Watson-Crick-like pairs in CCUG repeats: evidence for tautomeric shifts or protonation.

RNA transcripts that include expanded CCUG repeats are associated with myotonic dystrophy type 2. Crystal structures of two CCUG-containing oligomers show that the RNA strands associate into slipped duplexes that contain noncanonical C-U pairs that have apparently undergone tautomeric transition or protonation resulting in an unusual Watson-Crick-like pairing. The overhanging ends of the duplexes interact forming U-U pairs, which also show tautomerism. Duplexes consisting of CCUG repeats are thermodynamically less stable than the trinucleotide repeats involved in the TRED genetic disorders, but introducing LNA residues increases their stability and raises the melting temperature of the studied oligomers by ∼10°C, allowing detailed crystallographic studies. Quantum mechanical calculations were performed to test the possibility of the tautomeric transitions or protonation within the noncanonical pairs. The results indicate that tautomeric or ionic shifts of nucleobases can manifest themselves in biological systems, supplementing the canonical "rules of engagement."

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.

Modeling of the catalytic core of Arabidopsis thaliana Dicer-like 4 protein and its complex with double-stranded RNA.

Plant Dicer-like proteins (DCLs) belong to the Ribonuclease III (RNase III) enzyme family. They are involved in the regulation of gene expression and antiviral defense through RNA interference pathways. A model plant, Arabidopsis thaliana encodes four DCL proteins (AtDCL1-4) that produce different classes of small regulatory RNAs. Our studies focus on AtDCL4 that processes double-stranded RNAs (dsRNAs) into 21 nucleotide trans-acting small interfering RNAs. So far, little is known about the structures of plant DCLs and the complexes they form with dsRNA. In this work, we present models of the catalytic core of AtDCL4 and AtDCL4-dsRNA complex constructed by computational methods. We built a homology model of the catalytic core of AtDCL4 comprising Platform, PAZ, Connector helix and two RNase III domains. To assemble the AtDCL4-dsRNA complex two modeling approaches were used. In the first method, to establish conformations that allow building a consistent model of the complex, we used Normal Mode Analysis for both dsRNA and AtDCL4. The second strategy involved template-based approach for positioning of the PAZ domain and manual arrangement of the Connector helix. Our results suggest that the spatial orientation of the Connector helix, Platform and PAZ relative to the RNase III domains is crucial for measuring dsRNA of defined length. The modeled complexes provide information about interactions that may contribute to the relative orientations of these domains and to dsRNA binding. All these information can be helpful for understanding the mechanism of AtDCL4-mediated dsRNA recognition and binding, to produce small RNA of specific size.

Dynamics and stability of GCAA tetraloops with 2-aminopurine and purine substitutions.

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 tetraloop's 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 glycosyl 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 A's 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.

Antivirally active ribavirin analogues--4,5-disubstituted 1,2,3-triazole nucleosides: biological evaluation against certain respiratory viruses and computational modelling.

BACKGROUND: Ribavirin is a broad-spectrum antiviral agent that derives some of its activity from inhibition of cellular inosine monophosphate dehydrogenase (IMPDH), resulting in lower guanosine triphosphate (GTP) levels. Here we report the biological activities of three ribavirin analogues. METHODS: Antiviral activities of test compounds were performed by in vitro cytopathic effect inhibition assays against influenza A (H1N1, H3N2 and H5N1), influenza B, measles, parainfluenza type 3 (PIV-3) and respiratory syncytial viruses. Compounds were modelled into the ribavirin 5'-monophosphate binding site of the crystallographic structure of the human type II IMPDH (hIMPDH2) ternary complex. Effects of compounds on intracellular GTP levels were performed by strong anion exchange HPLC analysis. RESULTS: Of the three compounds evaluated, the 5-ethynyl nucleoside (ETCAR) exhibited virus-inhibitory activities (at 1.2-20 µM, depending upon the virus) against most of the viruses, except for weak activity against PIV-3 (62 µM). Antiviral activity of ETCAR was similar to ribavirin; however, cytotoxicity of ETCAR was greater than ribavirin. Replacing the 5-ethynyl group with a 5-propynyl or bromo substituent (BrCAR) considerably reduced antiviral activity. Computational studies of ternary complexes of hIMPDH2 enzyme with 5'-monophosphates of the compounds helped rationalize the observed differences in biological activity. All compounds suppressed GTP levels in cells; additionally, BrCAR suppressed adenosine triphosphate and elevated uridine triphosphate levels. CONCLUSIONS: Three compounds related to ribavirin inhibited IMPDH and had weak to moderate antiviral activity. Cytotoxicity adversely affected the antiviral selectivity of ETCAR. As with ribavirin, reduction in intracellular GTP may play a role in virus inhibition.

