Base Pair Step Parameters Research Articles

Computational Investigation of Bending Properties of RNA AUUCU, CCUG, CAG, and CUG Repeat Expansions Associated With Neuromuscular Disorders.

Expansions of RNA AUUCU, CCUG, CAG, and CUG repeats cause spinocerebellar ataxia type 10, myotonic dystrophy type 2, Huntington’s disease, and myotonic dystrophy type 1, respectively. By performing extensive molecular dynamic simulations, we investigated the bending propensities and conformational landscapes adopted by 3×3, 2×2, and 1×1 internal loops observed in RNA AUUCU, CCUG, CAG, and CUG repeat expansions using model systems having biologically relevant repeat sizes. We show that the conformational variability experienced by these loops is more complex than previous reports where a variety of unconventional hydrogen bonds are formed. At the global scale, strong bending propensity was observed in r(AUUCU)10, r(CCUG)15, r(CAG)20, and r(CUG)20, and, to a lesser extent, in r(AUUCU)4, r(CCUG)10, r(CAG)10, and r(CUG)10. Furthermore, RNA CAG repeats exhibit a tendency toward bent states with more than 50% of observed conformations having bending angles greater than 50°, while RNA CUG repeats display relatively linear-like conformations with extremely bent conformations accounting for less than 25% of the observed structures. Conformations experienced by RNA AUUCU repeats are a combination of strongly bent and kinked structures. The bent states in RNA CCUG repeats mostly fall into the moderately bent category with a marginal ensemble experiencing extreme bending. The general pattern observed in all the bent structures indicates the collapse of the major groove width as the mechanical trigger for bending, which is caused by alteration of base pair step parameters at multiple locations along the RNA due to local distortions at the loop sites. Overextension is also observed in all the RNA repeats that is attributed to widening of the major groove width as well as undertwisting phenomenon. This information and the rich structural repository could be applied for structure based small molecule design targeting disease-causing RNAs. The bending propensities of these constructs, at the global level, could also have implications on how expanded RNA repeats interact with proteins.

Genome Dashboards: Framework and Examples

Genomics is a sequence-based informatics science and a three-dimensional-structure-based material science. However, in practice, most genomics researchers utilize sequence-based informatics approaches or three-dimensional-structure-based material science techniques, not both. This division is, at least in part, the result of historical developments rather than a fundamental necessity. The underlying computational tools, experimental techniques, and theoretical models were developed independently. The primary result presented here is a framework for the unification of informatics- and physics-based data associated with DNA, nucleosomes, and chromatin. The framework is based on the mathematical representation of geometrically exact rods and the generalization of DNA basepair step parameters. Data unification enables researchers to integrate computational, experimental, and theoretical approaches for the study of chromatin biology. The framework can be implemented using model-view-controller design principles, existing genome browsers, and existing molecular visualization tools. We developed a minimal, web-based genome dashboard, G-Dash-min, and applied it to two simple examples to demonstrate the usefulness of data unification and proof of concept. Genome dashboards developed using the framework and design principles presented here are extensible and customizable and are therefore more broadly applicable than the examples presented. We expect a number of purpose-specific genome dashboards to emerge as a novel means of investigating structure-function relationships for genomes that range from basepairs to entire chromosomes and for generating, validating, and testing mechanistic hypotheses.

Role of Inosine⁻Uracil Base Pairs in the Canonical RNA Duplexes.

Adenosine to inosine (A–I) editing is the most common modification of double-stranded RNA (dsRNA). This change is mediated by adenosine deaminases acting on RNA (ADARs) enzymes with a preference of U>A>C>G for 5′ neighbor and G>C=A>U or G>C>U=A for 3′ neighbor. A–I editing occurs most frequently in the non-coding regions containing repetitive elements such as ALUs. It leads to disruption of RNA duplex structure, which prevents induction of innate immune response. We employed standard and biased molecular dynamics (MD) simulations to analyze the behavior of RNA duplexes with single and tandem inosine–uracil (I–U) base pairs in different sequence context. Our analysis showed that the I–U pairs induce changes in base pair and base pair step parameters and have different dynamics when compared with standard canonical base pairs. In particular, the first I–U pair from tandem I–U/I–U systems exhibited increased dynamics depending on its neighboring 5′ base. We discovered that UII sequence, which is frequently edited, has lower flexibility compared with other sequences (AII, GII, CII), hence it only modestly disrupts dsRNA. This might indicate that the UAA motifs in ALUs do not have to be sufficiently effective in preventing immune signaling.

