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

Pseudouridine (Ψ) and N1-methylpseudouridine (m1Ψ) are among 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 depends on the accuracy of the secondary structure prediction, which in turn depends on the nearest neighbor (NN) thermodynamic parameters for the standard and modified residues. Here, we propose a simple approach based on molecular dynamics simulations and linear interaction energy (LIE) approximation that is able to predict the NN free energy parameters for U-A, Ψ-A and m1Ψ-A pairs in reasonable 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 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.

RNA tertiary structure prediction using RNAComposer in CASP15.

As CASP15 participants, in the new category of 3D RNA structure prediction, we applied expert modeling with the support of our proprietary system RNAComposer. Although RNAComposer is primarily known as an automated web server, its features allow it to be used interactively, for example, for homology-based modeling or assembling models from user-provided structural elements. In the paper, we present various scenarios of applying the system to predict the 3D RNA structures that we employed. Their combination with expert input, comparative analysis of models, and routines to select representative resultant structures form a ready-for-reuse workflow. With selected examples, we demonstrate its application for the in silico modeling of natural and synthetic RNA molecules targeted in CASP15.

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 ßOL15 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.

Computational Pipeline for Reference-Free Comparative Analysis of RNA 3D Structures Applied to SARS-CoV-2 UTR Models.

RNA is a unique biomolecule that is involved in a variety of fundamental biological functions, all of which depend solely on its structure and dynamics. Since the experimental determination of crystal RNA structures is laborious, computational 3D structure prediction methods are experiencing an ongoing and thriving development. Such methods can lead to many models; thus, it is necessary to build comparisons and extract common structural motifs for further medical or biological studies. Here, we introduce a computational pipeline dedicated to reference-free high-throughput comparative analysis of 3D RNA structures. We show its application in the RNA-Puzzles challenge, in which five participating groups attempted to predict the three-dimensional structures of 5'- and 3'-untranslated regions (UTRs) of the SARS-CoV-2 genome. We report the results of this puzzle and discuss the structural motifs obtained from the analysis. All simulated models and tools incorporated into the pipeline are open to scientific and academic use.

RNAspider: a webserver to analyze entanglements in RNA 3D structures.

Advances in experimental and computational techniques enable the exploration of large and complex RNA 3D structures. These, in turn, reveal previously unstudied properties and motifs not characteristic for small molecules with simple architectures. Examples include entanglements of structural elements in RNA molecules and knot-like folds discovered, among others, in the genomes of RNA viruses. Recently, we presented the first classification of entanglements, determined by their topology and the type of entangled structural elements. Here, we introduce RNAspider - a web server to automatically identify, classify, and visualize primary and higher-order entanglements in RNA tertiary structures. The program applies to evaluate RNA 3D models obtained experimentally or by computational prediction. It supports the analysis of uncommon topologies in the pseudoknotted RNA structures. RNAspider is implemented as a publicly available tool with a user-friendly interface and can be freely accessed at https://rnaspider.cs.put.poznan.pl/.

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 (χIDRP) 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 (χND) 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.

Inosine and its methyl derivatives: Occurrence, biogenesis, and function in RNA.

Inosine is one of the most common post-transcriptional modifications. Since its discovery, it has been noted for its ability to contribute to non-Watson-Crick interactions within RNA. Rapidly accumulating evidence points to the widespread generation of inosine through hydrolytic deamination of adenosine to inosine by different classes of adenosine deaminases. Three naturally occurring methyl derivatives of inosine, i.e., 1-methylinosine, 2'-O-methylinosine and 1,2'-O-dimethylinosine are currently reported in RNA modification databases. These modifications are expected to lead to changes in the structure, folding, dynamics, stability and functions of RNA. The importance of the modifications is indicated by the strong conservation of the modifying enzymes across organisms. The structure, binding and catalytic mechanism of the adenosine deaminases have been well-studied, but the underlying mechanism of the catalytic reaction is not very clear yet. Here we extensively review the existing data on the occurrence, biogenesis and functions of inosine and its methyl derivatives in RNA. We also included the structural and thermodynamic aspects of these modifications in our review to provide a detailed and integrated discussion on the consequences of A-to-I editing in RNA and the contribution of different structural and thermodynamic studies in understanding its role in RNA. We also highlight the importance of further studies for a better understanding of the mechanisms of the different classes of deamination reactions. Further investigation of the structural and thermodynamic consequences and functions of these modifications in RNA should provide more useful information about their role in different diseases.

Entanglements of structure elements revealed in RNA 3D models.

