DNA Breathing Research Articles

DNA breathing integration with deep learning foundational model advances genome-wide binding prediction of human transcription factors.

It was previously shown that DNA breathing, thermodynamic stability, as well as transcriptional activity and transcription factor (TF) bindings are functionally correlated. To ascertain the precise relationship between TF binding and DNA breathing, we developed the multi-modal deep learning model EPBDxDNABERT-2, which is based on the Extended Peyrard-Bishop-Dauxois (EPBD) nonlinear DNA dynamics model. To train our EPBDxDNABERT-2, we used chromatin immunoprecipitation sequencing (ChIP-Seq) data comprising 690 ChIP-seq experimental results encompassing 161 distinct TFs and 91 human cell types. EPBDxDNABERT-2 significantly improves the prediction of over 660 TF-DNA, with an increase in the area under the receiver operating characteristic (AUROC) metric of up to 9.6% when compared to the baseline model that does not leverage DNA biophysical properties. We expanded our analysis to in vitro high-throughput Systematic Evolution of Ligands by Exponential enrichment (HT-SELEX) dataset of 215 TFs from 27 families, comparing EPBD with established frameworks. The integration of the DNA breathing features with DNABERT-2 foundational model, greatly enhanced TF-binding predictions. Notably, EPBDxDNABERT-2, trained on a large-scale multi-species genomes, with a cross-attention mechanism, improved predictive power shedding light on the mechanisms underlying disease-related non-coding variants discovered in genome-wide association studies.

Programmable Plasmonic Hydrogel Thermometers Actuated by DNA Breathing

AbstractNanothermometry explores the use of temperature‐dependent properties of materials for remote and sensitive thermal readout at the nanoscale. The currently established nanothermometers are largely limited by uncontrollable nanoparticle flocculation, complex experimental setup, and narrow working temperature range. Here it is shown that gold nanoparticles (e.g., spheres, rods) embedded in a hydrogel can afford sensitive, durable, and range‐tunable temperature sensing via the terminal breathing of the nanoparticle surface‐grafted DNA. The realization of the plasmonic hydrogel thermometer with a thermal sensitivity of ≤2 °C relies on the dynamically modulable interparticle spacing by thermo‐responsive terminal base pairing/unpairing of the surface DNA. By altering the alcoholic ratio of the hydrogel, the temperature‐response range can be continuously regulated based on solvent‐mediated DNA base pairing. Compared with the colloidal counterpart, importantly, the hydrogel thermometer exhibits greatly improved thermal sensing capability (e.g., repeatability ≥50 times) while possessing excellent durability. Given the excellent durability, high sensitivity, and programmable temperature response range, the thermometers actuated by DNA breathing for advanced uses in human sensing and optoelectronics are within reach.

Epigenetic Histone Modifications H3K36me3 and H4K5/8/12/16ac Induce Open Polynucleosome Conformations via Different Mechanisms

Nucleosomes are the basic compaction unit of chromatin and nucleosome structure and their higher-order assemblies regulate genome accessibility. Many post-translational modifications alter nucleosome dynamics, nucleosome-nucleosome interactions, and ultimately chromatin structure and gene expression. Here, we investigate the role of two post-translational modifications associated with actively transcribed regions, H3K36me3 and H4K5/8/12/16ac, in the contexts of tri-nucleosome arrays that provide a tractable model system for quantitative single-molecule analysis, while enabling us to probe nucleosome-nucleosome interactions. Direct visualization by AFM imaging reveals that H3K36me3 and H4K5/8/12/16ac nucleosomes adopt significantly more open and loose conformations than unmodified nucleosomes. Similarly, magnetic tweezers force spectroscopy shows a reduction in DNA outer turn wrapping and nucleosome-nucleosome interactions for the modified nucleosomes. The results suggest that for H3K36me3 the increased breathing and outer DNA turn unwrapping seen in mononucleosomes propagates to more open conformations in nucleosome arrays. In contrast, the even more open structures of H4K5/8/12/16ac nucleosome arrays do not appear to derive from the dynamics of the constituent mononucleosomes, but are driven by reduced nucleosome-nucleosome interactions, suggesting that stacking interactions can overrule DNA breathing of individual nucleosomes. We anticipate that our methodology will be broadly applicable to reveal the influence of other post-translational modifications and to observe the activity of nucleosome remodelers.

