Anomalous Laterally Stressed Kinetically Trapped DNA Surface Conformations.

HIGHLIGHTS: DNA kinking is inevitable for the highly anisotropic 1D-1D electrostatic interaction with the one-dimensionally periodically charged surface. The double helical structure of the DNA kinetically trapped on positively charged monomolecular films comprising the lamellar templates is strongly laterally stressed and extremely perturbed at the nanometer scale. The DNA kinetic trapping is not a smooth 3D-> 2D conformational flattening but is a complex nonlinear in-plane mechanical response (bending, tensile and unzipping) driven by the physics beyond the scope of the applicability of the linear worm-like chain approximation. Up to now, the DNA molecule adsorbed on a surface was believed to always preserve its native structure. This belief implies a negligible contribution of lateral surface forces during and after DNA adsorption although their impact has never been elucidated. High-resolution atomic force microscopy was used to observe that stiff DNA molecules kinetically trapped on monomolecular films comprising one-dimensional periodically charged lamellar templates as a single layer or as a sublayer are oversaturated by sharp discontinuous kinks and can also be locally melted and supercoiled. We argue that kink/anti-kink pairs are induced by an overcritical lateral bending stress (> 30 pNnm) inevitable for the highly anisotropic 1D-1D electrostatic interaction of DNA and underlying rows of positive surface charges. In addition, the unexpected kink-inducing mechanical instability in the shape of the template-directed DNA confined between the positively charged lamellar sides is observed indicating the strong impact of helicity. The previously reported anomalously low values of the persistence length of the surface-adsorbed DNA are explained by the impact of the surface-induced low-scale bending. The sites of the local melting and supercoiling are convincingly introduced as other lateral stress-induced structural DNA anomalies by establishing a link with DNA high-force mechanics. The results open up the study in the completely unexplored area of the principally anomalous kinetically trapped DNA surface conformations in which the DNA local mechanical response to the surface-induced spatially modulated lateral electrostatic stress is essentially nonlinear. The underlying rich and complex in-plane nonlinear physics acts at the nanoscale beyond the scope of applicability of the worm-like chain approximation.

DNA melting and energetics of the double helix.

Studying melting and energetics of the DNA double helix has been one of the major topics of molecular biophysics over the past six decades. The main objective of this article is to overview the current state of the field and to emphasize that there are still serious gaps in our understanding of the issue. We start with a concise description of the commonly accepted theoretical model of the DNA melting. We then concentrate on studies devoted to the comparison with experiment of theoretically predicted melting profiles of DNAs with known sequences. For long DNA molecules, such comparison is significant from the basic-science viewpoint while an accurate theoretical description of melting of short duplexes is necessary for various very important applications in biotechnology. Several sets of DNA melting parameters, proposed within the framework of the nearest neighbor model, are compared and analyzed. The analysis leads to a conclusion that in case of long DNA molecules the consensus set of nearest neighbor parameters describes well the experimental melting profiles. Unexpectedly, for short DNA duplexes the same set of parameters hardly yields any improvement as compared to the simplest model, which completely ignores the effect of heterogeneous stacking. Possible causes of this striking observation are discussed. We then overview the issue of separation of base-pairing and base-stacking contributions into the double helix stability. The recent experimental attempts to solve the problem are extensively analyzed. It is concluded that the double helix is essentially stabilized by stacking interaction between adjacent base pairs. Base pairing between complementary pairs does not appreciably contribute into the duplex stability. In the final section of the article, kinetic aspects of the DNA melting phenomenon are discussed. The main emphasis is made on the hysteresis effects often observed in melting of long DNA molecules. It is argued that the phenomenon can be well described via an accurate theoretical treatment of the random-walk model of melting kinetics of an isolated helical segment in DNA.

Fluctuations in the DNA double helix: A critical review.