CgHeliParm: analysis of dsDNA helical parameters for coarse-grained MARTINI molecular dynamics simulations.

We introduce cgHeliParm, a python program that provides the conformational analysis of Martini-based coarse-grained double strand DNA molecules. The software calculates the helical parameters such as base, base pair and base pair step parameters. cgHeliParm can be used for the analysis of coarse grain Martini molecular dynamics trajectories without transformation into atomistic models. This package works with Python 2.7 on MacOS and Linux. The program is freely available for download from https://github.com/ifaust83/cgheliparm. Together with the main script, the base reference files CG_X_std.lib, a number of examples and R scripts are also available from the same website. A tutorial on the use and application is also available at http://cgmartini.nl/index.php/tutorials-general-introduction/tutorial-martini-dna. i.faustino@rug.nl. Supplementary data are available at Bioinformatics online.

Molecular Dynamics Simulation Workflow for Nucleosome Studies

Nucleosomes are the building blocks of eukaryote genomes. Given that a nucleosome contains 147 base pairs of DNA, the DNA sequence complexity is ∼ 4147. With the advent of genome wide association studies attention can be focused on one nucleosome. Today's supercomputers and simulation tools are sufficiently powerful to conduct comparative studies of ensembles representing 10's to 100's of mononucleosomes in a few days. Here we demonstrate a workflow that characterizes 21 different positions of 601: 10 upstream, 10 downstream and the ideal position in atomic detail. Atomic models were constructed by automatically docking the 601-177 fragment to the histone core of 1KX5. In all 21 simulations, the superhelix geometry evolves in less than 100 ns to a conformation that is significantly different from the original 1KX5 geometry. We previously demonstrated that all x-ray structures of the nucleosome exhibit a specific distribution of Roll, Slide, and Twist. Twelve Fourier components are necessary and sufficient to describe the DNA base pair step parameters required to recreate a nucleosome superhelix in atomic resolution. Here we employ our Fourier filtering technique to show that 100 ns is sufficient to observe global conformational changes associated with positioning and mispositioning the 601-177 fragment. Specifically, ideal positioning yields a DNA superhelix conformation with a significantly different distribution of Slide than mispositioned nucleosomes. The phase and amplitude of Slide associated with wave number 14, approximately the DNA helix repeat 146bp/14, is strongly influenced by nucleosome mispositioning. Thus we have validated a workflow that allows us to efficiently simulate and analyze nucleosomes and demonstrated that simulations as short as 100 ns are sufficient to investigate sequence specific properties of the nucleosome. All simulation and analysis data is published via our iBIOMES server at http://www.latech.edu/∼bishop.

Stacking interactions involving non-Watson-Crick basepairs: dispersion corrected density functional theory studies.

The G:A basepair, stabilized by hydrogen bonding through the sugar edge of guanine and Hoogsteen edge of adenine in trans orientation (G:A S:HT), appears very frequently in the solved RNA structures and is very stable. We have carried out stacking energy analyses of two sequences, namely C:G W:WC::G:A S:HT and G:C W:WC::G:A S:HT (':' represents basepairing and '::' represents stacking interactions), formed by this non-Watson-Crick basepair, by DFT-D. We have scanned nearly the complete phase space by modeling structures of these sequences using different values of the basepair orientation parameters to determine the most stable conformations. It is found that the most stable conformations are rather far from the most frequently found orientations. We have considered the effect of sugar-phosphate backbone connectivity as an energy penalty arising from deformation of pseudo bond lengths between C1' atoms of successive bases along the strands. Augmentation of stacking energy from DFT-D by this coarse grain energy gives predicted structures extremely similar to the experimentally determined ones. It has been observed that the best stacking with small twist values is associated with positive roll and negative slide values, which are similar to their values in A-RNA structures for most sequences. Among the two base pair steps, C:G W:WC::G:A S:HT appears to be more stable in terms of stacking energy as compared to G:C W:WC::G:A S:HT possibly due to larger stacking overlap in the former one.

