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Coarse-grained models have emerged as valuable tools to simulate long DNA molecules while maintaining computational efficiency. These models aim at preserving interactions among coarse-grained variables in a manner that mirrors the underlying atomistic description. We explore here a method for testing coarse-grained vs all-atom models using stiffness matrices in Fourier space (q-stiffnesses), which are particularly suited to probe DNA elasticity at different length scales. We focus on a class of coarse-grained rigid base DNA models known as cgDNA and its most recent version, cgDNA+. Our analysis shows that while cgDNA+ closely follows the q-stiffnesses of the all-atom model, the original cgDNA shows some deviations for twist and bending variables, which are rather strong in the q → 0 (long length scale) limit. The consequence is that while both cgDNA and cgDNA+ give a suitable description of local elastic behavior, the former misses some effects that manifest themselves at longer length scales. In particular, cgDNA performs poorly on twist stiffness, with a value much lower than expected for long DNA molecules. Conversely, the all-atom and cgDNA+ twist are strongly length scale dependent: DNA is torsionally soft at a few base pair distances but becomes more rigid at distances of a few dozen base pairs. Our analysis shows that the bending persistence length in all-atom and cgDNA+ is somewhat overestimated., (© 2024 Author(s). Published under an exclusive license by AIP Publishing.)

Recent advances in experimental fluorescence microscopy allow high accuracy determination (resolution of 50 nm) of the three-dimensional physical location of multiple (up to ∼10^{2}) tagged regions of the chromosome. We investigate publicly available microscopy data for two loci of the human Chr21 obtained from multiplexed fluorescence in situ hybridization (FISH) methods for different cell lines and treatments. Inspired by polymer physics models, our analysis centers around distance distributions between different tags with the aim being to unravel the chromatin conformational arrangements. We show that for any specific genomic site, there are (at least) two different conformational arrangements of chromatin, implying coexisting distinct topologies which we refer to as phase α and phase β. These two phases show different scaling behaviors: the former is consistent with a crumpled globule, while the latter indicates a confined, but more extended conformation, such as a looped domain. The identification of these distinct phases sheds light on the coexistence of multiple chromatin topologies and provides insights into the effects of cellular context and/or treatments on chromatin structure.

Proteins often regulate their activities via allostery-or action at a distance-in which the binding of a ligand at one binding site influences the affinity for another ligand at a distal site. Although less studied than in proteins, allosteric effects have been observed in experiments with DNA as well. In these experiments two or more proteins bind at distinct DNA sites and interact indirectly with each other, via a mechanism mediated by the linker DNA molecule. We develop a mechanical model of DNA/protein interactions which predicts three distinct mechanisms of allostery. Two of these involve an enthalpy-mediated allostery, while a third mechanism is entropy driven. We analyze experiments of DNA allostery and highlight the distinctive signatures allowing one to identify which of the proposed mechanisms best fits the data.

All-atom simulations have become increasingly popular to study conformational and dynamical properties of nucleic acids as they are accurate and provide high spatial and time resolutions. This high resolution, however, comes at a heavy computational cost, and, within the time scales of simulations, nucleic acids weakly fluctuate around their ideal structure exploring a limited set of conformations. We introduce the RBB-NA algorithm (available as a package in the Open Source Library PLUMED), which is capable of controlling rigid base parameters in all-atom simulations of nucleic acids. With suitable biasing potentials, this algorithm can "force" a DNA or RNA molecule to assume specific values of the six rotational (tilt, roll, twist, buckle, propeller, opening) and/or the six translational parameters (shift, slide, rise, shear, stretch, stagger). The algorithm enables the use of advanced sampling techniques to probe the structure and dynamics of locally strongly deformed nucleic acids. We illustrate its performance showing some examples in which DNA is strongly twisted, bent, or locally buckled. In these examples, RBB-NA reproduces well the unconstrained simulations data and other known features of DNA mechanics, but it also allows one to explore the anharmonic behavior characterizing the mechanics of nucleic acids in the high deformation regime.

