Sequence-dependent Variations Research Articles

Data-driven prediction of geometry- and toolpath sequence-dependent intra-layer process conditions variations in laser powder bed fusion

Geometrical features and toolpath sequence are two important factors that cause process condition variations, such as variations in the meltpool temperature or meltpool size, that might lead to undesired material properties in the laser powder bed fusion (LPBF) process. Due to the high dynamics and complex physics of the LPBF process, it is difficult to predict variations in process conditions with simulations alone. Advances in measurement technology and computational technologies open up new possibilities for smart manufacturing. In this paper, a data-driven method to predict intra-layer variations in the processing conditions that source from the toolpath sequence and part geometry is presented. The approach is demonstrated using two-color on-axis pyrometer measurements. Three demonstration cases are presented in which it is demonstrated (1) how the trained predictive model can be used as a filter to ease the interpretation of process variations and discover patterns related to toolpath and part geometry, and (2) how to generate predictions that can be used for feedforward control, i.e., for adjusting laser power or scanning speed along the toolpath using a meltpool temperature prediction model generated based on on-axis measurements. Results show that the developed prediction model is able to meaningfully predict process variations resulted from toolpath sequence and geometry. Predictions are aligned with the results from the related work of others and for the case of 180° laser path turnarounds in our high-speed X-ray imaging experiments. The potential issues related to the current maturity status of the process and measuring equipment that could in practice affect the performance of the proposed solutions are also discussed.

Loop-seq: A high-throughput technique to measure the mesoscale mechanical properties of DNA.

The local mechanical properties of the DNA polymer influence molecular processes in biology that require mechanical deformations of DNA. Lack of suitable high-throughput experimental techniques had precluded measuring how these properties might vary with sequence along the vast lengths of genomes. Here, we present a detailed protocol for a recently developed experimental technique called loop-seq, which measures at least one local mechanical property of DNA-its propensity to cyclize-in genome-scale throughput. Loop-seq has been used to obtain experimentally derived genome-wide maps of a physical property of DNA. Such measurements have revealed that diverse DNA-deforming processes involved in chromatin organization at various genomic loci are regulated by the genetically encoded, sequence-dependent variations in the mechanical properties of DNA.

Understanding how natural sequence variation in serum albumin proteins affects conformational stability and protein adsorption

Serum albumins are evolutionary conserved proteins that are found in many animal species, and purified forms are widely used in biotechnology applications, such as components within surface passivation coatings and drug delivery systems. As such, there has long been interest in studying how serum albumins adsorb onto solid supports, although existing studies are limited to one or two species. Herein, we comprehensively investigated three serum albumins of bovine (BSA), human (HSA), and rat (RSA) origin, and discovered striking differences in their conformational stabilities and adsorption properties. Together with bioinformatics analysis, dynamic light scattering (DLS) and circular dichroism (CD) spectroscopy measurements revealed that the proteins form different types of macromolecular assemblies in solution. BSA and HSA existed as individual monomers while RSA formed multimers, and each protein exhibited sequence-dependent variations in conformational stability as well. Quartz crystal microbalance-dissipation (QCM-D) and localized surface plasmon resonance (LSPR) experiments further showed that BSA and HSA proteins adsorb to form well-packed adlayers, and the extent of protein uptake and spreading depended on their unique conformational stabilities. Conversely, RSA adsorption resulted in sparse adlayers and appreciably less spreading of the adsorbed multimers, as confirmed by attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy experiments. Together, our findings demonstrate that significant differences in conformational stability and adsorption behavior exist even between evolutionary conserved serum albumins with high sequence and structural similarity and illustrate how rational engineering of protein structures and stabilities, guided by insights from nature, might be useful to design protein-based coatings for various biointerfacial science applications.

Sequence-Dependent Deviations of Constrained DNA from Canonical B-Form.

Decades of crystallographic and NMR studies have produced canonical structural models of short DNA. However, no experimental method so far has been able to test these models in vivo, where DNA is long and constrained by interactions with membranes, proteins, and other molecules. Here, we employ high-resolution frequency-modulation AFM to image single long poly(dA)-poly(dT), poly(dG)-poly(dC), and lambda DNA molecules interacting with an underlying substrate that emulates the effect of biological constraints on molecular structure. We find systematic sequence-dependent variations in groove dimensions, indicating that the structure of DNA subject to realistic interactions may differ profoundly from canonical models. These findings highlight the value of AFM as a unique, single molecule characterization tool.

