RNA molecules play a key role in countless biochemical processes. RNA interactions, which are of highly diverse nature, are determined by the fact that RNA is a highly negatively charged polyelectrolyte, which leads to intimate interactions with an ion atmosphere. Although RNA molecules are formally single-stranded, canonical (Watson-Crick) duplexes are key components of folded RNAs. A double-stranded (ds) RNA is also important for the design of RNA-based nanostructures and assemblies. Despite the fact that the description of canonical dsRNA is considered the least problematic part of RNA modeling, the imperfect shape and flexibility of dsRNA can lead to imbalances in the simulations of larger RNAs and RNA-containing assemblies. We present a comprehensive set of molecular dynamics (MD) simulations of four canonical A-RNA duplexes. Our focus was directed toward the characterization of the influence of varying ion concentrations and of the size of the solvation box. We compared several water models and four RNA force fields. The simulations showed that the A-RNA shape was most sensitive to the RNA force field, with some force fields leading to a reduced inclination of the A-RNA duplexes. The ions and water models played a minor role. The effect of the box size was negligible, and even boxes with a small fraction of the bulk solvent outside the RNA hydration sphere were sufficient for the simulation of the dsRNA.

• MeSH
• ionty chemie   MeSH
• konformace nukleové kyseliny MeSH
• RNA * chemie   MeSH
• simulace molekulární dynamiky * MeSH
• voda chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ionty MeSH
• RNA * MeSH
• voda MeSH

With both catalytic and genetic functions, ribonucleic acid (RNA) is perhaps the most pluripotent chemical species in molecular biology, and its functions are intimately linked to its structure and dynamics. Computer simulations, and in particular atomistic molecular dynamics (MD), allow structural dynamics of biomolecular systems to be investigated with unprecedented temporal and spatial resolution. We here provide a comprehensive overview of the fast-developing field of MD simulations of RNA molecules. We begin with an in-depth, evaluatory coverage of the most fundamental methodological challenges that set the basis for the future development of the field, in particular, the current developments and inherent physical limitations of the atomistic force fields and the recent advances in a broad spectrum of enhanced sampling methods. We also survey the closely related field of coarse-grained modeling of RNA systems. After dealing with the methodological aspects, we provide an exhaustive overview of the available RNA simulation literature, ranging from studies of the smallest RNA oligonucleotides to investigations of the entire ribosome. Our review encompasses tetranucleotides, tetraloops, a number of small RNA motifs, A-helix RNA, kissing-loop complexes, the TAR RNA element, the decoding center and other important regions of the ribosome, as well as assorted others systems. Extended sections are devoted to RNA-ion interactions, ribozymes, riboswitches, and protein/RNA complexes. Our overview is written for as broad of an audience as possible, aiming to provide a much-needed interdisciplinary bridge between computation and experiment, together with a perspective on the future of the field.

• MeSH
• DNA chemie   MeSH
• katalýza MeSH
• konformace nukleové kyseliny * MeSH
• počítačová simulace MeSH
• RNA chemie   MeSH
• simulace molekulární dynamiky * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• DNA MeSH
• RNA MeSH

The sarcin-ricin RNA motif (SR motif) is one of the most prominent recurrent RNA building blocks that occurs in many different RNA contexts and folds autonomously, that is, in a context-independent manner. In this study, we combined bioinformatics analysis with explicit-solvent molecular dynamics (MD) simulations to better understand the relation between the RNA sequence and the evolutionary patterns of the SR motif. A SHAPE probing experiment was also performed to confirm the fidelity of the MD simulations. We identified 57 instances of the SR motif in a nonredundant subset of the RNA X-ray structure database and analyzed their base pairing, base-phosphate, and backbone-backbone interactions. We extracted sequences aligned to these instances from large rRNA alignments to determine the frequency of occurrence for different sequence variants. We then used a simple scoring scheme based on isostericity to suggest 10 sequence variants with a highly variable expected degree of compatibility with the SR motif 3D structure. We carried out MD simulations of SR motifs with these base substitutions. Nonisosteric base substitutions led to unstable structures, but so did isosteric substitutions which were unable to make key base-phosphate interactions. The MD technique explains why some potentially isosteric SR motifs are not realized during evolution. We also found that the inability to form stable cWW geometry is an important factor in the case of the first base pair of the flexible region of the SR motif. A comparison of structural, bioinformatics, SHAPE probing, and MD simulation data reveals that explicit solvent MD simulations neatly reflect the viability of different sequence variants of the SR motif. Thus, MD simulations can efficiently complement bioinformatics tools in studies of conservation patterns of RNA motifs and provide atomistic insight into the role of their different signature interactions.

