Joanna Sarzynska

Single Stranded Loops of Quadruplex DNA As Key Benchmark for Testing Nucleic Acids Force Fields.

We have carried out a set of explicit solvent molecular dynamics (MD) simulations on two DNA quadruplex (G-DNA) molecules, namely the antiparallel d(G4T4G4)2 dimeric quadruplex with diagonal loops and the parallel-stranded human telomeric monomolecular quadruplex d[AGGG(TTAGGG)3] with three propeller loops. The main purpose of the paper was testing of the capability of the MD simulation technique to describe single-stranded topologies of G-DNA loops, which represent a very challenging task for computational methods. The total amount of conventional and locally enhanced sampling (LES) simulations analyzed in this study exceeds 1.5 μs, while we tested several versions of the AMBER force field (parm99, parmbsc0, and a version with modified glycosidic χ torsion profile) and the CHARMM27 force field. Further, we compared minimal salt and excess salt simulations. Postprocessing MM-PBSA (Molecular Mechanics, Poisson-Boltzmann, Surface Area) free energy calculations are also reported. None of the presently available force fields is accurate enough in describing the G-DNA loops. The imbalance is best seen for the propeller loops, as their experimental structure is lost within a few ns of standard simulations with all force fields. Among them, parmbsc0 provides results that are clearly closest to the experimental target values but still not in full agreement. This confirms that the improvement of the γ torsional profile penalizing the γ trans substates in the parmbsc0 parametrization was a step in the right direction, albeit not sufficient to treat all imbalances. The modified χ parametrization appears to rigidify the studied systems but does not change the ultimate outcome of the present simulations. The structures obtained in simulations with the modified χ profile are predetermined by its combination with either parm99 or parmbsc0. Experimental geometries of diagonal loops of d(G4T4G4)2 are stable in standard simulations on the ∼10 ns time scale but are becoming progressively lost in longer and LES simulations. In addition, the d(G4T4G4)2 quadruplex contains, besides the three genuine binding sites for cations in the channel of its stem, also an ion binding site at each stem-loop junction. This arrangement of five cations in the quadruplex core region is entirely unstable in all 24 simulations that we attempted. Overall, our results confirm that G-DNA loops represent one of the most difficult targets for molecular modeling approaches and should be considered as reference structures in any future studies aiming to develop or tune nucleic acids force fields.

Conformations of Flanking Bases in HIV-1 RNA DIS Kissing Complexes Studied by Molecular Dynamics

Explicit solvent molecular dynamics simulations (in total almost 800 ns including locally enhanced sampling runs) were applied with different ion conditions and with two force fields (AMBER and CHARMM) to characterize typical geometries adopted by the flanking bases in the RNA kissing-loop complexes. We focus on flanking base positions in multiple x-ray and NMR structures of HIV-1 DIS kissing complexes and kissing complex from the large ribosomal subunit of Haloarcula marismortui. An initial x-ray open conformation of bulged-out bases in HIV-1 DIS complexes, affected by crystal packing, tends to convert to a closed conformation formed by consecutive stretch of four stacked purine bases. This is in agreement with those recent crystals where the packing is essentially avoided. We also observed variants of the closed conformation with three stacked bases, while nonnegligible populations of stacked geometries with bulged-in bases were detected, too. The simulation results reconcile differences in positions of the flanking bases observed in x-ray and NMR studies. Our results suggest that bulged-out geometries are somewhat more preferred, which is in accord with recent experiments showing that they may mediate tertiary contacts in biomolecular assemblies or allow binding of aminoglycoside antibiotics.

Effects of Base Substitutions in an RNA Hairpin from Molecular Dynamics and Free Energy Simulations

Contributions of individual interactions in the GGCGCAAGCC hairpin containing a GCAA tetraloop were studied by computer simulations using base substitutions. The G in the first tetraloop position was replaced by inosine (I) or adenosine (A), and the G in the C-G basepair closing the tetraloop was replaced by I. These substitutions eliminate particular hydrogen bonds proposed in the nuclear magnetic resonance model of the GCAA tetraloop. Molecular dynamics simulations of the GCAA tetraloop in aqueous solvent displayed a well-defined hydrogen pattern between the first and last loop nucleotides (G and A) stabilized by a bridging water molecule. Substitution of G-->I in the basepair closing the tetraloop did not significantly influence the loop structure and dynamics. The ICAA loop maintained the overall structure, but displayed variation in the hydrogen-bond network within the tetraloop itself. Molecular dynamics simulations of the ACAA loop led to conformational heterogeneity of the resulting structures. Changes of hairpin formation free energy associated with substitutions of individual bases were calculated by the free energy perturbation method. The calculated decrease of the hairpin stability upon G-->I substitution in the C-G basepair closing the tetraloop was in good agreement with experimental thermodynamic data. Our theoretical estimates for G-->I and G-->A mutations located in the tetraloop suggest larger loop destabilization than corresponding experimental results. The extent of conformational sampling of the structures resulting from base substitutions and its impact on the calculated free energy was discussed.