Passing faces: sequence-dependent variations in the perceptual processing of emotional faces

ABSTRACTThere is broad evidence that contextual factors influence the processing of emotional facial expressions. Yet temporal-dynamic aspects, inter alia how face processing is influenced by the specific order of neutral and emotional facial expressions, have been largely neglected. To shed light on this topic, we recorded electroencephalogram from 168 healthy participants while they performed a gender-discrimination task with angry and neutral faces. Our event-related potential (ERP) analyses revealed a strong emotional modulation of the N170 component, indicating that the basic visual encoding and emotional analysis of a facial stimulus happen, at least partially, in parallel. While the N170 and the late positive potential (LPP; 400–600 ms) were only modestly affected by the sequence of preceding faces, we observed a strong influence of face sequences on the early posterior negativity (EPN; 200–300 ms). Finally, the differing response patterns of the EPN and LPP indicate that these two ERPs represent distinct processes during face analysis: while the former seems to represent the integration of contextual information in the perception of a current face, the latter appears to represent the net emotional interpretation of a current face.

Discrimination among protein variants using an unfoldase-coupled nanopore.

Previously we showed that the protein unfoldase ClpX could facilitate translocation of individual proteins through the α-hemolysin nanopore. This results in ionic current fluctuations that correlate with unfolding and passage of intact protein strands through the pore lumen. It is plausible that this technology could be used to identify protein domains and structural modifications at the single-molecule level that arise from subtle changes in primary amino acid sequence (e.g., point mutations). As a test, we engineered proteins bearing well-characterized domains connected in series along an ∼700 amino acid strand. Point mutations in a titin immunoglobulin domain (titin I27) and point mutations, proteolytic cleavage, and rearrangement of beta-strands in green fluorescent protein (GFP), caused ionic current pattern changes for single strands predicted by bulk phase and force spectroscopy experiments. Among these variants, individual proteins could be classified at 86-99% accuracy using standard machine learning tools. We conclude that a ClpXP-nanopore device can discriminate among distinct protein domains, and that sequence-dependent variations within those domains are detectable.

All-atom crystal simulations of DNA and RNA duplexes

BackgroundMolecular dynamics simulations can complement experimental measures of structure and dynamics of biomolecules. The quality of such simulations can be tested by comparisons to models refined against experimental crystallographic data. MethodsWe report simulations of DNA and RNA duplexes in their crystalline environment. The calculations mimic the conditions for PDB entries 1D23 [d(CGATCGATCG)2] and 1RNA [(UUAUAUAUAUAUAA)2], and contain 8 unit cells, each with 4 copies of the Watson–Crick duplex; this yields in aggregate 64μs of duplex sampling for DNA and 16μs for RNA. ResultsThe duplex structures conform much more closely to the average structure seen in the crystal than do structures extracted from a solution simulation with the same force field. Sequence-dependent variations in helical parameters, and in groove widths, are largely maintained in the crystal structure, but are smoothed out in solution. However, the integrity of the crystal lattice is slowly degraded in both simulations, with the result that the interfaces between chains become heterogeneous. This problem is more severe for the DNA crystal, which has fewer inter-chain hydrogen bond contacts than does the RNA crystal. ConclusionsCrystal simulations using current force fields reproduce many features of observed crystal structures, but suffer from a gradual degradation of the integrity of the crystal lattice. General significanceThe results offer insights into force-field simulations that test their ability to preserve weak interactions between chains, which will be of importance also in non-crystalline applications that involve binding and recognition.This article is part of a Special Issue entitled Recent developments of molecular dynamics.

Sequence dependent variations in RNA duplex are related to non-canonical hydrogen bond interactions in dinucleotide steps

BackgroundSequence determines the three-dimensional structure of RNAs, and thereby plays an important role in carrying out various biological functions. RNA duplexes containing Watson-Crick (WC) basepairs, interspersed with non-Watson-Crick basepairs, are the dominant structural unit and form the scaffold for the 3-dimensional structure of RNA. It is therefore crucial to understand the geometric variation in the dinucleotide steps that form the helices. We have carried out a detailed analysis of the dinucleotide steps formed by AU and GC Watson-Crick basepairs in RNA structures (both free and protein bound) and compared the results to that seen in DNA. Further, the effect of protein binding on these steps was examined by comparing steps in free RNA structures with protein bound RNA structures.ResultsCharacteristic sequence dependent geometries are observed for the RR, RY and YR type of dinucleotide steps in RNA. Their geometric parameters show correlated variations that are different from those observed in B-DNA helices. Subtle, but statistically significant differences are seen in roll, slide and average propeller-twist values, between the dinucleotide steps of free RNA and protein bound RNA structures. Many non-canonical cross-strand and intra-strand hydrogen bonds were identified that can stabilise the RNA dinucleotide steps, among which YR steps show presence of many new unreported interactions.ConclusionsOur work provides for the first time a detailed analysis of the conformational preferences exhibited by Watson-Crick basepair containing steps in RNA double helices. Overall, the WC dinucleotide steps show considerable conformational variability. Furthermore, we have identified hydrogen bond interactions in several of the dinucleotide steps that could play a role in determining the preferred geometry, in addition to the intra-basepair hydrogen bonds and stacking interactions. Protein binding affects the conformation of the steps that are in direct contact, as well as allosterically affect the steps that are not in direct physical contact.