DNA supercoiling is a key regulatory mechanism that orchestrates DNA readout, recombination, and genome maintenance. DNA-binding proteins often mediate these processes by bringing two distant DNA sites together, thereby inducing (transient) topological domains. In order to understand the dynamics and molecular architecture of protein-induced topological domains in DNA, quantitative and time-resolved approaches are required. Here, we present a methodology to determine the size and dynamics of topological domains in supercoiled DNA in real time and at the single-molecule level. Our approach is based on quantifying the extension fluctuations-in addition to the mean extension-of supercoiled DNA in magnetic tweezers (MT). Using a combination of high-speed MT experiments, Monte Carlo simulations, and analytical theory, we map out the dependence of DNA extension fluctuations as a function of supercoiling density and external force. We find that in the plectonemic regime, the extension variance increases linearly with increasing supercoiling density and show how this enables us to determine the formation and size of topological domains. In addition, we demonstrate how the transient (partial) dissociation of DNA-bridging proteins results in the dynamic sampling of different topological states, which allows us to deduce the torsional stiffness of the plectonemic state and the kinetics of protein-plectoneme interactions. We expect our results to further the understanding and optimization of magnetic tweezer measurements and to enable quantification of the dynamics and reaction pathways of DNA processing enzymes in the context of physiologically relevant forces and supercoiling densities., (© The Author(s) 2022. Published by Oxford University Press on behalf of National Academy of Sciences.)

The real-time identification of protein biomarkers is crucial for the development of point-of-care and portable devices. Here, we use a PlyAB biological nanopore to detect haemoglobin (Hb) variants. Adult haemoglobin (HbA) and sickle cell anaemia haemoglobin (HbS), which differ by just one amino acid, were distinguished in a mixture with more than 97 % accuracy based on individual blockades. Foetal Hb, which shows a larger sequence variation, was distinguished with near 100 % accuracy. Continuum and Brownian dynamics simulations revealed that Hb occupies two energy minima, one near the inner constriction and one at the trans entry of the nanopore. Thermal fluctuations, the charge of the protein, and the external bias influence the dynamics of Hb within the nanopore, which in turn generates the unique ionic current signal in the Hb variants. Finally, Hb was counted from blood samples, demonstrating that direct discrimination and quantification of Hb from blood using nanopores, is feasible., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Plectonemes are intertwined helically looped domains which form when a DNA molecule is supercoiled, i.e., over- or underwound. They are ubiquitous in cellular DNA, and their physical properties have attracted significant interest both from the experimental side and from the modeling side. In this paper, we investigate fluctuations of the end-point distance z of supercoiled linear DNA molecules subject to external stretching forces. Our analysis is based on a two-phase model, which describes the supercoiled DNA as composed of a stretched phase and a plectonemic phase. A variety of mechanisms are found to contribute to extension fluctuations, characterized by the variance 〈Δz^{2}〉. We find the dominant contribution to 〈Δz^{2}〉 to originate from phase-exchange fluctuations, the transient shrinking and expansion of plectonemes, which is accompanied by an exchange of molecular length between the two phases. We perform Monte Carlo simulations of the twistable wormlike chain and analyze the fluctuation of various quantities, the results of which are found to agree with the two-phase model predictions. Furthermore, we show that the extension and its variance at high forces are very well captured by the two-phase model, provided that one goes beyond quadratic approximations.