Effect of temperature on the structure and hydration layer of TATA-box DNA: A molecular dynamics simulation study

DNA within the living cells experiences a diverse range of temperature, ranging from freezing condition to hot spring water. How the structure, the mechanical properties of DNA, and the solvation dynamics around DNA changes with the temperature is important to understand the functionality of DNA under those acute temperature conditions. In that notion, we have carried out molecular dynamics simulations of a DNA oligomer, containing TATA-box sequence for three different temperatures (250K, 300K and 350K). We observed that the structure of the DNA, in terms of backbone torsion angles, sugar pucker, base pair parameters, and base pair step parameters, did not show any unusual properties within the studied range of temperatures, but significant structural alteration was noticed between BI and BII forms at higher temperature. As expected, the flexibility of the DNA, in terms of the torsional rigidity and the bending rigidity is highly temperature dependent, confirming that flexibility increases with increase in temperature. Additionally, the groove widths of the studied DNA showed temperature sensitivity, specifically, the major groove width decreases and the minor groove width increases, respectively, with the increase in temperature. We observed that at higher temperature, water around both the major and the minor groove of the DNA is less structured. However, the water dynamics around the minor groove of the DNA is more restricted as compared to the water around the major groove throughout the studied range of temperatures, without any anomalous behavior.

Analysis of stacking overlap in nucleic acid structures: algorithm and application.

RNA contains different secondary structural motifs like pseudo-helices, hairpin loops, internal loops, etc. in addition to anti-parallel double helices and random coils. The secondary structures are mainly stabilized by base-pairing and stacking interactions between the planar aromatic bases. The hydrogen bonding strength and geometries of base pairs are characterized by six intra-base pair parameters. Similarly, stacking can be represented by six local doublet parameters. These dinucleotide step parameters can describe the quality of stacking between Watson-Crick base pairs very effectively. However, it is quite difficult to understand the stacking pattern for dinucleotides consisting of non canonical base pairs from these parameters. Stacking interaction is a manifestation of the interaction between two aromatic bases or base pairs and thus can be estimated best by the overlap area between the planar aromatic moieties. We have calculated base pair overlap between two consecutive base pairs as the buried van der Waals surface between them. In general, overlap values show normal distribution for the Watson-Crick base pairs in most double helices within a range from 45 to 50Å(2) irrespective of base sequence. The dinucleotide steps with non-canonical base pairs also are seen to have high overlap value, although their twist and few other parameters are rather unusual. We have analyzed hairpin loops of different length, bulges within double helical structures and pseudo-continuous helices using our algorithm. The overlap area analyses indicate good stacking between few looped out bases especially in GNRA tetraloop, which was difficult to quantitatively characterise from analysis of the base pair or dinucleotide step parameters. This parameter is also seen to be capable to distinguish pseudo-continuous helices from kinked helix junctions.

Rigid-body molecular dynamics of DNA inside a nucleosome

The majority of eukaryotic DNA, about three quarter, is wrapped around histone proteins forming so-called nucleosomes. To study nucleosomal DNA we introduce a coarse-grained molecular dynamics model based on sequence-dependent harmonic rigid base pair step parameters of DNA and nucleosomal binding sites. Mixed parametrization based on all-atom molecular dynamics and crystallographic data of protein-DNA structures is used for the base pair step parameters. The binding site parameters are adjusted by experimental B-factor values of the nucleosome crystal structure. The model is then used to determine the energy cost for placing a twist defect into the nucleosomal DNA which allows us to use Kramers theory to calculate nucleosome sliding caused by such defects. It is shown that the twist defect scenario together with the sequence-dependent elasticity of DNA can explain the slow time scales observed for nucleosome mobility along DNA. With this method we also show how the twist defect mechanism leads to a higher mobility of DNA in the presence of sin mutations near the dyad axis. Finally, by performing simulations on 5s rDNA, 601, and telomeric base pair sequences, it is demonstrated that the current model is a powerful tool to predict nucleosome positioning.