Mapping the interactions of selected antibiotics and their Cu2+ complexes with the antigenomic δ ribozyme.

The interactions of selected antibiotics with the trans-acting antigenomic delta ribozyme were mapped. Ribozyme with two oligonucleotide substrates was used, one uncleavable with deoxycytidine at the cleavage site, mimicking the initial state of ribozyme, and the other with an all-RNA substrate mimicking, after cleavage, the product state. Mapping was performed with a set of RNA structural probing methods: Pb(2+) -induced cleavage, nuclease digestion, and the selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) approach. The experimental results combined with molecular modeling revealed different binding sites for neomycin B, amikacin and actinomycin D inside the ribozyme structure. Neomycin B, an aminoglycoside antibiotic, which strongly inhibited the catalytic properties of delta ribozyme, was bound to the pocket formed by the P1 stem, the P1.1 pseudoknot, and the J4/2 junction. Amikacin showed less effective binding to the ribozyme catalytic core, resulting in weak inhibition. Complexes of these aminoglycosides with Cu(2+) ions were bound to the same ribozyme regions, but more effectively, showing lower Kd values. On the other hand, the Cu(2+) complex of the cyclopeptide antibiotic actinonomycin D was preferentially intercalated into the P2 and the P4 double-stranded region, and was three times more potent in ribozyme inhibition than the free antibiotic. In addition, some differences in SHAPE reactivities between the ribozyme forms containing all-RNA and deoxycytidine-modified substrates in the J4/2 region were detected, pointing to different ribozyme conformations before and after the cleavage event.

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.

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.

Thermodynamic stability of RNA structures formed by CNG trinucleotide repeats. Implication for prediction of RNA structure.

Trinucleotide repeat expansion diseases (TREDs) are correlated with elongation of CNG DNA and RNA repeats to pathological level. This paper shows, for the first time, complete data concerning thermodynamic stabilities of RNA with CNG trinucleotide repeats. Our studies include the stability of oligoribonucleotides composed of two to seven of CAG, CCG, CGG, and CUG repeats. The thermodynamic parameters of helix propagation correlated with the presence of multiple N-N mismatches within CNG RNA duplexes were also determined. Moreover, the total stability of CNG RNA hairpins, as well as the contribution of trinucleotide repeats placed only in the stem or loop regions, was evaluated. The improved thermodynamic parameters allow to predict much more accurately the thermodynamic stabilities and structures of CNG RNAs.

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.

The apical loop of the HIV-1 TAR RNA hairpin is stabilized by a cross-loop base pair.

The TAR hairpin of the HIV-1 RNA genome is indispensable for trans-activation of the viral promoter and virus replication. The TAR structure has been studied extensively, but most attention has been directed at the three-nucleotide bulge that constitutes the binding site of the viral Tat protein. In contrast, the conformational properties of the apical loop have remained elusive. We performed biochemical studies and molecular dynamics simulations, which indicate that the TAR loop is structured and stabilized by a cross-loop base pair between residues C30 and G34. Mutational disruption of the cross-loop base pair results in reduced Tat response of the LTR promoter, which can be rescued by compensatory mutations that restore the base pair. Thus, Tat-mediated transcriptional activation depends on the structure of the TAR apical loop. The C30-G34 cross-loop base pair classes TAR in a growing family of hairpins with a structured loop that was recently identified in ribosomal RNA, tRNA, and several viral and cellular mRNAs.