Can We Accurately Describe the Structure of Adenine Tracts in B-DNA? Reference Quantum-Chemical Computations Reveal Overstabilization of Stacking by Molecular Mechanics

Sequence-dependent local variations of helical parameters, structure, and flexibility are crucial for molecular recognition processes involving B-DNA. A-tracts, i.e., stretches of several consecutive adenines in one strand that are in phase with the DNA helical repeat, mediate significant DNA bending. During the past few decades, there have been intense efforts to understand the sequence dependence of helical parameters in DNA. Molecular dynamics (MD) simulations can provide valuable insights into the molecular mechanism behind the relationship between sequence and structure. However, although recent improvements in empirical force fields have helped to capture many sequence-dependent B-DNA properties, several problems remain, such as underestimation of the helical twist and suspected underestimation of the propeller twist in A-tracts. Here, we employ reference quantum mechanical (QM) calculations, explicit solvent MD, and bioinformatics to analyze the underestimation of propeller twisting of A-tracts in simulations. Although we did not identify a straightforward explanation, we discovered two imbalances in the empirical force fields. The first was overestimation of stacking interactions accompanied by underestimation of base-pairing energy, which we attribute to anisotropic polarizabilities that are not reflected by the isotropic force fields. This may lead to overstacking with potentially important consequences for MD simulations of nucleic acids. The second observed imbalance was steric clash between A(N1) and T(N3) nitrogens of AT base pairs in force-field descriptions, resulting in overestimation of the AT pair stretch in MD simulations. We also substantially extend the available set of benchmark estimated CCSD(T)/CBS data for B-DNA base stacking and provide a code that allows the generation of diverse base-stacking geometries suitable for QM computations with predefined intra- and interbase pair parameters.

Mapping the Transition State for DNA Bending by IHF

How DNA-bending proteins recognize their specific sites on DNA remains elusive, particularly for proteins that use indirect readout, which relies on sequence-dependent variations in DNA flexibility/bendability. The question remains as to whether the protein bends the DNA (protein-induced bending) or, alternatively, “prebent” DNA conformations are thermally accessible, which the protein captures to form the specific complex (conformational capture). To distinguish between these mechanisms requires characterization of reaction intermediates and, in particular, snapshots of the transition state along the recognition pathway. We present such a snapshot, from measurements of DNA bending dynamics in complex with Escherichia coli integration host factor (IHF), an architectural protein that bends specific sites on λ-DNA in a U-turn by creating two sharp kinks in DNA. Fluorescence resonance energy transfer measurements in response to laser temperature-jump perturbation monitor DNA bending. We find that nicks or mismatches that enhance DNA flexibility at the site of the kinks show 3- to 4-fold increase in DNA bending rates that reflect a 4- to 11-fold increase in binding affinities, while sequence modifications away from the kink sites, as well as mutations in IHF designed to destabilize the complex, have negligible effect on DNA bending rates despite >250-fold decrease in binding affinities. These results support the scenario that the bottleneck in the recognition step for IHF is spontaneous kinking of cognate DNA to adopt a partially prebent conformation and point to conformational capture as the underlying mechanism of initial recognition, with additional protein-induced bending occurring after the transition state.