A critical overview of the extensive literature on fluctuations in the DNA double helix is presented. Both theory and experiment are comprehensively reviewed and analyzed. Fluctuations, which open up the DNA double helix making bases accessible for hydrogen exchange and chemical modification, are the main focus of the review. Theoretical descriptions of the DNA fluctuations are discussed with special emphasis on most popular among them: the nonlinear-dynamic Peyrard-Bishop-Dauxois (PBD) model and the empirical two-state (or helix-coil) model. The experimental data on the issue are comprehensibly overviewed in the historical retrospective with main emphasis on the hydrogen exchange data and formaldehyde kinetics. The theoretical descriptions are critically evaluated from the viewpoint of their applicability to describe DNA in water environment and from the viewpoint of agreement of their predictions with the reliable experimental data. The presented analysis makes it possible to conclude that, while the two-state model is most adequate from theoretical viewpoint and its predictions, based on an empirical parametrization, agree with experimental data very well, the PBD model is inapplicable to DNA in water from theoretical viewpoint on one hand and it makes predictions totally incompatible with reliable experimental data on the other. In particular, it is argued that any oscillation movements of nucleotides, assumed by the PBD model, are severely damped in water, that no "bubbles", which the PBD model predicts, exist in reality in linear DNA well below the melting range and the lifetime of an open state in DNA is actually 5 orders of magnitude longer than the value predicted by the PBD model.

PNA openers and their applications for bacterial DNA diagnostics.

The unique ability of triplex-forming PNAs to invade the double helix has made it possible to develop a highly specific and sensitive approach for bacterial detection. The method uses short, about 20-bp-long, signature sequences presented as a single copy in the bacterial genome. Bacterial cells are fixed on slides and the PD-loop structure is assembled on the signature site with the help of PNA openers, which includes the circular probe. The sensitivity of the method is achieved via Rolling Circle Amplification (RCA) of the circular probe. The obtained amplicon is detected using short ssDNA decorator probes carrying fluorophores and via standard fluorescent microscopy.

Strong bending of the DNA double helix.

During the past decade, the issue of strong bending of the double helix has attracted a lot of attention. Here, we overview the major experimental and theoretical developments in the field sorting out reliably established facts from speculations and unsubstantiated claims. Theoretical analysis shows that sharp bends or kinks have to facilitate strong bending of the double helix. It remains to be determined what is the critical curvature of DNA that prompts the appearance of the kinks. Different experimental and computational approaches to the problem are analyzed. We conclude that there is no reliable evidence that any anomalous behavior of the double helix happens when DNA fragments in the range of 100 bp are circularized without torsional stress. The anomaly starts at the fragment length of about 70 bp when sharp bends or kinks emerge in essentially every molecule. Experimental data and theoretical analysis suggest that kinks may represent openings of isolated base pairs, which had been experimentally detected in linear DNA molecules. The calculation suggests that although the probability of these openings in unstressed DNA is close to 10(-5), it increases sharply in small DNA circles reaching 1 open bp per circle of 70 bp.

RNA topology.

A new variety on non-coding RNA has been discovered by several groups: circular RNA (circRNA). This discovery raises intriguing questions about the possibility of the existence of knotted RNA molecules and the existence of a new class of enzymes changing RNA topology, RNA topoisomerases.

Bending of short DNA helices.

In their recent Science paper, Vafabakhsh and Ha claim that DNA duplexes at the range of 100 bp experience anomalous flexibility, much greater than the flexibility of large DNA molecules. ( 1) However, careful reevaluation of their data leads to the conclusion that the presented data do not warrant the authors' claim.

Computational mapping reveals dramatic effect of Hoogsteen breathing on duplex DNA reactivity with formaldehyde.

Formaldehyde has long been recognized as a hazardous environmental agent highly reactive with DNA. Recently, it has been realized that due to the activity of histone demethylation enzymes within the cell nucleus, formaldehyde is produced endogenously, in direct vicinity of genomic DNA. Should it lead to extensive DNA damage? We address this question with the aid of a computational mapping method, analogous to X-ray and nuclear magnetic resonance techniques for observing weakly specific interactions of small organic compounds with a macromolecule in order to establish important functional sites. We concentrate on the leading reaction of formaldehyde with free bases: hydroxymethylation of cytosine amino groups. Our results show that in B-DNA, cytosine amino groups are totally inaccessible for the formaldehyde attack. Then, we explore the effect of recently discovered transient flipping of Watson-Crick (WC) pairs into Hoogsteen (HG) pairs (HG breathing). Our results show that the HG base pair formation dramatically affects the accessibility for formaldehyde of cytosine amino nitrogens within WC base pairs adjacent to HG base pairs. The extensive literature on DNA interaction with formaldehyde is analyzed in light of the new findings. The obtained data emphasize the significance of DNA HG breathing.