Mechanical properties of nucleic acids play an important role in many biological processes that often involve physical deformations of these molecules. At sufficiently long length scales (say, above ∼20-30 base pairs), the mechanics of DNA and RNA double helices is described by a homogeneous Twistable Wormlike Chain (TWLC), a semiflexible polymer model characterized by twist and bending stiffnesses. At shorter scales, this model breaks down for two reasons: the elastic properties become sequence-dependent and the mechanical deformations at distal sites get coupled. We discuss in this paper the origin of the latter effect using the framework of a non-local Twistable Wormlike Chain (nlTWLC). We show, by comparing all-atom simulations data for DNA and RNA double helices, that the non-local couplings are of very similar nature in these two molecules: couplings between distal sites are strong for tilt and twist degrees of freedom and weak for roll. We introduce and analyze a simple double-stranded polymer model that clarifies the origin of this universal distal couplings behavior. In this model, referred to as the ladder model, a nlTWLC description emerges from the coarsening of local (atomic) degrees of freedom into angular variables that describe the twist and bending of the molecule. Different from its local counterpart, the nlTWLC is characterized by a length-scale-dependent elasticity. Our analysis predicts that nucleic acids are mechanically softer at the scale of a few base pairs and are asymptotically stiffer at longer length scales, a behavior that matches experimental data.

Structural Maintenance of Chromosomes (SMC) complexes play essential roles in genome organization across all domains of life. To determine how the activities of these large (≈50 nm) complexes are controlled by ATP binding and hydrolysis, we developed a molecular dynamics model that accounts for conformational motions of the SMC and DNA. The model combines DNA loop capture with an ATP-induced 'power stroke' to translocate the SMC complex along DNA. This process is sensitive to DNA tension: at low tension (0.1 pN), the model makes loop-capture steps of average 60 nm and up to 200 nm along DNA (larger than the complex itself), while at higher tension, a distinct inchworm-like translocation mode appears. By tethering DNA to an experimentally-observed additional binding site ('safety belt'), the model SMC complex can perform loop extrusion (LE). The dependence of LE on DNA tension is distinct for fixed DNA tension vs. fixed DNA end points: LE reversal occurs above 0.5 pN for fixed tension, while LE stalling without reversal occurs at about 2 pN for fixed end points. Our model matches recent experimental results for condensin and cohesin, and makes testable predictions for how specific structural variations affect SMC function., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

We investigate the influence of nonlocal couplings on the torsional and bending elasticities of DNA. Such couplings have been observed in the past by several simulation studies. Here, we use a description of DNA conformations based on the variables tilt, roll, and twist. Our analysis of both coarse-grained (oxDNA) and all-atom models indicates that these share strikingly similar features: there are strong off-site couplings for tilt-tilt and twist-twist, while they are much weaker in the roll-roll case. By developing an analytical framework to estimate bending and torsional persistence lengths in nonlocal DNA models, we show how off-site interactions generate a length-scale-dependent elasticity. Based on the simulation-generated elasticity data, the theory predicts a significant length-scale-dependent effect on torsional fluctuations but only a modest effect on bending fluctuations. These results are in agreement with experiments probing DNA mechanics from single base pair to kilobase pair scales.

We present a synthetic nanoscale piston that uses chemical energy to perform molecular transport against an applied bias. Such a device comprises a 13 by 5 nm protein cylinder, embedded in a biological membrane enclosing a single-stranded DNA (ssDNA) rod. Hybridization with DNA cargo rigidifies the rod, allowing for transport of a selected DNA molecule across the nanopore. A strand displacement reaction from ssDNA fuel on the other side of the membrane then liberates the DNA cargo back into solution and regenerates the initial configuration. The entropic penalty of ssDNA confinement inside the nanopore drives DNA transport regardless of the applied bias. Multiple automated and reciprocating cycles are observed, in which the DNA piston moves through the 10 nm length of the nanopore. In every cycle, a single DNA molecule is transported across the nanopore against an external bias force, which is the hallmark of biological transporters.