Studies of DNA breathing in exciton-coupled (iCy3)2 dimer-labeled DNA constructs by polarization-sweep single-molecule fluorescence (PS-SMF) microscopy.

Local fluctuations of the sugar-phosphate backbones and bases of DNA (a form of DNA 'breathing') play a central role in the assembly of protein-DNA complexes. We present a single-molecule fluorescence method to sensitively measure the local conformational fluctuations of exciton-coupled cyanine [(iCy3)2] dimer-labeled DNA fork constructs in which the dimer probes are placed at varying positions relative to the DNA fork junction. These systems exhibit spectroscopic signals that are sensitive to the local conformations adopted by the sugar-phosphate backbones and bases immediately surrounding the dimer probe label positions. The (iCy3)2 dimer has one symmetric (+) and one anti-symmetric (-) exciton with respective transition dipole moments oriented perpendicular to one another. We excite single molecule samples using a continuous-wave, linearly polarized laser with its polarization direction rotated at a frequency of 1 MHz. The ensuing fluorescence signal is modulated as the laser polarization alternately excites the symmetric and anti-symmetric excitons of the (iCy3)2 dimer probe. Phase-sensitive detection of the signal at the photon-counting level provides information about the distribution of local conformations and conformational dynamics. We analyze our data using a kinetic network model, which we use to parametrize the free energy surface of the system. In addition to observing DNA breathing at and near ss-dsDNA junctions, the approach can be used to study the effects of proteins that bind and function at these sites.

Studies of DNA 'Breathing' by Polarization-Sweep Single-Molecule Fluorescence Microscopy of Exciton-Coupled (iCy3)2 Dimer-Labeled DNA Fork Constructs.

Local fluctuations of the sugar-phosphate backbones and bases of DNA (often called DNA 'breathing') play a variety of critical roles in controlling the functional interactions of the DNA genome with the protein complexes that regulate it. Here, we present a single-molecule fluorescence method that we have used to measure and characterize such conformational fluctuations at and near biologically important positions in model DNA replication fork constructs labeled with exciton-coupled cyanine [(iCy3)2] dimer probes. Previous work has shown that the constructs that we tested here exhibit a broad range of spectral properties at the ensemble level, and these differences can be structurally and dynamically interpreted using our present methodology at the single-molecule level. The (iCy3)2 dimer has one symmetric (+) and one antisymmetric (-) exciton, with the respective transition dipole moments oriented perpendicular to one another. We excite single-molecule samples using a continuous-wave linearly polarized laser, with the polarization direction continuously rotated at the frequency of 1 MHz. The ensuing fluorescence signal is modulated as the laser polarization alternately excites the symmetric and antisymmetric excitons of the (iCy3)2 dimer probe. Phase-sensitive detection of the modulated signal provides information about the distribution of local conformations and the conformational interconversion dynamics of the (iCy3)2 probe. We find that at most construct positions that we examined, the (iCy3)2 dimer-labeled DNA fork constructs can adopt four topologically distinct conformational macrostates. These results suggest that in addition to observing DNA breathing at and near ss-dsDNA junctions, our new methodology should be useful to determine which of these pre-existing macrostates are recognized by, bind to, and are stabilized by various genome-regulatory proteins.

Examining DNA breathing with pyDNA-EPBD.