Linked Control of Syllable Sequence and Phonology in Birdsong

The control of sequenced behaviors, including human speech, requires that the brain coordinate the production of discrete motor elements with their concatenation into complex patterns. In birdsong, another sequential vocal behavior, the acoustic structure (phonology) of individual song elements, or "syllables," must be coordinated with the sequencing of syllables into a song. However, it is unknown whether syllable phonology is independent of the sequence in which a syllable is produced. We quantified interactions between phonology and sequence in Bengalese finch song by examining both convergent syllables, which can be preceded by at least two different syllables and divergent syllables, which can be followed by at least two different syllables. Phonology differed significantly based on the identity of the preceding syllable for 97% of convergent syllables and differed significantly with the identity of the upcoming syllable for 92% of divergent syllables. Furthermore, sequence-dependent phonological differences extended at least two syllables away from the convergent or divergent syllable. To determine whether these phenomena reflect differences in central control, we analyzed premotor neural activity in the robust nucleus of the arcopallium (RA). Activity associated with a syllable varied significantly depending on the sequence in which the syllable was produced, suggesting that sequence-dependent variations in premotor activity contribute to sequence-dependent differences in phonology. Moreover, these data indicate that RA activity could contribute to the sequencing of syllables. Together, these results suggest that, rather than being controlled independently, the sequence and phonology of birdsong are intimately related, as is the case for human speech.

Assembly Mechanisms of RNA Pseudoknots are Determined by the Stabilities of the Constituent Secondary Structures

Understanding how RNA molecules navigate their rugged folding landscapes holds the key to describing their roles in a variety of cellular functions. To dissect RNA folding at the molecular level, we performed simulations of three pseudoknots (MMTV and SRV-1 from viral genomes and the hTR pseudoknot from human telomerase) using coarse-grained models. The melting temperatures from the specific heat profiles are in good agreement with the available experimental data for MMTV and hTR. The equilibrium free energy profiles, which predict the structural transitions that occur at each melting temperature, are used to propose that the relative stabilities of the isolated helices control their folding mechanisms. Kinetic simulations, which corroborate the inferences drawn from the free energy profiles, show that MMTV folds by a hierarchical mechanism with parallel paths i.e., formation of one of the helices nucleates the assembly of the rest of the structure. The SRV-1 pseudoknot folds in a highly cooperative manner and assembles in a single step in which the preformed helices coalesce nearly simultaneously to form the tertiary structure. Folding occurs by multiple pathways in the hTR pseudoknot, whose isolated structural elements have similar stabilities. In one of the paths, tertiary interactions are established prior to the formation of the secondary structures. Our work shows that there are significant sequence-dependent variations in the folding landscapes of RNA molecules with similar fold. We also establish that assembly mechanisms can be predicted using the stabilities of the isolated secondary structures.

Assembly mechanisms of RNA pseudoknots are determined by the stabilities of constituent secondary structures

Understanding how RNA molecules navigate their rugged folding landscapes holds the key to describing their roles in a variety of cellular functions. To dissect RNA folding at the molecular level, we performed simulations of three pseudoknots (MMTV and SRV-1 from viral genomes and the hTR pseudoknot from human telomerase) using coarse-grained models. The melting temperatures from the specific heat profiles are in good agreement with the available experimental data for MMTV and hTR. The equilibrium free energy profiles, which predict the structural transitions that occur at each melting temperature, are used to propose that the relative stabilities of the isolated helices control their folding mechanisms. Kinetic simulations, which corroborate the inferences drawn from the free energy profiles, show that MMTV folds by a hierarchical mechanism with parallel paths, i.e., formation of one of the helices nucleates the assembly of the rest of the structure. The SRV-1 pseudoknot, which folds in a highly cooperative manner, assembles in a single step in which the preformed helices coalesce nearly simultaneously to form the tertiary structure. Folding occurs by multiple pathways in the hTR pseudoknot, the isolated structural elements of which have similar stabilities. In one of the paths, tertiary interactions are established before the formation of the secondary structures. Our work shows that there are significant sequence-dependent variations in the folding landscapes of RNA molecules with similar fold. We also establish that assembly mechanisms can be predicted using the stabilities of the isolated secondary structures.

Dynamics of locking of peptides onto growing amyloid fibrils

Sequence-dependent variations in the growth mechanism and stability of amyloid fibrils, which are implicated in a number of neurodegenerative diseases, are poorly understood. We have carried out extensive all-atom molecular dynamics simulations to monitor the structural changes that occur upon addition of random coil (RC) monomer fragments from the yeast prion Sup35 and Abeta-peptide onto a preformed fibril. Using the atomic resolution structures of the microcrystals as the starting points, we show that the RC --> beta-strand transition for the Sup35 fragment occurs abruptly over a very narrow time interval, whereas the acquisition of strand content is less dramatic for the hydrophobic-rich Abeta-peptide. Expulsion of water, resulting in the formation of a dry interface between 2 adjacent sheets of the Sup35 fibril, occurs in 2 stages. Ejection of a small number of discrete water molecules in the second stage follows a rapid decrease in the number of water molecules in the first stage. Stability of the Sup35 fibril is increased by a network of hydrogen bonds involving both backbone and side chains, whereas the marginal stability of the Abeta-fibrils is largely due to the formation of weak dispersion interaction between the hydrophobic side chains. The importance of the network of hydrogen bonds is further illustrated by mutational studies, which show that substitution of the Asn and Gln residues to Ala compromises the Sup35 fibril stability. Despite the similarity in the architecture of the amyloid fibrils, the growth mechanism and stability of the fibrils depend dramatically on the sequence.