Computational methods to predict RNA 3D structure have more and more practical applications in molecular biology and medicine. Therefore, it is crucial to intensify efforts to improve the accuracy and quality of predicted three-dimensional structures. A significant role in this is played by the RNA-Puzzles initiative that collects, evaluates, and shares RNAs built computationally within currently nearly 30 challenges. RNA-Puzzles datasets, subjected to multi-criteria analysis, allow revealing the strengths and weaknesses of computer prediction methods. Here, we study the issue of entangled RNA fragments in the predicted RNA 3D structure models. By entanglement, we mean an arrangement of two structural elements such that one of them passes through the other. We propose the classification of entanglements driven by their topology and components. It distinguishes two general classes, interlaces and lassos, and subclasses characterized by element types-loops, dinucleotide steps, open single-stranded fragments-and puncture multiplicity. Our computational pipeline for entanglement detection, applied for 1,017 non-redundant models from RNA-Puzzles, has shown the frequency of different entanglements and allowed identifying 138 structures with intersected assemblies.

The model structure of the hammerhead ribozyme formed by RNAs of reciprocal chirality.

RNA-based tools are frequently used to modulate gene expression in living cells. However, the stability and effectiveness of such RNA-based tools is limited by cellular nuclease activity. One way to increase RNA's resistance to nucleases is to replace its D-ribose backbone with L-ribose isomers. This modification changes chirality of an entire RNA molecule to L-form giving it more chance of survival when introduced into cells. Recently, we have described the activity of left-handed hammerhead ribozyme (L-Rz, L-HH) that can specifically hydrolyse RNA with the opposite chirality at a predetermined location. To understand the structural background of the RNA specific cleavage in a heterochiral complex, we used circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy as well as performed molecular modelling and dynamics simulations of homo- and heterochiral RNA complexes. The active ribozyme-target heterochiral complex showed a mixed chirality as well as low field imino proton NMR signals. We modelled the 3D structures of the oligoribonucleotides with their ribozyme counterparts of reciprocal chirality. L- or D-ribozyme formed a stable, homochiral helix 2, and two short double heterochiral helixes 1 and 3 of D- or L-RNA strand thorough irregular Watson-Crick base pairs. The formation of the heterochiral complexes is supported by the result of simulation molecular dynamics. These new observations suggest that L-catalytic nucleic acids can be used as tools in translational biology and diagnostics.

How bioinformatics resources work with G4 RNAs.

Quadruplexes (G4s) are of interest, which increases with the number of identified G4 structures and knowledge about their biomedical potential. These unique motifs form in many organisms, including humans, where their appearance correlates with various diseases. Scientists store and analyze quadruplexes using recently developed bioinformatic tools-many of them focused on DNA structures. With an expanding collection of G4 RNAs, we check how existing tools deal with them. We review all available bioinformatics resources dedicated to quadruplexes and examine their usefulness in G4 RNA analysis. We distinguish the following subsets of resources: databases, tools to predict putative quadruplex sequences, tools to predict secondary structure with quadruplexes and tools to analyze and visualize quadruplex structures. We share the results obtained from processing specially created RNA datasets with these tools. Contact: mszachniuk@cs.put.poznan.pl Supplementary information: Supplementary data are available at Briefings in Bioinformatics online.

Molecular Dynamics Simulation of the Conformational Preferences of Pseudouridine Derivatives: Improving the Distribution in the Glycosidic Torsion Space.

There are only four derivatives of pseudouridine (Ψ) that are known to occur naturally in RNA as post-transcriptional modifications. We have studied the conformational consequences of pseudouridylation and further modifications using replica exchange molecular dynamics simulations at the nucleoside level, and the simulated conformational preferences were compared with the available experimental (NMR) data. We found that the existing AMBER FF99-derived parameters for these nucleosides did not reproduce the observed experimental features and while the recommended bsc0 correction could be combined with these parameters leading to an improvement in the description of sugar pucker distributions, the χOL3 correction could not be applied to these nucleosides as such because of base isomerization. On the other hand, the revised χ torsion parameters (χIDRP) for Ψ developed earlier by us (Deb, I., J. Comput. Chem., 2016, 37, 1576-1588) in combination with the AMBER provided parameters and the revised γ torsion parameters generated conformational distributions, which generally were in better agreement with the experimental data. A significant shift of the distribution of base orientation toward the syn conformation was observed with our revised parameter sets compared to the large excess of anti conformation predicted by the FF99 parameters. Overall, our observations indicated that our revised set of parameters (χIDRP) for Ψ were also able to generate conformational distributions for all of the derivatives of Ψ in better agreement with the experimental data.

The origin of the high stability of 3'-terminal uridine tetrads: contributions of hydrogen bonding, stacking interactions, and steric factors evaluated using modified oligonucleotide analogs.

