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

LNA-induced dynamic stability in a therapeutic aptamer:insights from molecular dynamics simulations

Modulation of structural and thermodynamic properties of nucleic acids with synthetic modifications is a promising area of research with possible applications in nanotechnology and nanotherapeutics. Locked nucleic acid (LNA) is one such modification in which the C4’ and O2’ atoms of the sugar moiety are connected through a methylene bridge. The LNA modified DNA aptamer RNV66, and its unmodified counterpart V7t1, both of which target the vascular endothelial growth factor (VEGF) implicated in oncogenic angiogenesis, have a G-rich tract that can fold into G-quadruplex structures. However, it is not understood why V7t1 has a polymorphic structure while its LNA modified counterpart RNV66 has a unique quadruplex fold with higher nuclease resistance, thermal stability and greater binding affinity for VEGF. In this work, we have performed extensive molecular dynamics simulations of RNV66 and V7t1 to study and compare the structural and dynamic consequences of the insertion of LNAs. It was observed that the increase in dynamic stability was significant in the presence of LNA residues and our protocol for combining different torsional parameters using OL15 for the DNA aptamer and parm99_LNA along with parmbsc0 and βOL15 for the LNAs nicely reproduced the experimentally observed conformational features of RNV66. Our observations would help in further theoretical studies in understanding the lack of frustration in the folding of the LNA modified aptamer and its higher affinity for VEGF. Communicated by Ramaswamy H. Sarma.</p

Predicting nearest neighbor free energies of modified RNA with LIE: Results for pseudouridine and N1-methylpseudouridine within RNA duplexes

Pseudouridine and N1-methylpseudouridine are the key modifications in the field of mRNA therapeutics and vaccine research. The accuracy of the design and development of therapeutic RNAs containing such modifications requires the accuracy of the secondary structure prediction, that depends on the nearest neighbor (NN) thermodynamic parameters for the standard and modified residues. The development of such NN thermodynamic parameters requires expensive and time-consuming experimental studies. There were some earlier attempts to predict the NN free energies of modified RNA using computational methods but those are either computationally expensive or not accurate enough. Here, we propose a new protocol based on MD simulations, which is able to predict the NN free energy parameters (ΔG◦37) for U-A, Ψ-A and m1Ψ-A pairs in general agreement with the recent experimental reports. We report the NN thermodynamic parameters for different U, Ψ and m1Ψ base pairs, which might be helpful for a deeper understanding of the effect of these modifications in RNA. The presence of m1Ψ resulted in more stable NN pairs compared to those containing U or Ψ. The predicted NN free energy parameters in this study are able to closely reproduce the folding free energies of duplexes containing internal Ψ for which the thermodynamic data were available. Additionally, we report the predicted folding free energies for the duplexes containing internal m1Ψ

Data-informed reparameterization of modified RNA and the effect of explicit water models:application to pseudouridine and derivatives

Pseudouridine is one of the most abundant post-transcriptional modifications in RNA. We have previously shown that the FF99-derived parameters for pseudouridine and some of its naturally occurring derivatives in the AMBER distribution either alone or in combination with the revised γ torsion parameters (parmbsc0) failed to reproduce their conformational characteristics observed experimentally (Deb et al. in J Chem Inf Model 54:1129–1142, 2014; Deb et al. in J Comput Chem 37:1576–1588, 2016; Dutta et al. in J Chem Inf Model 60:4995–5002, 2020). However, the application of the recommended bsc0 correction did lead to an improvement in the description not only of the distribution in the γ torsional space but also of the sugar pucker distributions. In an earlier study, we examined the transferability of the revised glycosidic torsion parameters (χ(IDRP)) for Ψ to its derivatives. We noticed that although these parameters in combination with the AMBER FF99-derived parameters and the revised γ torsional parameters resulted in conformational properties of these residues that were in better agreement with experimental observations, the sugar pucker distributions were still not reproduced accurately. Here we report a new set of partial atomic charges for pseudouridine, 1-methylpseudouridine, 3-methylpseudouridine and 2′-O-methylpseudouridine and a new set of glycosidic torsional parameters (χ(ND)) based on chosen glycosidic torsional profiles that most closely corresponded to the NMR data for conformational propensities and studied their effect on the conformational distributions using REMD simulations at the individual nucleoside level. We have also studied the effect of the choice of water model on the conformational characteristics of these modified nucleosides. Our observations suggest that the current revised set of parameters and partial atomic charges describe the sugar pucker distributions for these residues more accurately and that the choice of a suitable water model is important for the accurate description of their conformational properties. We have further validated the revised sets of parameters by studying the effect of substitution of uridine with pseudouridine within single stranded RNA oligonucleotides on their conformational and hydration characteristics. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10822-022-00447-4

Data-informed reparameterization of modified RNA and the effect of explicit water models: Application to pseudouridine and derivatives

Pseudouridine is the most abundant post-transcriptional modification in RNA. We have previously shown that the FF99-derived parameters for pseudouridine and some of its naturally occurring derivatives in the AMBER distribution either alone or in combination with the revised torsion parameters (parmbsc0) failed to reproduce their conformational characteristics observed experimentally (Deb I, et al. J. Chem. Inf. Model. 2014, 54 (4):1129–1142; Deb I, et al. J. Comput. Chem., 2016, 37:1576−1588; Dutta N, et al. J. Chem. Inf. Model. 2020, 60 (10):4995–5002). However, the application of the recommended bsc0 correction did lead to an improvement in the description not only of the distribution in the torsional space but also of the sugar pucker distributions. In an earlier study, we examined the transferability of the revised glycosidic torsion parameters (IDRP) for Ψ to its derivatives. We noticed that although these parameters in combination with the AMBER FF99-derived parameters and the revised torsional parameters resulted in conformational properties of these residues that were in better agreement with experimental observations, the sugar pucker distributions were still not reproduced accurately. Here we report a new set of partial atomic charges for pseudouridine, 1-methylpseudouridine, 3-methylpseudouridine and 2′-O-methylpseudouridine and a new set of glycosidic torsional parameters (ND) based on chosen glycosidic torsional profiles that most closely corresponded to the NMR data for conformational propensities and studied their effect on the conformational distributions using REMD simulations at the individual nucleoside level. We have also studied the effect of the choice of water model on the conformational characteristics of these modified nucleosides. Our observations suggest that the current revised set of parameters and partial atomic charges describe the sugar pucker distributions for these residues more accurately and that the choice of a suitable water model is important for the accurate description of their conformational properties

Reparameterizations of the χ Torsion and Lennard-Jones σ Parameters Improve the Conformational Characteristics of Modified Uridines

The currently available force field parameters for modified RNA residues in AMBER show significant deviations in conformational properties from experimental observations. The examination of the transferability of the recently revised torsion parameters revealed that there was an overall improvement in the conformational properties for some of the modifications but the improvements were still insufficient in describing the sugar pucker preferences (J. Chem. Inf. Model. 2014, 54, 1129-1142). Here, we report an approach for the development and fine tuning of the AMBER force field parameters for 2-thiouridine, 4-thiouridine, and pseudouridine with diverse conformational preferences. The χ torsion parameters were reparameterized at the individual nucleoside level. The effect of combining the revised γ torsion parameter and modifying the Lennard-Jones σ parameters were also tested by directly comparing the conformational preferences obtained from our extensive molecular dynamics simulations with those from experimental observations.</p