Biomolecular conformational transitions are usually modeled as barrier crossings in a free energy landscape. The transition paths connect two local free energy minima and transition path times (TPT) are the actual durations of the crossing events. The simplest model employed to analyze TPT and to fit empirical data is that of a stochastic particle crossing a parabolic barrier. Motivated by some disagreement between the value of the barrier height obtained from the TPT distributions as compared to the value obtained from kinetic and thermodynamic analyses, we investigate here TPT for barriers which deviate from the symmetric parabolic shape. We introduce a continuous set of potentials, that starting from a parabolic shape, can be made increasingly asymmetric by tuning a single parameter. The TPT distributions obtained in the asymmetric case are very well-fitted by distributions generated by parabolic barriers. The fits, however, provide values for the barrier heights and diffusion coefficients which deviate from the original input values. We show how these findings can be understood from the analysis of the eigenvalues spectrum of the Fokker-Planck equation and highlight connections with experimental results.

DNA surface-hybridization biosensors utilize the selective hybridization of target sequences in solution to surface-immobilized probes. In this process, the target is usually assumed to be in excess, so that its concentration does not significantly vary while hybridizing to the surface-bound probes. If the target is initially at low concentrations and/or if the number of probes is very large, and they have high affinity for the target, the DNA in solution may become depleted. In this paper we analyze the equilibrium and kinetics of hybridization of DNA biosensors in the case of strong target depletion, by extending the Langmuir adsorption model. We focus, in particular, on the detection of a small amount of a single-nucleotide "mutant" sequence (concentration c 2 ) in a solution, which differs by one or more nucleotides from an abundant "wild-type" sequence (concentration c 1 ≫ c 2 ). We show that depletion can give rise to a strongly enhanced sensitivity of the biosensors. Using representative values of rate constants and hybridization free energies, we find that in the depletion regime one could detect relative concentrations c 2 / c 1 that are up to 3 orders of magnitude smaller than in the conventional approach. The kinetics is surprisingly rich and exhibits a nonmonotonic adsorption with no counterpart in the no-depletion case. Finally, we show that, alongside enhanced detection sensitivity, this approach offers the possibility of sample enrichment, by substantially increasing the relative amount of the mutant over the wild-type sequence.

By combining analytical results and simulations of various coarse-grained models, we investigate the minimal energy shape of DNA minicircles which are torsionally constrained by an imposed over or undertwist. We show that twist-bend coupling, a cross interaction term discussed in the recent DNA literature, induces minimal energy shapes with a periodic alternation of parts with high and low curvature resembling rounded polygons. We briefly discuss the possible experimental relevance of these findings. We finally show that the twist and bending energies of minicircles are governed by renormalized stiffness constants, rather than the bare ones. This has important consequences for the analysis of experiments involving circular DNA meant to determine DNA elastic constants.

The simplest model of DNA mechanics describes the double helix as a continuous rod with twist and bend elasticity. Recent work has discussed the relevance of a little-studied coupling G between twisting and bending, known to arise from the groove asymmetry of the DNA double helix. Here the effect of G on the statistical mechanics of long DNA molecules subject to applied forces and torques is investigated. We present a perturbative calculation of the effective torsional stiffness C_{eff} for small twist-bend coupling. We find that the "bare" G is "screened" by thermal fluctuations, in the sense that the low-force, long-molecule effective free energy is that of a model with G=0 but with long-wavelength bending and twisting rigidities that are shifted by G-dependent amounts. Using results for torsional and bending rigidities for freely fluctuating DNA, we show how our perturbative results can be extended to a nonperturbative regime. These results are in excellent agreement with numerical calculations for Monte Carlo "triad" and molecular dynamics "oxDNA" models, characterized by different degrees of coarse graining, validating the perturbative and nonperturbative analyses. While our theory is in generally good quantitative agreement with experiment, the predicted torsional stiffness does systematically deviate from experimental data, suggesting that there are as-yet-uncharacterized aspects of DNA twisting-stretching mechanics relevant to low-force, long-molecule mechanical response, which are not captured by widely used coarse-grained models.