The two strands of the DNA double helix locally and spontaneously separate and recombine in living cells due to the inherent thermal DNA motion. This dynamics results in transient openings in the double helix and is referred to as "DNA breathing" or "DNA bubbles." The propensity to form local transient openings is important in a wide range of biological processes, such as transcription, replication, and transcription factors binding. However, the modeling and computer simulation of these phenomena, have remained a challenge due to the complex interplay of numerous factors, such as, temperature, salt content, DNA sequence, hydrogen bonding, base stacking, and others. We present pyDNA-EPBD, a parallel software implementation of the Extended Peyrard-Bishop-Dauxois (EPBD) nonlinear DNA model that allows us to describe some features of DNA dynamics in detail. The pyDNA-EPBD generates genomic scale profiles of average base-pair openings, base flipping probability, DNA bubble probability, and calculations of the characteristically dynamic length indicating the number of base pairs statistically significantly affected by a single point mutation using the Markov Chain Monte Carlo algorithm. pyDNA-EPBD is supported across most operating systems and is freely available at https://github.com/lanl/pyDNA_EPBD. Extensive documentation can be found at https://lanl.github.io/pyDNA_EPBD/.

The effects of viral nucleosome-like particles on DNA compaction, dynamics, and accessibility.

Melbourne viruses are large DNA-based viruses that organize their genome using histone-like proteins that act as “fused” histones with homologies similar to the histone dimers found in eukaryotic nucleosomes. Cryo-EM studies have suggested that these viral nucleosome-like particles have a weak binding affinity that causes their wrapped DNA structure to have more open conformations than their eukaryotic counterparts. However, these studies provide limited insight into the dynamics and stability of the DNA in these viral structures and do not reveal how differences between the viral and eukaryotic histone-like proteins affect large-scale structural properties such as DNA unwrapping, DNA accessibility, and protein core stability. To probe the dynamical differences between the eukaryotic nucleosome and viral nucleosome-like particle and whether this equally affects differing DNA types, we have performed a series of all-atom molecular dynamics simulations on nucleosome systems with different protein core and DNA sequences. Our results show that differences between the viral and eukaryotic structures create weaker overall stability of the viral system and promote DNA breathing and oscillations of the DNA ends around the protein core regardless of DNA composition. While the presence of normal histone tails adds to nucleosome stability, replacing eukaryotic histone tails with viral histone connectors also creates a more dynamic and unstable system. Together, our results show that the evolutionarily diverse mechanisms of compacting DNA in eukaryotic and viral systems result in disparate DNA compaction and accessibility in these systems.

Studies of Local DNA Backbone Conformation and Conformational Disorder Using Site-Specific Exciton-Coupled Dimer Probe Spectroscopy.

The processes of genome expression, regulation, and repair require direct interactions between proteins and DNA at specific sites located at and near single-stranded-double-stranded DNA (ssDNA-dsDNA) junctions. Here, we review the application of recently developed spectroscopic methods and analyses that combine linear absorbance and circular dichroism spectroscopy with nonlinear 2D fluorescence spectroscopy to study the local conformations and conformational disorder of the sugar-phosphate backbones of ssDNA-dsDNA fork constructs that have been internally labeled with exciton-coupled cyanine (iCy3)2 dimer probes. With the application of these methods, the (iCy3)2 dimer can serve as a reliable probe of the mean local conformations and conformational distributions of the sugar-phosphate backbones of dsDNA at various critical positions. The results of our studies suggest a possible structural framework for understanding the roles of DNA breathing in driving the processes of protein-DNA complex assembly and function.

Nonlinear physics opens a new paradigm for accurate transcription start site prediction

There is evidence that DNA breathing (spontaneous opening of the DNA strands) plays a relevant role in the interactions of DNA with other molecules, and in particular in the transcription process. Therefore, having physical models that can predict these openings is of interest. However, this source of information has not been used before either in transcription start sites (TSSs) or promoter prediction. In this article, one such model is used as an additional information source that, when used by a machine learning (ML) model, improves the results of current methods for the prediction of TSSs. In addition, we provide evidence on the validity of the physical model, as it is able by itself to predict TSSs with high accuracy. This opens an exciting avenue of research at the intersection of statistical mechanics and ML, where ML models in bioinformatics can be improved using physical models of DNA as feature extractors.