Conformational Dynamics of a 5S rRNA Hairpin Domain Containing Loop D and a Single Nucleotide Bulge

Molecular modeling and molecular dynamics have been employed to study the conformation and flexibility of a 15-nucleotide fragment of the plant 5S rRNA containing loop D and a single uridine bulge. Two different model built initial structures were used: one with the bulge localized inside the helical stem and another with the bulge pointing out from the helix. Several independent 700-ps-long trajectories in aqueous solution with Na(+) conterions were produced for each starting structure. The bulge nucleotide inside the helix stayed in two main conformations, both of which affected the geometry of the stem part opposite the bulge. When the bulge nucleotide was located outside the helix, we found high base mobility and local backbone flexibility. The dynamics of the hydrogen bond network and conformational changes from a direct to a water mediated hydrogen bond in the sheared G-A basepair in the tetraloop was described. Our results correlate with lead ion induced cleavage patterns in 5S rRNA. Sites resistant to nonspecific lead cleavage appeared in all our simulations as the most rigid fragments independent of the localization of the bulge nucleotide.

New functionality of RNAComposer: an application to shape the axis of miR160 precursor structure

RNAComposer is a fully automated, web-interfaced system for RNA 3D structure prediction, freely available at http://rnacomposer.cs.put.poznan.pl/ and http://rnacomposer.ibch.poznan.pl/. Its main components are: manually curated database of RNA 3D structure elements, highly efficient computational engine and user-friendly web application. In this paper, we demonstrate how the latest additions to the system allow the user to significantly affect the process of 3D model composition on several computational levels. Although in general our method is based on the knowledge of secondary structure topology, currently the RNAComposer offers a choice of six incorporated programs for secondary structure prediction. It also allows to apply a conditional search in the database of 3D structure elements and introduce user-provided elements into the final 3D model. This new functionality contributes to a significant improvement of the predicted 3D model reliability and it facilitates a better model adjustment to the experimental data. This is exemplified based on the RNAComposer application for modelling of the 3D structures of precursors of the miR160 family members.

Conformational transitions of flanking purines in HIV-1 RNA dimerization initiation site kissing complexes studied by CHARMM explicit solvent molecular dynamics

Dimerization of HIV‐1 genomic RNA is initiated by kissing loop interactions at the Dimerization Initiation Site (DIS). Dynamics of purines that flank the 5′ ends of the loop–loop helix in HIV‐1 DIS kissing complex were explored using explicit solvent molecular dynamics (MD) simulations with the CHARMM force field. Multiple MD simulations (200 ns in total) of X‐ray structures for HIV‐1 DIS Subtypes A, B, and F revealed conformational variability of flanking purines. In particular, the flanking purines, which in the starting X‐ray structures are bulged‐out and stack in pairs, formed a consecutive stack of four bulged‐out adenines at the beginning of several simulations. This conformation is seen in the crystal structure of DIS Subtype F with no interference from crystal packing, and was frequently reported in our preceding MD studies performed with the AMBER force field. However, as CHARMM simulations progressed, the four continuously stacked adenines showed conformational transitions from the bulged‐out into the bulged‐in geometries. Although such an arrangement has not been seen in any X‐ray structure, it has been suggested by a recent NMR investigation. In CHARMM simulations, in the longer time scale, the flanking purines display the tendency to move to bulged‐in conformations. This is in contrast with the AMBER simulations, which indicate a modest prevalence for bulged‐out flanking base positions in line with the X‐ray data. The simulations also suggest that the intermolecular stacking between purines from the opposite hairpins can additionally stabilize the kissing complex.

Conformational preferences of modified uridines: comparison of AMBER derived force fields.

