and small molecules, specifically cisplatin, will also be presented. Cisplatin has a strong affinity for DNA: it binds to the N7 of two neighboring purines with preference to neighboring guanines. This binding causes a kink in the DNA which destabilizes it. This research will contribute to a better understanding of the way in which small molecules, like cisplatin, affect the stability of DNA and how such interactions are affected by high electric fields. Ultimately, these studies may provide the foundation upon which future electrochemical biosensors can be developed.

Molecular mechanism of promoter opening by RNA polymerase III.

SummaryRNA polymerase III (Pol III) assembles together with transcription factor IIIB (TFIIIB) on different promoter types to initiate the transcription of small, structured RNAs. Here, we present structures of Pol III pre-initiation complexes comprising the 17-subunit Pol III and hetero-trimeric transcription factor TFIIIB with subunits TATA-binding protein (TBP), B-related factor 1 (Brf1) and B double prime 1 (Bdp1) bound to a natural promoter in different functional states. Electron cryo-microscopy (cryo-EM) reconstructions varying from 3.7 Å to 5.5 Å resolution include two early intermediates in which the DNA duplex is closed, an open DNA complex and an initially transcribing complex with RNA in the active site. Our structures reveal an extremely tight and multivalent interaction of TFIIIB with promoter DNA and explain how TFIIIB recruits Pol III. TFIIIB and Pol III subunit C37 together activate the intrinsic transcription factor-like activity of the Pol III-specific heterotrimer to initiate melting of double-stranded DNA in a mechanism similar as used in the Pol II system.

Unwinding Induced Melting of Double-Stranded DNA Studied by Free Energy Simulations.

DNA unwinding plays a major role in many biological processes, such as replication, transcription, and repair. It can lead to local melting and strand separation and can serve as a key mechanism to promote access to the separate strands of a double-stranded DNA. While DNA unwinding has been investigated extensively by DNA cyclization and single-molecule studies on a length-scale of kilo base pairs, it is neither fully understood at the base pair level nor at the level of molecular interactions. By employing a torque acting on the termini of DNA oligonucleotides during molecular dynamics free energy simulations, we locally unwind the central part of a DNA beyond an elastic (harmonic) regime. The simulations reproduce experimental results on the twist elasticity in the harmonic regime (characterized by a mostly quadratic free energy change with respect to changes in twist) and a deformation up to 7° was found as a limit of the harmonic response. Beyond this limit the free energy increase per twist change dropped dramatically coupled to local base pair disruptions and significant deformation of the nucleic acid backbone structure. Restriction of the DNA bending flexibility resulted in a stiffer harmonic response and an earlier onset of the anharmonic response. Whereas local melting with a complete disruption of base pairing and flipping of nucleotides was observed in case of an AT rich central segment strong backbone changes and changes in the stacking arrangements were observed in case of a GC rich segment. Unrestrained MD simulations starting from locally melted DNA reformed regular B-DNA after 50-300 ns simulation time. The simulations may have important implications for understanding DNA recognition processes coupled with significant structural alterations.

Manipulation of double-stranded DNA melting by force.

By integrating elasticity-as described by the Gaussian network model-with bond binding energies that distinguish between different base-pair identities and stacking configurations, we study the force induced melting of a double-stranded DNA (dsDNA). Our approach is a generalization of our previous study of thermal dsDNA denaturation [J. Chem. Phys. 145, 144101 (2016)JCPSA60021-960610.1063/1.4964285] to that induced by force at finite temperatures. It allows us to obtain semimicroscopic information about the opening of the chain, such as whether the dsDNA opens from one of the ends or from the interior, forming an internal bubble. We study different types of force manipulation: (i) "end unzipping," with force acting at a single end base pair perpendicular to the helix, (ii) "midunzipping," with force acting at a middle base pair perpendicular to the helix, and (iii) "end shearing," where the force acts at opposite ends along the helix. By monitoring the free-energy landscape and probability distribution of intermediate denaturation states, we show that different dominant intermediate states are stabilized depending on the type of force manipulation used. In particular, the bubble state of the sequence L60B36, which we have previously found to be a stable state during thermal denaturation, is absent for end unzipping and end shearing, whereas very similar bubbles are stabilized by midunzipping, or when the force location is near the middle of the chain. Ours results offer a simple tool for stabilizing bubbles and loops using force manipulations at different temperatures, and may implicate on the mechanism in which DNA enzymes or motors open regions of the chain.