DNA breathes Hoogsteen.

A recent claim is discussed that Watson-Crick pairs in the naked duplex DNA spontaneously flip into Hoogsteen pairs under ordinary conditions. The claim is considered within the historical retrospective and is put into the broader context of DNA biophysics.

PNA-based microbial pathogen identification and resistance marker detection: An accurate, isothermal rapid assay based on genome-specific features.

With the rapidly growing availability of the entire genome sequences of microbial pathogens, there is unmet need for increasingly sensitive systems to monitor the gene-specific markers for diagnosis of bacteremia that enables an earlier detection of causative agent and determination of drug resistance. To address these challenges, a novel FISH-type genomic sequence-based molecular technique is proposed that can identify bacteria and simultaneously detect antibiotic resistance markers for rapid and accurate testing of pathogens. The approach is based on a synergistic combination of advanced Peptide Nucleic Acid (PNA)-based technology and signal-enhancing Rolling Circle Amplification (RCA) reaction to achieve a highly specific and sensitive assay. A specific PNA-DNA construct serves as an exceedingly selective and very effective biomarker, while RCA enhances detection sensitivity and provide with a highly multiplexed assay system. Distinct-color fluorescent decorator probes are used to identify about 20-nucleotide-long signature sequences in bacterial genomic DNA and/or key genetic markers of drug resistance in order to identify and characterize various pathogens. The technique's potential and its utility for clinical diagnostics are illustrated by identification of S. aureus with simultaneous discrimination of methicillin-sensitive (MSSA) versus methicillin-resistant (MRSA) strains. Overall these promising results hint to the adoption of PNA-based rapid sensitive detection for diagnosis of other clinically relevant organisms. Thereby, new assay enables significantly earlier administration of appropriate antimicrobial therapy and may, thus have a positive impact on the outcome of the patient.

Sequence specificity at targeting double-stranded DNA with a γ-PNA oligomer modified with guanidinium G-clamp nucleobases.

γ-PNA, a new class of peptide nucleic acids, promises to overcome previous sequence limitations of double-stranded DNA (dsDNA) targeting with PNA. To check the potential of γ-PNA, we have synthesized a biotinylated, pentadecameric γ-PNA of mixed sequence carrying three guanidinium G-clamp nucleobases. We have found that strand invasion reactions of the γ-PNA oligomer to its fully complementary target within dsDNA occurs with significantly higher binding rates than to targets containing single mismatches. Association of the PNA oligomer to mismatched targets does not go to completion but instead reaches a stationary level at or below 60%, even at conditions of very low ionic strength. Initial binding rates to both matched and mismatched targets experience a steep decrease with increasing salt concentration. We demonstrate that a linear DNA target fragment with the correct target sequence can be purified from DNA mixtures containing mismatched target or unrelated genomic DNA by affinity capture with streptavidin-coated magnetic beads. Similarly, supercoiled plasmid DNA is obtained with high purity from an initial sample mixture that included a linear DNA fragment with the fully complementary sequence. Based on the results obtained in this study we believe that γ-PNA has a great potential for specific targeting of chosen duplex DNA sites in a sequence-unrestricted fashion.

Labeling of unique sequences in double-stranded DNA at sites of vicinal nicks generated by nicking endonucleases.

We describe a new approach for labeling of unique sequences within dsDNA under nondenaturing conditions. The method is based on the site-specific formation of vicinal nicks, which are created by nicking endonucleases (NEases) at specified DNA sites on the same strand within dsDNA. The oligomeric segment flanked by both nicks is then substituted, in a strand displacement reaction, by an oligonucleotide probe that becomes covalently attached to the target site upon subsequent ligation. Monitoring probe hybridization and ligation reactions by electrophoretic mobility retardation assay, we show that selected target sites can be quantitatively labeled with excellent sequence specificity. In these experiments, predominantly probes carrying a target-independent 3' terminal sequence were employed. At target labeling, thus a branched DNA structure known as 3'-flap DNA is obtained. The single-stranded terminus in 3'-flap DNA is then utilized to prime the replication of an externally supplied ssDNA circle in a rolling circle amplification (RCA) reaction. In model experiments with samples comprised of genomic lambda-DNA and human herpes virus 6 type B (HHV-6B) DNA, we have used our labeling method in combination with surface RCA as reporter system to achieve both high sequence specificity of dsDNA targeting and high sensitivity of detection. The method can find applications in sensitive and specific detection of viral duplex DNA.