We derive an analytical expression for the transition path time (TPT) distribution for a one-dimensional particle crossing a parabolic barrier. The solution is expressed in terms of the eigenfunctions and eigenvalues of the associated Fokker-Planck equation. The particle exhibits anomalous dynamics generated by a power-law memory kernel, which includes memoryless Markovian dynamics as a limiting case. Our result takes into account absorbing boundary conditions, extending existing results obtained for free boundaries. We show that TPT distributions obtained from numerical simulations are in excellent agreement with analytical results, while the typically employed free boundary conditions lead to a systematic overestimation of the barrier height. These findings may be useful in the analysis of experimental results on the transition path time. A web tool to perform this analysis is freely available.

Recent work indicates that twist-bend coupling plays an important role in DNA micromechanics. Here we investigate its effect on bent DNA. We provide an analytical solution of the minimum-energy shape of circular DNA, showing that twist-bend coupling induces sinusoidal twist waves. This solution is in excellent agreement with both coarse-grained simulations of minicircles and nucleosomal DNA data, which is bent and wrapped around histone proteins in a superhelical conformation. Our analysis shows that the observed twist oscillation in nucleosomal DNA, so far attributed to the interaction with the histone proteins, is an intrinsic feature of free bent DNA, and should be observable in other protein-DNA complexes.

The capture and translocation of biomolecules through nanometer-scale pores are processes with a potentially large number of applications, and hence they have been intensively studied in recent years. The aim of this paper is to review existing models of the capture process by a nanopore, together with some recent experimental data of short single- and double-stranded DNA captured by the Cytolysin A (ClyA) nanopore. ClyA is a transmembrane protein of bacterial origin which has been recently engineered through site-specific mutations, to allow the translocation of double- and single-stranded DNA. A comparison between theoretical estimations and experiments suggests that for both cases the capture is a reaction-limited process. This is corroborated by the observed salt dependence of the capture rate, which we find to be in quantitative agreement with the theoretical predictions.

It is well established that many physical properties of DNA at sufficiently long length scales can be understood by means of simple polymer models. One of the most widely used elasticity models for DNA is the twistable worm-like chain (TWLC), which describes the double helix as a continuous elastic rod with bending and torsional stiffness. An extension of the TWLC, which has recently received some attention, is the model by Marko and Siggia, who introduced an additional twist-bend coupling, expected to arise from the groove asymmetry. By performing computer simulations of two available versions of oxDNA, a coarse-grained model of nucleic acids, we investigate the microscopic origin of twist-bend coupling. We show that this interaction is negligible in the oxDNA version with symmetric grooves, while it appears in the oxDNA version with asymmetric grooves. Our analysis is based on the calculation of the covariance matrix of equilibrium deformations, from which the stiffness parameters are obtained. The estimated twist-bend coupling coefficient from oxDNA simulations is G=30±1 nm. The groove asymmetry induces a novel twist length scale and an associated renormalized twist stiffness κ t ≈80 nm, which is different from the intrinsic torsional stiffness C≈110 nm. This naturally explains the large variations on experimental estimates of the intrinsic stiffness performed in the past.

Recent magnetic tweezers experiments have reported systematic deviations of the twist response of double-stranded DNA from the predictions of the twistable wormlike chain model. Here we show, by means of analytical results and computer simulations, that these discrepancies can be resolved if a coupling between twist and bend is introduced. We obtain an estimate of 40±10  nm for the twist-bend coupling constant. Our simulations are in good agreement with high-resolution, magnetic-tweezers torque data. Although the existence of twist-bend coupling was predicted long ago [J. Marko and E. Siggia, Macromolecules 27, 981 (1994)MAMOBX0024-929710.1021/ma00082a015], its effects on the mechanical properties of DNA have been so far largely unexplored. We expect that this coupling plays an important role in several aspects of DNA statics and dynamics.