Binding of the Single-Stranded DNA Binding Protein (gp32) of T4 Bacteriophage Induces Position-Specific Local Conformational Changes in DNA Lattices that can be Monitored by Fluorescent Probes

The single-stranded (ss) DNA binding protein (gp32) of the bacteriophage T4 DNA replication system plays a major role in integrating the functions of the sub-assemblies of the replication complex by interacting with different replication proteins. Substitution of fluorescent base analogues at defined positions in nucleic acid constructs can be used to study local conformational changes of the nucleic acid bases at specific sites, and Cy3/Cy5 dye-pairs inserted into the sugar-phosphate backbones can track the backbone motions of the DNA lattice during gp32 binding. By monitoring the fluorescence, circular dichroism, absorbance and FRET signals of these base and backbone probes, equilibrium and dynamic measurements can provide information about the molecular details of replication mechanisms. In the present study we use 6-methyl isoxanthopterin (6-MI), a fluorescent base analogue of guanine, to monitor local conformational changes of DNA lattices on gp32 binding. We find that aromatic amino acid residues of gp32 stacks with the bases of ssDNA lattices, depending on the specific positions of the bases within the binding domain of the gp32 molecule, and that Molecular Dynamics simulations are consistent with the experimental results. These results may shed light on how the hydrophobic pockets found in many protein-nucleic acid interaction complexes may stabilize the intercalation or stacking of aromatic amino acid residues with nucleic acid bases. Using internally labeled cyanine dyes, we have also investigated the double-stranded DNA melting properties of gp32 protein at a replication fork junction, and our results suggest that gp32 can enhance breathing at a fork junction if the ssDNA arm at the fork is of sufficient length to permit formation of cooperatively-bound gp32 clusters at the junction.

Single-Molecule DNA Melting Bubble Formation and Single-Strand Binding Protein Interaction

Exposed single-stranded DNA (ssDNA) in cells is threatened by degradation from nucleases (DNases) and prone to form secondary structures, which is why cells use single-strand binding proteins (SSB) to protect ssDNA. The most common example is exposure of the Okazaki fragments during replication, or double-stranded DNA melting induced by negative superhelicity, i.e. underwinding of double-stranded DNA caused by replication or transcription. Magnetic tweezers are the tool of choice to mimic and study the influence of superhelicity on DNA structure at the single-molecule level owing to the direct ability to apply torsional stress to double-stranded DNA. Here, we study the conditions of ssDNA exposure and formation from double-stranded DNA (dsDNA) samples with negatively supercoiled single DNA molecules with magnetic tweezers.Tracking double-stranded DNA extension with nanometer precision allows a direct observation of plectonemic coils formed during positive or negative supercoiling. Here, we use magnetic tweezers to set defined states of negative superhelicity with externally applied force. We can thus control the onset and behavior of DNA double strand separation (melting) and then vary parameters surrounding the formation of DNA melting bubbles. We find a strong SSB interaction with transient DNA bubbles even at low forces and superhelicities. SSB DNA bubble interaction is strongly force-dependent and additional superhelicity can displace SSB.

Quantification of topological coupling between DNA superhelicity and G-quadruplex formation.

It has been proposed that new transcription modulations can be achieved via topological coupling between duplex DNA and DNA secondary structures, such as G-quadruplexes, in gene promoters through superhelicity effects. Limited by available methodologies, however, such a coupling has not been quantified directly. In this work, using novel magneto-optical tweezers that combine the nanometer resolution of optical tweezers and the easy manipulation of magnetic tweezers, we found that the flexibility of DNA increases with positive superhelicity (σ). More interestingly, we found that the population of G-quadruplex increases linearly from 2.4% at σ = 0.1 to 12% at σ = -0.03. The population then rapidly increases to a plateau of 23% at σ < -0.05. The rapid increase coincides with the melting of double-stranded DNA, suggesting that G-quadruplex formation is correlated with DNA melting. Our results provide evidence for topology-mediated transcription modulation at the molecular level. We anticipate that these high-resolution magneto-optical tweezers will be instrumental in studying the interplay between the topology and activity of biological macromolecules from a mechanochemical perspective.

Dynamic Force Spectroscopy of Photoswitch-Modified DNA

We apply a combination of photoswitch-modified DNA and AFM-based pulling measurements to study the force-induced melting of double-stranded DNA in the unzipping geometry. We measure the differences in peak rupture force for azobenzene-modified DNA, as the incorporated azobenzenes are photoswitched reversibly between the trans and the cis form. Fitting our rupture force versus loading rate data, we obtain off rate (koff) at zero force values in the range of ∼10 s(-1). We show that the change in peak rupture force and koff induced by destabilizing the DNA duplex depends on the position of the destabilizing azobenzene photoswitch relative to the force-loading site. When the azobenzenes are proximal to the unzipping end, the decrease in peak force and koff upon azobenzene photoisomerization is significantly larger than when the azobenzene is distal to the site of force loading. We interpret these results as experimental evidence supporting the picture that the destabilization of a double-stranded DNA by a photoswitch isomerization is localized to a small bubble around the photoswitch.