High-precision mapping reveals rare N6-deoxyadenosine methylation in the mammalian genome

N6-deoxyadenosine methylation (6mA) is the most widespread type of DNA modification in prokaryotes and is also abundantly distributed in some unicellular eukaryotes. However, 6mA levels are remarkably low in mammals. The lack of a precise and comprehensive mapping method has hindered more advanced investigations of 6mA. Here, we report a new method MM-seq (modification-induced mismatch sequencing) for genome-wide 6mA mapping based on a novel detection principle. We found that modified DNA bases are prone to form a local open region that allows capture by antibody, for example, via a DNA breathing or base-flipping mechanism. Specified endonuclease or exonuclease can recognize the antibody-stabilized mismatch-like structure and mark the exact modified sites for sequencing readout. Using this method, we examined the genomic positions of 6mA in bacteria (E. coli), green algae (C. reinhardtii), and mammalian cells (HEK239T, Huh7, and HeLa cells). In contrast to bacteria and green algae, human cells possess a very limited number of 6mA sites which are sporadically distributed across the genome of different cell types. After knocking out the RNA m6A methyltransferase METTL3 in mouse ES cells, 6mA becomes mostly diminished. Our results imply that rare 6mA in the mammalian genome is introduced by RNA m6A machinery via a non-targeted mechanism.

Breathers-like rogue wave trains induced by nonlinear dynamics of DNA breathing

Breathers-like rogue wave trains induced by nonlinear dynamics of DNA breathing

Determining local nucleic acid base conformations by fourier transform spectroscopy of 6-methyl isoxanthopterin (6-MI) substituted DNA fork constructs

Base stacking is centrally involved in controlling the structure of ‘Watson-Crick’ double-stranded (ds) DNA. However, the local conformations adopted by nucleic acid bases as a consequence of DNA breathing, and their roles in protein-DNA binding, are not well understood. Here we employ the fluorescent base analogue 6-methyl isoxanthopterin (6-MI), which displays a chemical structure and base-pairing interactions similar to guanine, to probe the local conformations of nucleic acid bases at site-specific positions at and near single-stranded (ss)-dsDNA junctions.

Parallel DNA circuits by autocatalytic strand displacement and nanopore readout.

DNA nanotechnology provides a unique opportunity for molecular computation, with strand displacement reactions enabling controllable reorganization of nanostructures. Additional DNA strand exchange strategies with high selectivity for input will enable novel complex systems including biosensing applications. Herein, we propose an autocatalytic strand displacement (ACSD) circuit: initiated by DNA breathing and accelerated by a seesaw catalytic reaction, ACSD ensures that only the correct base sequence starts the catalytic cycle. Analogous to an electronic circuit with a variable resistor, two ACSD reactions with different rates are connected in parallel to mimic a parallel circuit containing branches with different resistances. Finally, we introduce a multiplexed nanopore sensing platform to report the output results of a parallel path selection system at the single-molecule level. By combining the ACSD strategy with fast and sensitive single-molecule nanopore readout, a new generation of DNA-based computing tools is established.

Histone dynamics mediate DNA unwrapping and sliding in nucleosomes

Nucleosomes are elementary building blocks of chromatin in eukaryotes. They tightly wrap ∼147 DNA base pairs around an octamer of histone proteins. How nucleosome structural dynamics affect genome functioning is not completely clear. Here we report all-atom molecular dynamics simulations of nucleosome core particles at a timescale of 15 microseconds. At this timescale, functional modes of nucleosome dynamics such as spontaneous nucleosomal DNA breathing, unwrapping, twisting, and sliding were observed. We identified atomistic mechanisms of these processes by analyzing the accompanying structural rearrangements of the histone octamer and histone-DNA contacts. Octamer dynamics and plasticity were found to enable DNA unwrapping and sliding. Through multi-scale modeling, we showed that nucleosomal DNA dynamics contribute to significant conformational variability of the chromatin fiber at the supranucleosomal level. Our study further supports mechanistic coupling between fine details of histone dynamics and chromatin functioning, provides a framework for understanding the effects of various chromatin modifications.