Many important processes in biology involve the translocation of a biopolymer through a nanometer-scale pore. Moreover, the electrophoretic transport of DNA across nanopores is under intense investigation for single-molecule DNA sequencing and analysis. Here, we show that the precise patterning of the ClyA biological nanopore with positive charges is crucial to observe the electrophoretic translocation of DNA at physiological ionic strength. Surprisingly, the strongly electronegative 3.3 nm internal constriction of the nanopore did not require modifications. Further, DNA translocation could only be observed from the wide entry of the nanopore. Our results suggest that the engineered positive charges are important to align the DNA in order to overcome the entropic and electrostatic barriers for DNA translocation through the narrow constriction. Finally, the dependencies of nucleic acid translocations on the Debye length of the solution are consistent with a physical model where the capture of double-stranded DNA is diffusion-limited while the capture of single-stranded DNA is reaction-limited.

Within a single infected individual, a virus population can have a high genomic variability. In the case of HIV, several mutations can be present even in a small genomic window of 20-30 nucleotides. For diagnostics purposes, it is often needed to resequence genomic subsets where crucial mutations are known to occur. In this article, we address this issue using DNA microarrays and inputs from hybridization thermodynamics. Hybridization signals from multiple probes are analysed, including strong signals from perfectly matching (PM) probes and a large amount of weaker cross-hybridization signals from mismatching (MM) probes. The latter are crucial in the data analysis. Seven coded clinical samples (HIV-1) are analyzed, and the microarray results are in full concordance with Sanger sequencing data. Moreover, the thermodynamic analysis of microarray signals resolves inherent ambiguities in Sanger data of mixed samples and provides additional clinically relevant information. These results show the reliability and added value of DNA microarrays for point-of-care diagnostic purposes.

Hybridization of nucleic acids on solid surfaces is a key process involved in high-throughput technologies such as microarrays and, in some cases, next-generation sequencing (NGS). A physical understanding of the hybridization process helps to determine the accuracy of these technologies. The goal of a widespread research program is to develop reliable transformations between the raw signals reported by the technologies and individual molecular concentrations from an ensemble of nucleic acids. This research has inputs from many areas, from bioinformatics and biostatistics, to theoretical and experimental biochemistry and biophysics, to computer simulations. A group of leading researchers met in Ploen Germany in 2011 to discuss present knowledge and limitations of our physico-chemical understanding of high-throughput nucleic acid technologies. This meeting inspired us to write this summary, which provides an overview of the state-of-the-art approaches based on physico-chemical foundation to modeling of the nucleic acids hybridization process on solid surfaces. In addition, practical application of current knowledge is emphasized.

Background: Several preprocessing methods are available for the analysis of Affymetrix Genechips arrays. The most popular algorithms analyze the measured fluorescence intensities with statistical methods. Here we focus on a novel algorithm, AffyILM, available from Bioconductor, which relies on inputs from hybridization thermodynamics and uses an extended Langmuir isotherm model to compute transcript concentrations. These concentrations are then employed in the statistical analysis. We compared the performance of AffyILM and other traditional methods both in the old and in the newest generation of GeneChips., Results: Tissue mixture and Latin Square datasets (provided by Affymetrix) were used to assess the performances of the differential expression analysis depending on the preprocessing strategy. A correlation analysis conducted on the tissue mixture data reveals that the median-polish algorithm allows to best summarize AffyILM concentrations computed at the probe-level. Those correlation results are equivalent to the best correlations observed using popular preprocessing methods relying on intensity values. The performances of each tested preprocessing algorithm were quantified using the Latin Square HG-U133A dataset, thanks to the comparison of differential analysis results with the list of spiked genes. The figures of merit generated illustrates that the performances associated to AffyILM(medianpolish), inferred from the present statistical analysis, are comparable to the best performing strategies previously reported., Conclusions: Converting probe intensities to estimates of target concentrations prior to the statistical analysis, AffyILM(medianpolish) is one of the best performing strategy currently available. Using hybridization theory, probe-level estimates of target concentrations should be identically distributed. In the future, a probe-level multivariate analysis of the concentrations should be compared to the univariate analysis of probe-set summarized expression data.