• MeSH
• konformace nukleové kyseliny MeSH
• nukleotidové motivy MeSH
• párování bází MeSH
• RNA ribozomální chemie   metabolismus   MeSH
• RNA chemie   metabolismus   MeSH
• rozpouštědla chemie   MeSH
• simulace molekulární dynamiky MeSH
• vodíková vazba MeSH
• výpočetní biologie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• RNA ribozomální MeSH
• RNA MeSH
• rozpouštědla MeSH

Riboswitches often occur in the 5'-untranslated regions of bacterial mRNA where they regulate gene expression. The preQ(1) riboswitch controls the biosynthesis of a hypermodified nucleoside queuosine in response to binding the queuosine metabolic intermediate. Structures of the ligand-bound and ligand-free states of the preQ(1) riboswitch from Thermoanaerobacter tengcongensis were determined recently by X-ray crystallography. We used multiple, microsecond-long molecular dynamics simulations (29 μs in total) to characterize the structural dynamics of preQ(1) riboswitches in both states. We observed different stabilities of the stem in the bound and free states, resulting in different accessibilities of the ribosome-binding site. These differences are related to different stacking interactions between nucleotides of the stem and the associated loop, which itself adopts different conformations in the bound and free states. We suggest that the loop not only serves to bind preQ(1) but also transmits information about ligand binding from the ligand-binding pocket to the stem, which has implications for mRNA accessibility to the ribosome. We explain functional results obscured by a high salt crystallization medium and help to refine regions of disordered electron density, which demonstrates the predictive power of our approach. Besides investigating the functional dynamics of the riboswitch, we have also utilized this unique small folded RNA system for analysis of performance of the RNA force field on the μs time scale. The latest AMBER parmbsc0χ(OL3) RNA force field is capable of providing stable trajectories of the folded molecule on the μs time scale. On the other hand, force fields that are not properly balanced lead to significant structural perturbations on the sub-μs time scale, which could easily lead to inappropriate interpretation of the simulation data.

• MeSH
• bakteriální RNA chemie   MeSH
• krystalografie rentgenová MeSH
• molekulární modely MeSH
• riboswitch * MeSH
• simulace molekulární dynamiky * MeSH
• Thermoanaerobacter chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• bakteriální RNA MeSH
• riboswitch * MeSH

Refinement of empirical force fields for nucleic acids requires their extensive testing using as wide range of systems as possible. However, finding unambiguous reference data is not easy. In this paper, we analyze four systems which we suggest should be included in standard portfolio of molecules to test nucleic acids force fields, namely, parallel and antiparallel stranded DNA guanine quadruplex stems, RNA quadruplex stem, and Z-DNA. We highlight parameters that should be monitored to assess the force field performance. The work is primarily based on 8.4 μs of 100-250 ns trajectories analyzed in detail followed by 9.6 μs of additional selected back up trajectories that were monitored to verify that the results of the initial analyses are correct. Four versions of the Cornell et al. AMBER force field are tested, including an entirely new parmχ(OL4) variant with χ dihedral specifically reparametrized for DNA molecules containing syn nucleotides. We test also different water models and ion conditions. While improvement for DNA quadruplexes is visible, the force fields still do not fully represent the intricate Z-DNA backbone conformation.

• Publikační typ
• časopisecké články MeSH

The L1 stalk is a key mobile element of the large ribosomal subunit which interacts with tRNA during translocation. Here, we investigate the structure and mechanical properties of the rRNA H76/H75/H79 three-way junction at the base of the L1 stalk from four different prokaryotic organisms. We propose a coarse-grained elastic model and parameterize it using large-scale atomistic molecular dynamics simulations. Global properties of the junction are well described by a model in which the H76 helix is represented by a straight, isotropically flexible elastic rod, while the junction core is represented by an isotropically flexible spherical hinge. Both the core and the helix contribute substantially to the overall H76 bending fluctuations. The presence of wobble pairs in H76 does not induce any increased flexibility or anisotropy to the helix. The half-closed conformation of the L1 stalk seems to be accessible by thermal fluctuations of the junction itself, without any long-range allosteric effects. Bending fluctuations of H76 with a bulge introduced in it suggest a rationale for the precise position of the bulge in eukaryotes. Our elastic model can be generalized to other RNA junctions found in biological systems or in nanotechnology.