Real-Time Surface-Enhanced Raman Spectroscopy Monitoring of Surface pH during Electrochemical Melting of Double-Stranded DNA

The application of a negative potential ramp at a double-stranded DNA (dsDNA) functionalized electrode surface results in the gradual denaturation of the DNA in a process known as electrochemical melting. The underlying physical chemistry behind electrochemically driven DNA denaturation is not well understood, and one possible mechanism is a change in local pH at the electrode surface. We demonstrate that by coimmobilization of p-mercaptobenozic acid at a dsDNA-functionalized electrode surface, it is possible to monitor both DNA denaturation and the local pH simultaneously using surface-enhanced Raman spectroscopy. We find that the local pH at the electrode surface does not change as the applied potential is scanned negative and the dsDNA denatures. We therefore conclude that in these experiments electrochemical melting is not caused by electrochemically driven local pH changes.

Two-dimensional random walk and characteristics of helix-coil transition in DNA

On the basis of two-dimensional random walk model, a model of melting of double-stranded DNA is proposed. The expressions for the free energy, the helicity degree and the correlation function of nitrogenous base pair formation, remoted along the chain, are obtained. The temperature dependence of thermodynamic parameters of the model is analyzed. It is shown that the temperature dependence of the helicity degree is qualitatively similar to those obtained in experiments. It is established that the correlation function decays exponentially with increasing distance along the chain between the base pairs.

Temperature-induced melting of double-stranded DNA in the absence and presence of covalently bonded antitumour drugs: insight from molecular dynamics simulations

The difference in melting temperature of a double-stranded (ds) DNA molecule in the absence and presence of bound ligands can provide experimental information about the stabilization brought about by ligand binding. By simulating the dynamic behaviour of a duplex of sequence 5′-d(TAATAACGGATTATT)·5′-d(AATAATCCGTTATTA) in 0.1 M NaCl aqueous solution at 400 K, we have characterized in atomic detail its complete thermal denaturation profile in <200 ns. A striking asymmetry was observed on both sides of the central CGG triplet and the strand separation process was shown to be strongly affected by bonding in the minor groove of the prototypical interstrand crosslinker mitomycin C or the monofunctional tetrahydroisoquinolines trabectedin (Yondelis®), Zalypsis® and PM01183®. Progressive helix unzipping was clearly interspersed with some reannealing events, which were most noticeable in the oligonucleotides containing the monoadducts, which maintained an average of 6 bp in the central region at the end of the simulations. These significant differences attest to the demonstrated ability of these drugs to stabilize dsDNA, stall replication and transcription forks, and recruit DNA repair proteins. This stabilization, quantified here in terms of undisrupted base pairs, supports the view that these monoadducts can functionally mimic a DNA interstrand crosslink.

Smart Drug Delivery through DNA/Magnetic Nanoparticle Gates

Mesoporous silica nanoparticles can be modified to perform on-demand stimuli-responsive dosing of therapeutic molecules. The silica network was loaded with iron oxide superparamagnetic nanocrystals, providing the potential to perform targeting and magnetic resonance imaging. Single-stranded DNA was immobilized onto the material surface. The complementary DNA sequence was then attached to magnetic nanoparticles. The present work demonstrates that DNA/magnetic nanoparticle conjugates are able to cap the pores of the magnetic silica particles upon hybridization of both DNA strands. Progressive double-stranded DNA melting as a result of temperature increase gave rise to uncapping and the subsequent release of a mesopore-filled model drug, fluorescein. The reversibility of DNA linkage results in an "on-off" release mechanism. Moreover, the magnetic component of the whole system allows reaching hyperthermic temperatures (42-47 °C) under an alternating magnetic field. This feature leaves open the possibility of a remotely triggered drug delivery. Furthermore, due to its capacity to increase the temperature of the surrounding media, this multifunctional device could play an important role in the development of advanced drug delivery systems for thermochemotherapy against cancer.