We consider mixtures of two DNA sequences t and t' differing by a single nucleotide, which are analyzed by an Agilent custom DNA microarray. In particular we focus on the case in which t, the "wild type", is predominantly abundant and t' the "mutant" is at very low concentrations compared to t. We show that by using appropriately designed arrays it is possible to accurately quantify the presence of t' even at low relative concentrations (≈1%). The detection method is based on thermodynamic models of DNA hybridisation and on the analysis of a large number of hybridisation intensities from probes containing one or two mismatches with respect to t and t'., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background: One important preprocessing step in the analysis of microarray data is background subtraction. In high-density oligonucleotide arrays this is recognized as a crucial step for the global performance of the data analysis from raw intensities to expression values., Results: We propose here an algorithm for background estimation based on a model in which the cost function is quadratic in a set of fitting parameters such that minimization can be performed through linear algebra. The model incorporates two effects: 1) Correlated intensities between neighboring features in the chip and 2) sequence-dependent affinities for non-specific hybridization fitted by an extended nearest-neighbor model., Conclusion: The algorithm has been tested on 360 GeneChips from publicly available data of recent expression experiments. The algorithm is fast and accurate. Strong correlations between the fitted values for different experiments as well as between the free-energy parameters and their counterparts in aqueous solution indicate that the model captures a significant part of the underlying physical chemistry.

Background: An algorithm for the analysis of Affymetrix Genechips is presented. This algorithm, referred to as the Inverse Langmuir Method (ILM), estimates the binding of transcripts to complementary probes using DNA/RNA hybridization free energies, and the hybridization between partially complementary transcripts in solution using RNA/RNA free energies. The balance between these two competing reactions allows for the translation of background-subtracted intensities into transcript concentrations., Results: To validate the ILM, it is applied to publicly available microarray data from a multi-lab comparison study. Here, microarray experiments are performed on samples which deviate only in few genes. The log2 fold change between these two samples, as obtained from RT-PCR experiments, agrees well with the log2 fold change as obtained with the ILM, indicating that the ILM determines changes in the expression level accurately. We also show that the ILM allows for the identification of outlying probes, as it yields independent concentration estimates per probe., Conclusion: The ILM is robust and offers an interesting alternative to purely statistical algorithms for microarray data analysis.

Background: Affymetrix Genechips are characterized by probe pairs, a perfect match (PM) and a mismatch (MM) probe differing by a single nucleotide. Most of the data preprocessing algorithms neglect MM signals, as it was shown that MMs cannot be used as estimators of the non-specific hybridization as originally proposed by Affymetrix. The aim of this paper is to study in detail on a large number of experiments the behavior of the average PM/MM ratio. This is taken as an indicator of the quality of the hybridization and, when compared between different chip series, of the quality of the chip design., Results: About 250 different GeneChip hybridizations performed at the VIB Microarray Facility for Homo sapiens, Drosophila melanogaster, and Arabidopsis thaliana were analyzed. The investigation of such a large set of data from the same source minimizes systematic experimental variations that may arise from differences in protocols or from different laboratories. The PM/MM ratios are derived theoretically from thermodynamic laws and a link is made with the sequence of PM and MM probe, more specifically with their central nucleotide triplets., Conclusion: The PM/MM ratios subdivided according to the different central nucleotides triplets follow qualitatively those deduced from the hybridization free energies in solution. It is shown also that the PM and MM histograms are related by a simple scale transformation, in agreement with what is to be expected from hybridization thermodynamics. Different quantitative behavior is observed on the different chip organisms analyzed, suggesting that some organism chips have superior probe design compared to others.