Effects of H2A.B incorporation on nucleosome structures and dynamics

The H2A.B histone variant is an epigenetic regulator involved in transcriptional upregulation, DNA synthesis, and splicing that functions by replacing the canonical H2A histone in the nucleosome core particle. Introduction of H2A.B results in less compact nucleosome states with increased DNA unwinding and accessibility at the nucleosomal entry and exit sites. Despite being well characterized experimentally, the molecular mechanisms by which H2A.B incorporation alters nucleosome stability and dynamics remain poorly understood. To study the molecular mechanisms of H2A.B, we have performed a series of conventional and enhanced sampling molecular dynamics simulation of H2A.B- and canonical H2A-containing nucleosomes. Results of conventional simulations show that H2A.B weakens protein-protein and protein-DNA interactions at specific locations throughout the nucleosome. These weakened interactions result in significantly more DNA opening from both the entry and exit sites in enhanced sampling simulations. Furthermore, free energy profiles show that H2A.B-containing nucleosomes have significantly broader free wells and that H2A.B allows for sampling of states with increased DNA breathing, which are shown to be stable on the hundreds of nanoseconds timescale with further conventional simulations. Together, our results show the molecular mechanisms by which H2A.B creates less compacted nucleosome states as a means of increasing genetic accessibility and gene transcription.

Range-tunable plasmon switching of gold nanorods by terminal breathing of surface-grafted DNA in alcoholic solvents

A smart thermal plasmon switch of Au nanorods featured with tunable response temperature is developed by thermal-responsive terminal breathing of surface-grafted DNA in alcoholic solvents.

Plasmon switching of gold nanoparticles through thermo-responsive terminal breathing of surface-grafted DNA in hydrated ionic liquids.

Self-assembly performed in ionic liquids (ILs) as a unique solvent promises distinct functions and applications in sensors, therapeutics, and optoelectronic devices due to the rich interactions between nanoparticle building blocks and ILs. However, the general consideration that common nanoparticles are readily destabilized by counterions in an IL has largely prevented researchers from investigating controlled nanoparticle assembly in IL-based systems. This study explores the assembling behaviour of double-stranded (ds) DNA-functionalized gold nanoparticles (dsDNA-AuNPs) in hydrated ionic liquids. The DNA base pair stacking assembly of dsDNA-AuNPs occurs at a low IL concentration (<5%). However, a moderate ionic liquid concentration (5-40%) can de-hybridize dsDNA and leaves single-stranded (ss) DNA stabilizing the AuNPs. In concentrated ionic liquids (>40%), interestingly, the higher ionic strength leads to the assembly of DNA-AuNPs. The triphasic assembly trend is also generally observed regardless of the type of IL. By down-regulation of DNA's melting temperature with the IL, the assembly of DNA-AuNPs affords robust response to a lower temperature range, promising applications in plasmonic devices and range-tunable temperature sensors.

Ancillary Ligand in Ternary CuII Complexes Guides Binding Selectivity toward Minor-Groove DNA.

Copper-containing compounds known as Casiopeínas are biologically active molecules which show promising antineoplastic effects against several cancer types. Two possible hypotheses regarding the mode of action of the Casiopeínas have emerged from the experimental evidence: the generation of reactive oxygen species or the ability of the compounds to bind and interact with nucleic acids. Using robust molecular dynamics simulations, we investigate the interaction of four different Casiopeínas with the DNA duplex d(GCACGAACGAACGAACGC). The studied copper complexes contain either 4–7- or 5–6-substituted dimethyl phenanthroline as the primary ligand and either glycinate or acetylacetonate as the secondary ligand. For statistical significance and to reduce bias in the simulations, four molecules of each copper compound were manually placed at a distance of 10 Å away from the DNA and 20 independent molecular dynamics simulations were performed, each reaching at least 30 μs. This time scale allows us to reproduce expected DNA terminal base-pair fraying and also to observe intercalation/base-pair eversion events generated by the compounds interacting with DNA. The results reveal that the secondary ligand is the guide toward the mode of binding between the copper complex and DNA in which glycinate prefers minor-groove binding and acetylacetonate produces base-pair eversion and intercalation. The CuII complexes containing glycinate interact within the DNA minor groove which are stabilized principally by the hydrogen bonds formed between the amino group of the aminoacidate moiety, whereas the compounds with the acetylacetonate do not present a stable network of hydrogen bonds and the ligand interactions enhance DNA breathing dynamics that result in base-pair eversion.