On the parameterization of rigid base and basepair models of DNA from molecular dynamics simulations

A method is described to extract a complete set of sequence-dependent material parameters for rigid base and basepair models of DNA in solution from atomistic molecular dynamics simulations. The method is properly consistent with equilibrium statistical mechanics, leads to effective shape, stiffness and mass parameters, and employs special procedures for treating spontaneous torsion angle flips and H-bond breaks, both of which can have a significant effect on the results. The method is accompanied by various analytical consistency checks that can be used to assess the equilibration of statistical averages, and different modeling assumptions pertaining to the rigidity of the bases and basepairs and the locality of the quadratic internal energy. The practicability of the approach is verified by estimating complete parameter sets for the 16-basepair palindromic oligomer G(TA)(7)C simulated in explicit water and counterions. Our results indicate that the method is capable of resolving sequence-dependent variations in each of the material parameters. Moreover, they show that the assumptions of rigidity and locality hold rather well for the base model, but not for the basepair model. For the latter, it is shown that the non-local nature of the internal energy can be understood in terms of a certain compatibility relation involving Schur complements.

Sequence-Dependent Variations Associated with H2A/H2B Depletion of Nucleosomes

Mechanisms that can alter nucleosome structure to enhance DNA accessibility are of great interest because of their potential involvement in genomic processes. One such mechanism is H2A/H2B release from nucleosomes; it occurs in vivo and is involved in the in vitro activities of several transcription-associated complexes. Using fluorescence approaches based on Förster resonance energy transfer, we previously detected sequence-dependent structure/stability variations between 5S and two types of promoter nucleosomes (from yeast GAL10 or mouse mammary tumor virus promoters). Those variations included differing responses when nucleosomes were diluted to concentrations (sub-nM) known to produce H2A/H2B loss. Here, we show that treatment of these same three types of nucleosomes with the histone chaperone yNAP-1, which causes H2A/H2B release from nucleosomes in vitro, produces the same differential Förster resonance energy transfer responses, again demonstrating sequence-dependent variations associated with conditions that produce H2A/H2B loss. Single-molecule population data indicate that DNA dynamics on the particles produced by diluting nucleosomes to sub-nM concentrations follow two-state behavior. Rate information (determined by fluorescence correlation spectroscopy) suggests that these dynamics are enhanced in MMTV-B or GAL10 compared to 5S particles. Taken together, the results indicate that H2A/H2B loss has differing effects on 5S compared to these two promoter nucleosomes and the differences reflect sequence-dependent structure/stability variations in the depleted particles.

Sequence‐dependent Interactions of Cationic Naphthalimides and Polynucleotides

The binding interactions of three naphthalimide derivatives with heteropoly nucleic acids have been evaluated using fluorescence, absorption and circular dichroism spectroscopies. Mono- and bifunctionalized naphthalimides exhibit sequence-dependent variations in their affinity toward DNA. The heteropoly nucleic acids, [Poly(dA-dT)]2 and [Poly(dG-dC)]2, as well as calf thymus (CT) DNA, were used to understand the factors that govern binding strength and selectivity. Sequence selectivity was addressed by determining the binding constants as a function of polynucleotide composition according to the noncooperative McGhee-von Hippel binding model. Binding affinities toward [poly(dA-dT)](2) were the largest for spermine-substituted naphthalimides (Kb = 2-6 x 10(6) M(-1)). The association constants for complex formation between the cationic naphthalimides and [poly(dG-dC)]2 or CT DNA (58% A-T content) were 2-500 times smaller, depending on the naphthalimide-polynucleotide pair. The binding modes were also assessed using a combination of induced circular dichroism and salt effects to determine whether the naphthalimides associate with DNA through intercalative, electrostatic or groove-binding. The results show that the monofunctionalized spermine and pyridinium-substituted naphthalimides associate with DNA through electrostatic interactions. In contrast, intercalative interactions are predominant in the complex formed between the bifunctionalized spermine compound and all of the polynucleotides.