54 DNA conformation and energy in nucleosome core: a theoretical approach

In this work, we developed a mechanical model to address the problem of DNA structure and energy under deformation. DNA in nucleosome core particle is described as an example. The structure and energy of nucleosomal DNA is calculated based on its sequence and positioning state. Our theory is on the level of base pair step parameters. A quadratic elastic energy function describes the deformational energy of the molecule in which sequence dependency appears in a matrix of coupling constants. This rigidity matrix has been evaluated based on molecular dynamics simulation data and X-ray crystallography structures of DNA. The inferred structure has been calculated through minimization of the energy and the results show remarkable similarity with X-ray data. Figure 1 compares the base pair step parameters of a typical nucleosomal DNA (NCP147: PDB id 1kx5) with its inferred structure. Although there is no sequence-specific interaction of bases and the histone core, we found considerable sequence dependency for nucleosomal DNA positioning. The wrapping affinity for 5S rRNA, 601 positioning, TATA box, TGGA repeat, and NCP147 sequences are calculated and compared with the experimental data. The structural energy differences, ΔΔG, are in good agreement with the data obtained by calorimetric approaches for natural sequences (Table 1). We argue that structural energy determines the natural state of nucleosomal DNA and is the main reason for affinity differences in vitro. This theory can be utilized for the DNA structure and energy determination in protein–DNA complexes in general.

NMR characterisation of a highly conserved secondary structural RNA motif of Halobacterium halobium 23S rRNA

The highly conserved 29-mer RNA motif corresponding to the peptidyl transferase central circle region of the domain V of Halobacterium halobium 23S rRNA has been characterised by multidimensional NMR spectroscopy. The NMR structure has a good all atom average RMSD of 1.28 Å and a stable A-form helical conformation. The NMR structure differs from the X-ray crystal structure of an analogous motif, contained within the Escherichia coli ribosome, as none of the bases are flipped out and a number of non-canonical base pairs are formed in the solution structure. Thus in the observed NMR structure, the predicted A7 to U30 base pair is not seen and a non-canonical U6 to U30 base pair was formed in its place. Similarly the predicted A9 to U26 base pair was also not observed and another non-canonical A9 to A27 base pair was formed. It was also seen from the conformational analysis that the steps near the bulges had the greatest deviation from the canonical Watson-Crick base pair step parameters. Despite these differences, the 29-mer structure provides a working model of the RNA within the ribosome in a more natural solution state than that observed in the intact ribosome crystal structures, particularly around the A27 residue. The NMR structure determination of the 29-mer RNA motif provides a solid foundation for determining the NMR structure of the RNA-amicetin complex in the next step. To extend the above study, a fully (13)C and (15)N isotopically labelled 37-mer RNA version of the Halobacterium halobium RNA sample has been characterised using ultra high field 1 GHz spectroscopy. The results have been used to demonstrate the advantages conferred by the use of a 1 GHz spectrometer frequency over 800 MHz in terms of superior sensitivity and greater spectral dispersion achieved in the spectrum of the RNA.

Computational Studies on the Effects of DNA Methylation on its Structure and Dynamics

DNA methylation occurs in CpG dinucleotides when a methyl group is added at the carbon 5 position of the C base ring, and it has an important role in regulating gene expression in higher eukaryotes. It is known that when cytosine bases are methylated, gene is silenced, but the precise role of methylated DNA in this process is still debated, and the mechanism by which methylation affects gene expression remains poorly understood.One hypothesis is that the methylation state of a DNA segment can alter the positions of nucleosomes, which in turn, determine gene expression. Several studies point to a strong link between CpG methylation and nucleosome positioning, but these effects might be indirect because methylation influences the binding of other factors, which could in turn trigger nucleosome repositioning. Because methylation chemically modifies DNA, the methylation status of a DNA sequence could influence its flexibility and, thus, its affinity to the nucleosome. Recent experimental studies suggest that CpG methylation can decrease the ability of DNA to bend into the major groove at the methylated site, and can thereby influence nucleosome positioning.To test the idea that methylation affects the ability of DNA to bend, we performed MD simulations of short DNA segments using an all atom force field. DNA conformations were characterized using the six rigid body base-pair step parameters, the minor groove width and the hybridization state. These properties were measured and compared for dsDNA segments in both the unmethylated and methylated states. In addition, all atom trajectories for both the cases were analyzed to extract a new set of parameters for a coarse grain model for DNA that can now be used to study the effects of methylation in larger DNA systems such as nucleosomes.