DNA Transformation, Cell Epigenetic Landscape and Open Complex Dynamics in Cancer Development

Statistical thermodynamics allowed the formulation of mesoscopic approach of DNA transformation in course of the excitation of collective distortion modes (denaturation bubbles) associated with hydrogen bond breaking between the base pairs. Intermediate (non-continual limit) of DNA modeling (the Peyrard-Bishop model) is combined with the field description (generalized Ginzburg-Landau approach) to analyze the dynamics of collective open complex modes associated with mesodefects in the DNA ensemble. Collective modes dynamics describes different scenario of gene expression according to statistically predicted form of out-of-equilibrium potential (epigenetic landscape) reflecting specific type criticality of “soft matter” with mesodefects (open complexes) – the structural-scaling transition. Principal difference of thermodynamics of non-continual and continual models is thermalization conditions related to thermal fluctuations responsible for the DNA breathing (localized excitation with breather dynamics) and structural-scaling parameter responsible for spinodal decomposition of out-of-equilibrium potential metastability due to generation of open complex collective modes. Open complex collective modes have the nature of self-similar solutions (breathers, auto-solitary and blow-up modes) of open complex evolution equation accounting qualitative different types of potential metastabilities. Sub-sets of collective modes represent the phase variables of attractors associated with different scenario of expression dynamics, which allows the interpretation of multistability of the epigenetic landscape and the Huang diagram of gene expression. It was shown different epigenetic pathway in attractors phase space corresponding to normal and cancer expression scenario. These scenarios were supported by laser interference microscopy of living normal and cancer cells illustrating multi- and monofractal dynamics.

2’-methylklavuzon causes lipid-lowering effects on A549 non-small cell lung cancer cells and significant changes on DNA structure evidenced by fourier transform infrared spectroscopy

Various chemical agents are used in the treatment of Non-Small Cell Lung Cancer (NSCLC). 2′-methylklavuzon was proposed as a potential chemotherapeutic agent in cancer treatment based on its topoisomerase inhibition activity. In this study the cellular effects of 2′-methylklavuzon was evaluated on A549 cancer cells using FTIR spectroscopy. 2′-methylklavuzon induced significant changes on both the whole cell lyophilizates and the lipid extracts of the A549 lung cancer cells. 2′-methylklavuzon caused significant structural changes in A549 cell DNA structure: T, A and G DNA breathing modes are lost after the drug application indicating the loss of topoisomerase activity. The level of transcription and RNA synthesis was enhanced. 2′-methylklavuzon induced single stranded DNA formation evidenced by the increase in the ratio of asymmetric/symmetric phosphate stretching modes. 2′-methylklavuzon induced band shifts only in the asymmetric mode of phosphate bonds not in the symmetrical phosphate bond stretching. 2′-methylklavuzon induced A form of DNA topography. In addition to the changes in the DNA structure and transcription 2′-methylklavuzon also caused lipid-lowering effect in A549 cancer cells. 2′-methylklavuzon suppressed lipid unsaturation, however, it induced formation of lipids with ring structures. 2′-methylklavuzon suppressed phosphate-containing lipids significantly and decreased carbonyl containing lipids and cholesterol slightly. 2′-methylklavuzon caused increases in the hydrocarbon chain length. Overall, 2′-methylklavuzon can be used as a lipid-lowering compound in the treatment of NSCLC and other cancer therapies.