While designing oligonucleotide-based microarrays, cross-hybridization between surface-bound oligos and non-intended labeled targets is probably the most difficult parameter to predict. Although literature describes rules-of-thumb concerning oligo length, overall similarity, and continuous stretches, the final behavior is difficult to predict. The aim of this study was to investigate the effect of well-defined mismatches on hybridization specificity using CodeLink Activated Slides and to study quantitatively the relation between hybridization intensity and Gibbs free energy (DeltaG), taking the mismatches into account. Our data clearly showed a correlation between the hybridization intensity and DeltaG of the oligos over 3 orders of magnitude for the hybridization intensity, which could be described by the Langmuir model. As DeltaG was calculated according to the nearest-neighbor model, using values related to DNA hybridizations in solution, this study clearly shows that target-probe hybridizations on microarrays with a three-dimensional coating are in quantitative agreement with the corresponding reaction in solution. These results can be interesting for some practical applications. The correlation between intensity and DeltaG can be used in quality control of microarray hybridizations by designing probes and corresponding RNA spikes with a range of DeltaG values. Furthermore, this correlation might be of use to fine-tune oligonucleotide design algorithms in a way to improve the prediction of the influence of mismatching targets on microarray hybridizations.

The genes of eukaryotes are characterized by protein coding fragments, the exons, interrupted by introns, i.e., stretches of DNA which do not carry useful information for protein synthesis. We have analyzed the melting behavior of randomly selected human cDNA sequences obtained from genomic DNA by removing all introns. A clear correspondence is observed between exons and melting domains. This finding may provide new insights into the physical mechanisms underlying the evolution of genes.

We analyze the unbinding transition for a two-dimensional lattice polymer in which the constituent strands are mutually avoiding random walks. At low temperatures the strands are bound and form a single self-avoiding walk. We show that unbinding in this model is a strong first order transition. The entropic exponents associated with denaturated loops and end-segment distributions show sharp differences at the transition point and in the high temperature phase. Their values can be deduced from some exact arguments relying on a conformal mapping of copolymer networks into a fluctuating geometry, i.e., in the presence of quantum gravity. An excellent agreement between analytical and numerical estimates is observed for all cases analyzed.

Recent advances in the understanding of the melting behavior of double-stranded DNA with statistical mechanics methods lead to improved estimates of the weight factors for the dissociation events of the chains, in particular for interior loop melting. So far, in the modeling of DNA melting, the entropy of denaturated loops has been estimated from the number of configurations of a closed self-avoiding walk. It is well understood now that a loop embedded in a chain is characterized by a loop closure exponent c which is higher than that of an isolated loop. Here we report an analysis of DNA melting curves for sequences of a broad range of lengths (from 10 to 10(6) base pairs) calculated with a program based on the algorithms underlying MELTSIM. Using the embedded loop exponent we find that the cooperativity parameter is one order of magnitude bigger than current estimates. We argue that in the melting region the double helix persistence length is greatly reduced compared to its room temperature value, so that the use of the embedded loop closure exponent for real DNA sequences is justified.

For a model of DNA denaturation, exponents describing the distributions of denaturated loops and unzipped end segments are determined by exact enumeration and by Monte Carlo simulations in two and three dimensions. The loop distributions are consistent with first-order thermal denaturation in both cases. Results for end segments show a coexistence of two distinct power laws in the relative distributions, which is not foreseen by a recent approach in which DNA is treated as a homogeneous network of linear polymer segments. This unexpected feature, and the discrepancies with such an approach, are explained in terms of a refined scaling picture in which a precise distinction is made between network branches representing single-stranded and effective double-stranded segments.

The nature and the universal properties of DNA thermal denaturation are investigated by Monte Carlo simulations. For suitable lattice models we determine the exponent c describing the decay of the probability distribution of denaturated loops of length l, P approximately l(-c). If excluded volume effects are fully taken into account, c = 2.10(4) is consistent with a first order transition. The stiffness of the double stranded chain has the effect of sharpening the transition, if it is continuous, but not of changing its order and the value of the exponent c, which is also robust with respect to inclusion of specific base-pair sequence heterogeneities.