Changes in Thermodynamic Properties of DNA Base Pairs in Protein-DNA Recognition

The mechanism of protein-DNA recognition, particularly the induced fit mechanism, is poorly understood due to ineffective analysis of the protein-DNA complex crystal structures. It is expected that upon protein binding the DNA becomes structurally more rigid. However, a previous analysis (W.K. Olson, A. A. Gorin, X. Lu, L. M. Hock and V. Zhurkin, Proc. Natl. Acad. Sci. USA, 95, 11163 (1998)) indicates increase in the flexibility of the DNA segment complexed with protein. We have considered an ensemble of configurations from crystallographic data of the TBP-TATA box complex structures under a given thermodynamic condition. Analysis of the ensemble of structures of this complex indicates that the DNA deforms significantly to form specific hydrogen bonds and as a consequence, its structure attains more rigidity. We calculate the free energy profiles in term of the DNA base pair (bp) step parameters via the binding patterns in the ensemble of the given complex, and for free DNA bp steps as well. The rigidities estimated from these free energies for small deformations around the minimum indicate enhanced structural rigidities of DNA upon complexation with protein. Further, the changes in the thermodynamic properties of the bp steps upon complex formation have been estimated from the two sets of free energy profiles. These results indicate differential role played by different bp steps in the thermodynamic stabilization of the complex.

Applications of VDNA from Basic Geometry to Chromatin Folding

We recently introduced VDNA(1), a plugin for VMD, that allows users to construct arbitrarily complex models of double stranded DNA. VDNA is preinstalled in VMD 1.8.7 and has a number of predefined mathematical expressions that allow for investigation of the complex relations between the DNA base pair step parameters (Tilt, Roll, Twist, Shift, Slide, Rise) and spatial conformation. VDNA is capable of producing static images, ensembles and trajectories. Using VDNA we demonstrate some of the geometric relations between the DNA step parameters and Cartesian coordinates that do not readily yield to intuition. We also consider a number of biologically motivated models including: a model of thermal fluctuations in linear DNA, comparison of a Torsion Helix and a Shear Helix model of the nucleosome(2), and models for regular and irregularly folded chromatin. Finally we demonstrate how VDNA can be used to convert nucleosome footprints from a chromatin map into a three dimensional chromatin fold. Such folding provides an immediate answer as to whether or not the footprints yield a conformation that is sterically allowed or if extra-nucleosomal proteins, non-canonical nucleosome conformations or thermal effects should also be considered. The most up to date version of VDNA is available from http://dna.ccs.tulane.edu/vdna. Acknowledgement: This work supported in part by NIH R01-GM076356. (1) T. C. Bishop. VDNA: The Virtual DNA plug-in for VMD, Bioinformatics (2009). (2) T. C. Bishop. Geometry of the nucleosomal DNA superhelix, Biophys. J. 95, 1007-1017 (2008).

A systematic molecular dynamics study of nearest-neighbor effects on base pair and base pair step conformations and fluctuations in B-DNA

It is well recognized that base sequence exerts a significant influence on the properties of DNA and plays a significant role in protein–DNA interactions vital for cellular processes. Understanding and predicting base sequence effects requires an extensive structural and dynamic dataset which is currently unavailable from experiment. A consortium of laboratories was consequently formed to obtain this information using molecular simulations. This article describes results providing information not only on all 10 unique base pair steps, but also on all possible nearest-neighbor effects on these steps. These results are derived from simulations of 50–100 ns on 39 different DNA oligomers in explicit solvent and using a physiological salt concentration. We demonstrate that the simulations are converged in terms of helical and backbone parameters. The results show that nearest-neighbor effects on base pair steps are very significant, implying that dinucleotide models are insufficient for predicting sequence-dependent behavior. Flanking base sequences can notably lead to base pair step parameters in dynamic equilibrium between two conformational sub-states. Although this study only provides limited data on next-nearest-neighbor effects, we suggest that such effects should be analyzed before attempting to predict the sequence-dependent behavior of DNA.