Sequence-Dependent Nucleosome Structure and Stability Variations Detected by Förster Resonance Energy Transfer

Nucleosomes, the basic unit of eukaryotic chromosome structure, cover most of the DNA in eukaryotes, including regulatory sequences. Here, a recently developed Förster resonance energy transfer approach is used to compare structure and stability features of sea urchin 5S nucleosomes and nucleosomes reconstituted on two promoter sequences that are nucleosomal in vivo, containing the yeast GAL10 TATA or the major transcription response elements from the mouse mammary tumor virus promoter. All three sequences form mononucleosomes with similar gel mobilities and similar stabilities at moderate salt concentrations. However, the two promoter nucleosomes differ from 5S nucleosomes in (1) diffusion coefficient values, which suggest differences in nucleosome compaction, (2) intrinsic FRET efficiencies (in solution or in gels), and (3) the response of FRET efficiency to high (>or=600 mM) NaCl concentrations, subnanomolar nucleosome concentrations, and elevated temperatures (to 42 degrees C). These results indicate that nucleosome features can vary depending on the DNA sequence they contain and show that this fluorescence approach is sufficiently sensitive to detect such differences. Sequence-dependent variations in nucleosome structure or stability could facilitate specific nucleosome recognition, working together with other known genomic regulatory mechanisms. The variations in salt-, concentration-, and temperature-dependent responses all occur under conditions that have been shown previously to produce release of H2A-H2B dimers or terminal DNA from nucleosomes and could thus involve differences in those processes, as well as in other features.

Base triplet nonisomorphism strongly influences DNA triplex conformation: Effect of nonisomorphic G∗︁ GC and A∗︁ AT triplets and bending of DNA triplexes

Structural understanding of DNA triplexes is grossly inadequate despite their efficacy as therapeutic agents. Lack of structural similarity (isomorphism) of base triplets that figure in different DNA triplexes brings in an added complexity. Recently, we have shown that the residual twist (Deltat degrees ) and the radial difference (Deltar A) adequately define base triplet nonisomorphism in structural terms and allow assessment of their role in conferring stability as well as sequence-dependent structural variations in DNA triplexes. To further corroborate these, molecular dynamics (MD) simulations are carried out on DNA triplexes comprising nonisomorphic G* GC and A* AT base triplets under different sequential contexts. Base triplet nonisomorphism between G* GC and A* AT triplets is dominated by Deltat degrees (9.8 degrees ), in view of small Deltar (0.2 A), and is in contrast to G* GC and T* AT triplets where both Deltat degrees (10.6 degrees ) and Deltar (1.1A) are prominent. Results show that Deltat degrees alone enforces mechanistic influence on the triplex-forming purine strand so as to favor a zigzag conformation with alternating conformational features that include high (40 degrees ) and low (20 degrees ) helical twists, and high anti(G) and anti(A) glycosyl conformation. Higher thermal stability of this triplex compared to that formed with G* GC and T* AT triplets can be traced to enhanced base-stacking and counterion interactions. Surprisingly, it is found for the first time that the presence of a nonisomorphic G* GC or A* AT base triplet interrupting an otherwise mini A* AT or G* GC isomorphic triplex can induce a bend/curvature in a DNA triplex. These observations should prove useful in the design of triplex-forming oligonucleotides and in the understanding the binding affinities of this triplex with proteins.

Interfacial Water as a “Hydration Fingerprint” in the Noncognate Complex of BamHI

The molecular code of specific DNA recognition by proteins as a paradigm in molecular biology remains an unsolved puzzle primarily because of the subtle interplay between direct protein-DNA interaction and the indirect contribution from water and ions. Transformation of the nonspecific, low affinity complex to a specific, high affinity complex is accompanied by the release of interfacial water molecules. To provide insight into the conversion from the loose to the tight form, we characterized the structure and energetics of water at the protein-DNA interface of the BamHI complex with a noncognate sequence and in the specific complex. The fully hydrated models were produced with Grand Canonical Monte Carlo simulations. Proximity analysis shows that water distributions exhibit sequence dependent variations in both complexes and, in particular, in the noncognate complex they discriminate between the correct and the star site. Variations in water distributions control the number of water molecules released from a given sequence upon transformation from the loose to the tight complex as well as the local entropy contribution to the binding free energy. We propose that interfacial waters can serve as a “hydration fingerprint” of a given DNA sequence.