Single-stranded DNA binding activity of XPBI, but not XPBII, from Sulfolobus tokodaii causes double-stranded DNA melting

XPB helicase is the largest subunit of transcription factor IIH (TFIIH), a ten-subunit protein complex essential for transcription initiation and nucleotide excision repair (NER) in Eukarya. Two XPB homologues (XPBI and XPBII) are present in the genome of most crenarchaeota, one of the two major phyla of archaea; however, the biochemical properties have not been fully characterized and their cellular roles have not been clearly defined. Here, we report that XPBI from the hyperthermophilic crenarchaeon Sulfolobus tokodaii (StoXPBI) is able to destabilize double-stranded DNA (dsDNA) helix independent of ATP (designated as dsDNA melting activity). This activity is inhibited by single-stranded DNA (ssDNA) and relies on the unique N-terminal domain of StoXPBI, which is also likely responsible for the intrinsic strong ssDNA binding activity of StoXPBI as revealed by deletion analysis. We demonstrate that the ATPase activity of StoXPBII is remarkably stimulated by StoBax1, a nuclease partner of StoXPBII. The role of the unique dsDNA melting activity of XPBI in NER in archaea was discussed.

Validation of High-Resolution DNA Melting Analysis for Mutation Scanning of the Cystic Fibrosis Transmembrane Conductance Regulator ( CFTR) Gene

High-resolution melting analysis of polymerase chain reaction products for mutation scanning, which began in the early 2000s, is based on monitoring of the fluorescence released during the melting of double-stranded DNA labeled with specifically developed saturation dye, such as LC-Green. We report here the validation of this method to scan 98% of the coding sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. We designed 32 pairs of primers to amplify and analyze the 27 exons of the gene. Thanks to the addition of a small GC-clamp at the 5' ends of the primers, one single melting domain and one identical annealing temperature were obtained to co-amplify all of the fragments. A total of 307 DNA samples, extracted by the salt precipitation method, carrying 221 mutations and 21 polymorphisms, plus 20 control samples free from variations (confirmed by denaturing high-performance liquid chromatography analysis), was used. With the conditions described in this study, 100% of samples that carry heterozygous mutations and 60% of those with homozygous mutations were identified. The study of a cohort of 136 idiopathic chronic pancreatitis patients enabled us to prospectively evaluate this technique. Thus, high-resolution melting analysis is a robust and sensitive single-tube technique for screening mutations in a gene and promises to become the gold standard over denaturing high-performance liquid chromatography, particularly for highly mutated genes such as CFTR, and appears suitable for use in reference diagnostic laboratories.

A microfluidic platform using molecular beacon-based temperature calibration for thermal dehybridization of surface-bound DNA.

This work presents a simple microfluidic device with an integrated thin-film heater for studies of DNA hybridization kinetics and double-stranded DNA melting temperature measurements. The heating characteristics of the device were evaluated with a novel, noninvasive indirect technique using molecular beacons as temperature probes inside reaction chambers. This is the first microfluidic device in which thermal dehybridization of surface-bound oligonucleotides was performed for measurement of double-stranded DNA melting temperatures with +/- 1 degrees C precision. Surface modification and oligonucleotide immobilization were performed by continuously flowing reagents through the microchannels. The resulting reproducibility of oligonucleotide surface densities, at 9% RSD, was better than for the same modification chemistries on glass slides in unstirred reagent solutions (RSD=20%). Moreover, the surface density of immobilized DNA probe molecules could be varied controllably by changing the concentration of the reagent solution used for immobilization. Thus, excellent control of surface characteristics was made possible, something which is often difficult to achieve with larger devices. Solid-phase hybridization reactions, a fundamental aspect of microarray technologies often taking several hours in conventional systems, were reduced to minutes in this device. It was also possible to determine forward rate constants for hybridization, k. These varied from 820,000 to 72,000 M(-1) s(-1), decreasing as surface densities increased. Surface densities could therefore be optimized to obtain rapid hybridization using such an approach. Taken together, this combined microfluidic/small-volume heating approach represents a powerful tool for surface-based DNA analysis.

On-line melting of double-stranded DNA for analysis of single-stranded DNA using capillary electrophoresis

Capillary electrophoresis (CE) is a convenient, fast and non-radioactive method with possibilities for automatization. To analyse single-stranded DNA molecules in a more automated way, we developed a heating device to melt double-stranded DNA fragments in the capillary during electrophoresis. In this study we used this device to obtain single-stranded DNA, necessary for the detection of point mutations in DNA using the single-strand conformation polymorphism technique. Results show that double-stranded DNA molecules can be melted on-line into single-stranded DNA molecules, although not for 100%. In an attempt to find universal electrophoretic conditions for the analysis of single-stranded DNA, we investigated the influence of several parameters on the yield of single-stranded DNA molecules and on the resolution of the single-stranded DNA peaks. We demonstrate that this heating device is a technical adjustment of CE which contributes to more automated analyses of DNA fragments.