RNA G-quadruplexes fold almost exclusively into parallel-stranded structures and thus display much less structural diversity than their DNA counterparts. However, also among RNA G-quadruplexes peculiar structural elements can be found which are capable of reshaping the physico-chemical properties of the folded structure. A striking example is provided by a uridine tetrad (U-tetrad) placed on the 3'-terminus of the tetramolecular G-quadruplex. In this context, the U-tetrad adopts a unique conformation involving chain reversal and is responsible for a tremendous stabilization of the G-quadruplex (ΔTm up to 30°C). In this report, we attempt to rationalize the origin of this stabilizing effect by concurrent structural, thermal stability, and molecular dynamics studies of a series of G-quadruplexes with subtle chemical modifications at their 3'-termini. Our results provide detailed insights into the energetics of the "reversed" U-tetrad motif and the requirements for its formation. They point to the importance of the 2'OH to phosphate hydrogen bond and preferential stacking interactions for the formation propensity and stability of the motif.

Ensemble Allosteric Model for the Modified Wobble Hypothesis.

The residue 2-thiouridine (s2U) provides a remarkable example for the "modified wobble" hypothesis, which postulates that some post-transcriptional modifications at the wobble position of tRNAs restrict recognition of degenerate codons. Through extensive molecular dynamics simulations using our χIDRP force field parameters, we demonstrate how this modification shifts the conformational ensemble from a predominantly disordered, as in the case of an RNA pentamer (GUUUC), to a substantially ordered population in Gs2UUUC. Our simulations clearly showed that the van der Waals interaction of sulfur played a major role in driving the disorder-to-order transition. The conformational redistribution and the slowing down of the transition between the clusters within the population in the presence of s2U suggest ensemble allostery to be a key mechanism that may play a general role in the functioning of the wobble modifications of tRNAs.

RNA-Puzzles Round IV: 3D structure predictions of four ribozymes and two aptamers.

RNA-Puzzles is a collective endeavor dedicated to the advancement and improvement of RNA 3D structure prediction. With agreement from crystallographers, the RNA structures are predicted by various groups before the publication of the crystal structures. We now report the prediction of 3D structures for six RNA sequences: four nucleolytic ribozymes and two riboswitches. Systematic protocols for comparing models and crystal structures are described and analyzed. In these six puzzles, we discuss (i) the comparison between the automated web servers and human experts; (ii) the prediction of coaxial stacking; (iii) the prediction of structural details and ligand binding; (iv) the development of novel prediction methods; and (v) the potential improvements to be made. We show that correct prediction of coaxial stacking and tertiary contacts is essential for the prediction of RNA architecture, while ligand binding modes can only be predicted with low resolution and simultaneous prediction of RNA structure with accurate ligand binding still remains out of reach. All the predicted models are available for the future development of force field parameters and the improvement of comparison and assessment tools.

Topology-based classification of tetrads and quadruplex structures.

MOTIVATION: Quadruplexes attract the attention of researchers from many fields of bio-science. Due to a specific structure, these tertiary motifs are involved in various biological processes. They are also promising therapeutic targets in many strategies of drug development, including anticancer and neurological disease treatment. The uniqueness and diversity of their forms cause that quadruplexes show great potential in novel biological applications. The existing approaches for quadruplex analysis are based on sequence or 3D structure features and address canonical motifs only. RESULTS: In our study, we analyzed tetrads and quadruplexes contained in nucleic acid molecules deposited in Protein Data Bank. Focusing on their secondary structure topology, we adjusted its graphical diagram and proposed new dot-bracket and arc representations. We defined the novel classification of these motifs. It can handle both canonical and non-canonical cases. Based on this new taxonomy, we implemented a method that automatically recognizes the types of tetrads and quadruplexes occurring as unimolecular structures. Finally, we conducted a statistical analysis of these motifs found in experimentally determined nucleic acid structures in relation to the new classification. AVAILABILITY AND IMPLEMENTATION: https://github.com/tzok/eltetrado/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Computational and NMR studies of RNA duplexes with an internal pseudouridine-adenosine base pair.