• MeSH
• biomechanika MeSH
• konformace nukleové kyseliny MeSH
• ribozomální proteiny chemie   MeSH
• RNA ribozomální 23S chemie   MeSH
• simulace molekulární dynamiky MeSH
• velké podjednotky ribozomu archebakteriální chemie   MeSH
• velké podjednotky ribozomu bakteriální chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ribosomal protein L1 MeSH Prohlížeč
• ribozomální proteiny MeSH
• RNA ribozomální 23S MeSH

The RNA hairpin loops represent important RNA topologies with indispensable biological functions in RNA folding and tertiary interactions. 5'-UNCG-3' and 5'-GNRA-3' RNA tetraloops are the most important classes of RNA hairpin loops. Both tetraloops are highly structured with characteristic signature three-dimensional features and are recurrently seen in functional RNAs and ribonucleoprotein particles. Explicit solvent molecular dynamics (MD) simulation is a computational technique which can efficiently complement the experimental data and provide unique structural dynamics information on the atomic scale. Nevertheless, the outcome of simulations is often compromised by imperfections in the parametrization of simplified pairwise additive empirical potentials referred to also as force fields. We have pointed out in several recent studies that a force field description of single-stranded hairpin segments of nucleic acids may be particularly challenging for the force fields. In this paper, we report a critical assessment of a broad set of MD simulations of UUCG, GAGA, and GAAA tetraloops using various force fields. First, we utilized the three widely used variants of Cornell et al. (AMBER) force fields known as ff94, ff99, and ff99bsc0. Some simulations were also carried out with CHARMM27. The simulations reveal several problems which show that these force fields are not able to retain all characteristic structural features (structural signature) of the studied tetraloops. Then we tested four recent reparameterizations of glycosidic torsion of the Cornell et al. force field (two of them being currently parametrized in our laboratories). We show that at least some of the new versions show an improved description of the tetraloops, mainly in the syn glycosidic torsion region of the UNCG tetraloop. The best performance is achieved in combination with the bsc0 parametrization of the α/γ angles. Another critically important region to properly describe RNA molecules is the anti/high-anti region of the glycosidic torsion, where there are significant differences among the tested force fields. The tetraloop simulations are complemented by simulations of short A-RNA stems, which are especially sensitive to an appropriate description of the anti/high-anti region. While excessive accessibility of the high-anti region converts the A-RNA into a senseless "ladder-like" geometry, excessive penalization of the high-anti region shifts the simulated structures away from typical A-RNA geometry to structures with a visibly underestimated inclination of base pairs with respect to the helical axis.

• Publikační typ
• časopisecké články MeSH

The hairpin ribozyme is a prominent member of the group of small catalytic RNAs (RNA enzymes or ribozymes) because it does not require metal ions to achieve catalysis. Biochemical and structural data have implicated guanine 8 (G8) and adenine 38 (A38) as catalytic participants in cleavage and ligation catalyzed by the hairpin ribozyme, yet their exact role in catalysis remains disputed. To gain insight into dynamics in the active site of a minimal self-cleaving hairpin ribozyme, we have performed extensive classical, explicit-solvent molecular dynamics (MD) simulations on time scales of 50-150 ns. Starting from the available X-ray crystal structures, we investigated the structural impact of the protonation states of G8 and A38, and the inactivating A-1(2'-methoxy) substitution employed in crystallography. Our simulations reveal that a canonical G8 agrees well with the crystal structures while a deprotonated G8 profoundly distorts the active site. Thus MD simulations do not support a straightforward participation of the deprotonated G8 in catalysis. By comparison, the G8 enol tautomer is structurally well tolerated, causing only local rearrangements in the active site. Furthermore, a protonated A38H(+) is more consistent with the crystallography data than a canonical A38. The simulations thus support the notion that A38H(+) is the dominant form in the crystals, grown at pH 6. In most simulations, the canonical A38 departs from the scissile phosphate and substantially perturbs the structures of the active site and S-turn. Yet, we occasionally also observe formation of a stable A-1(2'-OH)...A38(N1) hydrogen bond, which documents the ability of the ribozyme to form this hydrogen bond, consistent with a potential role of A38 as general base catalyst. The presence of this hydrogen bond is, however, incompatible with the expected in-line attack angle necessary for self-cleavage, requiring a rapid transition of the deprotonated 2'-oxyanion to a position more favorable for in-line attack after proton transfer from A-1(2'-OH) to A38(N1). The simulations revealed a potential force field artifact, occasional but irreversible formation of "ladder-like", underwound A-RNA structure in one of the external helices. Although it does not affect the catalytic center of the hairpin ribozyme, further studies are under way to better assess possible influence of such force field behavior on long RNA simulations.