Fluorescence-based detection of short DNA sequences under non-denaturing conditions.

The ability of peptide nucleic acid (PNA) to open up duplex DNA in a highly sequence-specific manner makes it possible to detect short DNA sequences on the background of or within genomic DNA under non-denaturing conditions. To do so, chosen marker sites in double-stranded DNA are locally opened by a pair of PNA openers, thus transforming one strand within the target region (20-30 bp) into the single-stranded form. Onto this accessible DNA sequence a circular oligonucleotide probe is assembled, which serves as a template for rolling circle amplification (RCA). Both homogeneous and heterogeneous assay formats are investigated, as are different formats for fluorescence-based amplicon detection. Our recent data with immobilized analytes suggest that marker sequences in plasmid and bacterial chromosomal DNA can be successfully detected.

Ab initio DNA synthesis accelerated by endonuclease.

In the presence of an endonuclease that digests double-stranded DNA, DNA polymerase efficiently synthesizes and amplifies DNA from dNTPs in the absence of any added template and primer nucleic acid. This cut-polymerization DNA synthesis (Cut-grow) can be carried out under a wide range of temperature (4-85 degrees C) by many endonucleases and DNA polymerases. The high efficiency of Cut-grow results from an efficient exponential amplification involving digestion-elongation cycles. Our findings suggest that digestion of nucleic acids may play an important role during the evolution of genetic material for procreating the diversification of genetic information on the early earth.

Sequence-dependent base pair opening in DNA double helix.

Preservation of genetic information in DNA relies on shielding the nucleobases from damage within the double helix. Thermal fluctuations lead to infrequent events of the Watson-Crick basepair opening, or DNA "breathing", thus making normally buried groups available for modification and interaction with proteins. Fluctuational basepair opening implies the disruption of hydrogen bonds between the complementary bases and flipping of the base out of the helical stack. Prediction of sequence-dependent basepair opening probabilities in DNA is based on separation of the two major contributions to the stability of the double helix: lateral pairing between the complementary bases and stacking of the pairs along the helical axis. The partition function calculates the basepair opening probability at every position based on the loss of two stacking interactions and one base-pairing. Our model also includes a term accounting for the unfavorable positioning of the exposed base, which proceeds through a formation of a highly constrained small loop, or a ring. Quantitatively, the ring factor is found as an adjustable parameter from the comparison of the theoretical basepair opening probabilities and the experimental data on short DNA duplexes measured by NMR spectroscopy. We find that these thermodynamic parameters suggest nonobvious sequence dependent basepair opening probabilities.

Base-stacking and base-pairing contributions into thermal stability of the DNA double helix.

Two factors are mainly responsible for the stability of the DNA double helix: base pairing between complementary strands and stacking between adjacent bases. By studying DNA molecules with solitary nicks and gaps we measure temperature and salt dependence of the stacking free energy of the DNA double helix. For the first time, DNA stacking parameters are obtained directly (without extrapolation) for temperatures from below room temperature to close to melting temperature. We also obtain DNA stacking parameters for different salt concentrations ranging from 15 to 100 mM Na+. From stacking parameters of individual contacts, we calculate base-stacking contribution to the stability of A*T- and G*C-containing DNA polymers. We find that temperature and salt dependences of the stacking term fully determine the temperature and the salt dependence of DNA stability parameters. For all temperatures and salt concentrations employed in present study, base-stacking is the main stabilizing factor in the DNA double helix. A*T pairing is always destabilizing and G*C pairing contributes almost no stabilization. Base-stacking interaction dominates not only in the duplex overall stability but also significantly contributes into the dependence of the duplex stability on its sequence.