Pseudouridine (Ψ) is the most common chemical modification present in RNA. In general, Ψ increases the thermodynamic stability of RNA. However, the degree of stabilization depends on the sequence and structural context. To explain experimentally observed sequence dependence of the effect of Ψ on the thermodynamic stability of RNA duplexes, we investigated the structure, dynamics and hydration of RNA duplexes with an internal Ψ-A base pair in different nearest-neighbor sequence contexts. The structures of two RNA duplexes containing 5'-GΨC/3'-CAG and 5'-CΨG/3'-GAC motifs were determined using NMR spectroscopy. To gain insight into the effect of Ψ on duplex dynamics and hydration, we performed molecular dynamics (MD) simulations of RNA duplexes with 5'-GΨC/3'-CAG, 5'-CΨG/3'-GAC, 5'-AΨU/3'-UAA and 5'-UΨA/3'-AAU motifs and their unmodified counterparts. Our results showed a subtle impact from Ψ modification on the structure and dynamics of the RNA duplexes studied. The MD simulations confirmed the change in hydration pattern when U is replaced with Ψ. Quantum chemical calculations showed that the replacement of U with Ψ affected the intrinsic stacking energies at the base pair steps depending on the sequence context. The calculated intrinsic stacking energies help to explain the experimentally observed sequence dependent changes in the duplex stability from Ψ modification.

Unraveling the structural basis for the exceptional stability of RNA G-quadruplexes capped by a uridine tetrad at the 3' terminus.

Uridine tetrads (U-tetrads) are a structural element encountered in RNA G-quadruplexes, for example, in the structures formed by the biologically relevant human telomeric repeat RNA. For these molecules, an unexpectedly strong stabilizing influence of a U-tetrad forming at the 3' terminus of a quadruplex was reported. Here we present the high-resolution solution NMR structure of the r(UGGUGGU)4 quadruplex which, in our opinion, provides an explanation for this stabilization. Our structure features a distinctive, abrupt chain reversal just prior to the 3' uridine tetrad. Similar "reversed U-tetrads" were already observed in the crystalline phase. However, our NMR structure coupled with extensive explicit solvent molecular dynamics (MD) simulations identifies some key features of this motif that up to now remained overlooked. These include the presence of an exceptionally stable 2'OH to phosphate hydrogen bond, as well as the formation of an additional K+ binding pocket in the quadruplex groove.

Mutations in human AID differentially affect its ability to deaminate cytidine and 5-methylcytidine in ssDNA substrates in vitro.

Activation-induced cytidine deaminase (AID) is known for its established role in antibody production. AID induces the diversification of antibodies by deaminating deoxycytidine (C) within immunoglobulin genes. The capacity of AID to deaminate 5-methyldeoxycytidine (5 mC) and/or 5-hydroxymethyldeoxycytidine (5 hmC), and consequently AID involvement in active DNA demethylation, is not fully resolved. For instance, structural determinants of AID activity on different substrates remain to be identified. To better understand the latter issue, we tested how mutations in human AID (hAID) influence its ability to deaminate C, 5 mC, and 5 hmC in vitro. We showed that each of the selected mutations differentially affects hAID's ability to deaminate C and 5 mC. At the same time, we did not observe hAID activity on 5 hmC. Surprisingly, we found that the N51A hAID mutant, with no detectable activity on C, efficiently deaminated 5 mC, which may suggest different requirements for C and 5 mC deamination. Homology modeling and molecular dynamics simulations revealed that the pattern of enzyme-substrate recognition is one of the important factors determining enzyme activity on C and 5 mC. Consequently, we have proposed mechanisms that explain why wild type hAID more efficiently deaminates C than 5 mC in vitro and why 5 hmC is not deaminated.

RNA-Puzzles Round III: 3D RNA structure prediction of five riboswitches and one ribozyme.

RNA-Puzzles is a collective experiment in blind 3D RNA structure prediction. We report here a third round of RNA-Puzzles. Five puzzles, 4, 8, 12, 13, 14, all structures of riboswitch aptamers and puzzle 7, a ribozyme structure, are included in this round of the experiment. The riboswitch structures include biological binding sites for small molecules (S-adenosyl methionine, cyclic diadenosine monophosphate, 5-amino 4-imidazole carboxamide riboside 5'-triphosphate, glutamine) and proteins (YbxF), and one set describes large conformational changes between ligand-free and ligand-bound states. The Varkud satellite ribozyme is the most recently solved structure of a known large ribozyme. All puzzles have established biological functions and require structural understanding to appreciate their molecular mechanisms. Through the use of fast-track experimental data, including multidimensional chemical mapping, and accurate prediction of RNA secondary structure, a large portion of the contacts in 3D have been predicted correctly leading to similar topologies for the top ranking predictions. Template-based and homology-derived predictions could predict structures to particularly high accuracies. However, achieving biological insights from de novo prediction of RNA 3D structures still depends on the size and complexity of the RNA. Blind computational predictions of RNA structures already appear to provide useful structural information in many cases. Similar to the previous RNA-Puzzles Round II experiment, the prediction of non-Watson-Crick interactions and the observed high atomic clash scores reveal a notable need for an algorithm of improvement. All prediction models and assessment results are available at http://ahsoka.u-strasbg.fr/rnapuzzles/.

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.