• MeSH
• adenin chemie   MeSH
• guanin chemie   MeSH
• katalytická doména MeSH
• katalýza MeSH
• konformace nukleové kyseliny MeSH
• krystalografie rentgenová MeSH
• protony * MeSH
• RNA katalytická chemie   metabolismus   MeSH
• simulace molekulární dynamiky * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• adenin MeSH
• guanin MeSH
• hairpin ribozyme MeSH Prohlížeč
• protony * MeSH
• RNA katalytická MeSH

Helix 38 (H38) of the large ribosomal subunit, with a length of 110 A, reaches the small subunit through intersubunit bridge B1a. Previous cryo-EM studies revealed that the tip of H38 moves by more than 10 A from the non-ratcheted to the ratcheted state of the ribosome while mutational studies implicated a key role of flexible H38 in attenuation of translocation and in dynamical signaling between ribosomal functional centers. We investigate a region including the elbow-shaped kink-turn (Kt-38) in the Haloarcula marismortui archaeal ribosome, and equivalently positioned elbows in three eubacterial species, located at the H38 base. We performed explicit solvent molecular dynamics simulations on the H38 elbows in all four species. They are formed by at first sight unrelated sequences resulting in diverse base interactions but built with the same overall topology, as shown by X-ray crystallography. The elbows display similar fluctuations and intrinsic flexibilities in simulations indicating that the eubacterial H38 elbows are structural and dynamical analogs of archaeal Kt-38. We suggest that this structural element plays a pivotal role in the large motions of H38 and may act as fulcrum for the abovementioned tip motion. The directional flexibility inferred from simulations correlates well with the cryo-EM results.

• MeSH
• chlorid draselný chemie   MeSH
• Deinococcus genetika   MeSH
• elektronová kryomikroskopie MeSH
• Escherichia coli genetika   MeSH
• Haloarcula marismortui genetika   MeSH
• konformace nukleové kyseliny MeSH
• RNA ribozomální 23S chemie   MeSH
• simulace molekulární dynamiky MeSH
• sodík chemie   MeSH
• Thermus thermophilus genetika   MeSH
• velké podjednotky ribozomu archebakteriální chemie   MeSH
• velké podjednotky ribozomu bakteriální chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• chlorid draselný MeSH
• RNA ribozomální 23S MeSH
• sodík MeSH

Functional RNA molecules such as ribosomal RNAs frequently contain highly conserved internal loops with a 5'-UAA/5'-GAN (UAA/GAN) consensus sequence. The UAA/GAN internal loops adopt distinctive structure inconsistent with secondary structure predictions. The structure has a narrow major groove and forms a trans Hoogsteen/Sugar edge (tHS) A/G base pair followed by an unpaired stacked adenine, a trans Watson-Crick/Hoogsteen (tWH) U/A base pair and finally by a bulged nucleotide (N). The structure is further stabilized by a three-adenine stack and base-phosphate interaction. In the ribosome, the UAA/GAN internal loops are involved in extensive tertiary contacts, mainly as donors of A-minor interactions. Further, this sequence can adopt an alternative 2D/3D pattern stabilized by a four-adenine stack involved in a smaller number of tertiary interactions. The solution structure of an isolated UAA/GAA internal loop shows substantially rearranged base pairing with three consecutive non-Watson-Crick base pairs. Its A/U base pair adopts an incomplete cis Watson-Crick/Sugar edge (cWS) A/U conformation instead of the expected Watson-Crick arrangement. We performed 3.1 µs of explicit solvent molecular dynamics (MD) simulations of the X-ray and NMR UAA/GAN structures, supplemented by MM-PBSA free energy calculations, locally enhanced sampling (LES) runs, targeted MD (TMD) and nudged elastic band (NEB) analysis. We compared parm99 and parmbsc0 force fields and net-neutralizing Na(+) vs. excess salt KCl ion environments. Both force fields provide a similar description of the simulated structures, with the parmbsc0 leading to modest narrowing of the major groove. The excess salt simulations also cause a similar effect. While the NMR structure is entirely stable in simulations, the simulated X-ray structure shows considerable widening of the major groove, loss of base-phosphate interaction and other instabilities. The alternative X-ray geometry even undergoes conformational transition towards the solution 2D structure. Free energy calculations confirm that the X-ray arrangement is less stable than the solution structure. LES, TMD and NEB provide a rather consistent pathway for interconversion between the X-ray and NMR structures. In simulations, the incomplete cWS A/U base pair of the NMR structure is water mediated and alternates with the canonical A-U base pair, which is not indicated by the NMR data. Completion of full cWS A/U base pair is prevented by the overall internal loop arrangement. In summary, the simulations confirm that the UAA/GAN internal loop is a molecular switch RNA module that adopts its functional geometry upon specific tertiary contexts.

• Publikační typ
• časopisecké články MeSH