VDNA: The virtual DNA plug-in for VMD

Summary: The DNA inter base pair step parameters (Tilt, Roll, Twist, Shift, Slide, Rise) are a standard internal coordinate representation of DNA. In the absence of bend and shear, it is relatively easy to mentally visualize how Twist and Rise generate the familiar double helix. More complex structures do not readily yield to such intuition. For this reason, we developed a plug-in for VMD that accepts a set of mathematical expressions as input and generates a coarse-grained model of DNA as output. This feature of VDNA appears to provide a unique approach to DNA modeling. Predefined expressions include: linear, sheared, bent and circular DNA, and models of the nucleosome superhelix, chromatin, thermal motion and nucleosome unwrapping.Availability: VDNA is pre-installed in VMD, http://www.ks.uiuc.edu/Research/vmd. Updates are at http://dna.ccs.tulane.edu.Contact: bishop@tulane.edu

The post-SCF quantum chemistry characteristics of inter- and intra-strand stacking interactions in d(CpG) and d(GpC) steps found in B-DNA, A-DNA and Z-DNA crystals

The energies of intra- and inter-strand stacking interactions in model d(GpC) and d(CpG) two-base-pair steps were estimated by MP2/aug-cc-pVDZ single point calculations corrected for basis superposition errors. The stacked two-nucleobase pairs were constructed using experimental values of base pair and base step parameters taken from Nucleic Acid Database (http://ndbserver.rutgers.edu/). Three distinct polymorphic forms were analysed, namely A-, B- and Z-DNA. The applied methodology enables statistical analysis of structural and energetic diversities. The structural relationships between polymorphic forms are quite complex and depend on the sequence of pairs. The variability of parameters such as shift and tilt is almost the same irrespective of the polymorphic form and sequence of steps analysed. In contrast, shift and twist distributions easily discriminate all three polymorphic forms of DNA. Interestingly, despite significant structural diversities, the energies of the most frequent energy ranges are comparable irrespective of the polymorphic form and base sequence. There was observed compensation of inter- and intra-strand interactions, especially for d(GpC) and d(CpG) steps found in A- and B-DNA. Thus, among many other roles, these pairs act as a kind of energetic buffer, balancing the double helix.

Principal component analysis of DNA oligonucleotide structural data

The microstructure of a DNA helix is characterized by several base pair and base step parameters such as twist, rise, roll, propeller twist, etc., in addition to conformational parameters such as the backbone and the glycosidic torsion angles. Among these only a few, which are independent of all others and of each other, may be used to precisely characterize the helix. The problem however is to identify these independent parameters. We have used principal component analysis to identify a relatively small set of independent parameters, with which to characterize each DNA helix. We show that these principal components clearly discriminate between A and B DNA helical types. The calculations further suggest that the microstructure of a DNA helix is better characterized using dinucleotides.

Identification of conserved structural features at sequentially degenerate locations in transcription factor binding sites.

Many locations within transcription factor binding sites are not sequentially conserved and appear to be degenerate. We hypothesize that some of these positions contain essential structural codes that are recognized by the transcription factors that bind to them. The structural codes can be defined by base-pair step parameters that describe the relative displacement and orientation of two adjacent base pairs in a nucleic acid structure. We have developed a method, Identification of Conserved Structural Features (ICSF), which uses base-pair step parameters obtained from a collection of high-resolution DNA crystal structures to discover structural conservation that exists in the sequentially degenerate areas within a binding site and produce profiles of the structural features along the entire site. We have focused our study on the transcription factor binding sites in the JASPAR database and have found that one-third (P-value > or = 0.05) of the binding sites contain sequentially degenerate locations with highly conserved structural features as described by the base-pair step parameters. These results will help us to gain a better understanding of the process by which transcription factors recognize their binding sites and possibly lead to an improvement in our ability to find these sites in genomic sequences. ICSF is freely available to academic users at http://zlab.bu.edu/ICSF . http://zlab.bu.edu/ICSF .

Modeling DNA structure, elasticity, and deformations at the base-pair level.

We present a generic model for DNA at the base-pair level. We use a variant of the Gay-Berne potential to represent the stacking energy between the neighboring base pairs. The sugar-phosphate backbones are taken into account by semirigid harmonic springs with a nonzero spring length. The competition between these two interactions and the introduction of a simple geometrical constraint lead to a stacked right-handed B-DNA-like conformation. The mapping of the presented model to the Marko-Siggia and the stack-of-plates model enables us to optimize the free model parameters so as to reproduce the experimentally known observables such as persistence lengths, mean and mean-squared base-pair step parameters. For the optimized model parameters, we measured the critical force where the transition from B- to S-DNA occurs to be approximately 140 pN. We observe an overstretched S-DNA conformation with highly inclined bases which partially preserves the stacking of successive base pairs.