The widespread occurrence of modified residues in RNA sequences necessitates development of accurate parameters for these modifications for reliable modeling of RNA structure and dynamics. A comprehensive set of parameters for the 107 naturally occurring RNA modifications was proposed by Aduri et al. (J. Chem. Theory Comput. 2007, 3, 1464-1475) for the AMBER FF99 force field. In this work, we tested these parameters on a set of modified uridine residues, namely, dihydrouridine, 2-thiouridine, 4-thiouridine, pseudouridine, and uridine-5-oxyacetic acid, by performing molecular dynamics and replica exchange molecular dynamics simulations of these nucleosides. Although our simulations using the FF99 force field did not, in general, reproduce the experimentally observed conformational characteristics well, combination of the parameter set with recent revisions of the FF99 force field for RNA showed noticeable improvement for some of the nucleosides.

Dynamics and Stability of GCAA Tetraloops with 2-Aminopurine and Purine Substitutions

Abstract The contributions of various interactions in the GGCGCAAGCC hairpin containing a GCAA tetraloop were studied by computer simulations using the substitutions of functional groups. The guanosine (G) in the first tetraloop position or in the C-G closing base pair was replaced by 2-aminopurine (AP), and the individual tetraloops adenosines (A) were replaced by purine (PUR). These substitutions eliminated particular hydrogen bonds thought to stabilize the GCAA tetraloop. For each substitution, molecular dynamics (MD) simulations were carried out in an aqueous solution with sodium counterions, using the CHARMM27 force field. The MD simulations showed that the substitutions in the first (G→AP) and the third (A→PUR) position of the GCAA tetraloop did not significantly influence the conformation of the hairpin. A long-lived bridging water molecule observed in the GCAA loop was present in both modified loops. The substitutions made in the last loop position (A→PUR) or in the C-G base pair closing the tetraloop (G→AP) to some extent influenced the loop structure and dynamics. These loops did not display the long- lived bridging water molecules. When the second A in the GCAA loop was replaced by PUR, the first A in the loop was observed in the anti or in the syn orientation about the gly- cosyl bond. The G to AP substitution in C-G base pair led to a change of their arrangement from the Watson-Crick to wobble. The MD simulations of the hairpin with C-AP wobble closing base pair showed increased conformational dynamics of the hairpin. The changes of hairpin formation free energy associated with the substitutions of individual bases were calculated by the free energy perturbation method. Our theoretical estimates suggest a larger destabilization for the G to AP substitutions in GCAA loop than for the substitutions of individual As by PUR, which is in accordance with experimental tendency. The calculations predicted a similar free energy change for G to AP substitutions in the GCAA tetraloop and in the C-G closing base pair.

AmiRNA Designer — new method of artificial miRNA design

MicroRNAs (miRNAs) are small non-coding RNAs that have been found in most of the eukaryotic organisms. They are involved in the regulation of gene expression at the post-transcriptional level in a sequence specific manner. MiRNAs are produced from their precursors by Dicer-dependent small RNA biogenesis pathway. Involvement of miRNAs in a wide range of biological processes makes them excellent candidates for studying gene function or for therapeutic applications. For this purpose, different RNA-based gene silencing techniques have been developed. Artificially transformed miRNAs (amiRNAs) targeting one or several genes of interest represent one of such techniques being a potential tool in functional genomics. Here, we present a new approach to amiRNA*design, implemented as AmiRNA Designer software. Our method is based on the thermodynamic analysis of the native miRNA/miRNA* and miRNA/target duplexes. In contrast to the available automated tools, our program allows the user to perform analysis of natural miRNAs for the organism of interest and to create customized constraints for the design stage. It also provides filtering of the amiRNA candidates for the potential off-targets. AmiRNA Designer is freely available at http://www.cs.put.poznan.pl/arybarczyk/AmiRNA/.

Differences in the association abilities in aqueous solutions of cytidine, 2′-deoxycytidine and their phosphate salts studied by Fourier transform infrared spectroscopy

Abstract Fourier transform infrared (FT-IR) spectral investigations of Cyd and dCyd nucleosides and their phosphate salts were made in aqueous media in order to determine their association abilities. The observed effects on the FT-IR spectra of increasing concentrations of H 2 O and D 2 O solutions indicate drastically different association properties and hydration between the compounds studied in the ribo and deoxyribo series. The results show that examination of biomolecules in their natural environment (aqueous media) by FT-IR spectrometry is feasible and informative with regard to precrystallization phenomena.