RCDPeaks: memory-efficient density peaks clustering of long molecular dynamics.

MOTIVATION: Density Peaks is a widely spread clustering algorithm that has been previously applied to Molecular Dynamics (MD) simulations. Its conception of cluster centers as elements displaying both a high density of neighbors and a large distance to other elements of high density, particularly fits the nature of a geometrical converged MD simulation. Despite its theoretical convenience, implementations of Density Peaks carry a quadratic memory complexity that only permits the analysis of relatively short trajectories. RESULTS: Here, we describe DP+, an exact novel implementation of Density Peaks that drastically reduces the RAM consumption in comparison to the scarcely available alternatives designed for MD. Based on DP+, we developed RCDPeaks, a refined variant of the original Density Peaks algorithm. Through the use of DP+, RCDPeaks was able to cluster a one-million frames trajectory using less than 4.5 GB of RAM, a task that would have taken more than 2 TB and about 3× more time with the fastest and less memory-hunger alternative currently available. Other key features of RCDPeaks include the automatic selection of parameters, the screening of center candidates and the geometrical refining of returned clusters. AVAILABILITY AND IMPLEMENTATION: The source code and documentation of RCDPeaks are free and publicly available on GitHub (https://github.com/LQCT/RCDPeaks.git). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

MDSCAN: RMSD-based HDBSCAN clustering of long molecular dynamics.

MOTIVATION: The term clustering designates a comprehensive family of unsupervised learning methods allowing to group similar elements into sets called clusters. Geometrical clustering of molecular dynamics (MD) trajectories is a well-established analysis to gain insights into the conformational behavior of simulated systems. However, popular variants collapse when processing relatively long trajectories because of their quadratic memory or time complexity. From the arsenal of clustering algorithms, HDBSCAN stands out as a hierarchical density-based alternative that provides robust differentiation of intimately related elements from noise data. Although a very efficient implementation of this algorithm is available for programming-skilled users (HDBSCAN*), it cannot treat long trajectories under the de facto molecular similarity metric RMSD. RESULTS: Here, we propose MDSCAN, an HDBSCAN-inspired software specifically conceived for non-programmers users to perform memory-efficient RMSD-based clustering of long MD trajectories. Methodological improvements over the original version include the encoding of trajectories as a particular class of vantage-point tree (decreasing time complexity), and a dual-heap approach to construct a quasi-minimum spanning tree (reducing memory complexity). MDSCAN was able to process a trajectory of 1 million frames using the RMSD metric in about 21 h with <8 GB of RAM, a task that would have taken a similar time but more than 32 TB of RAM with the accelerated HDBSCAN* implementation generally used. AVAILABILITY AND IMPLEMENTATION: The source code and documentation of MDSCAN are free and publicly available on GitHub (https://github.com/LQCT/MDScan.git) and as a PyPI package (https://pypi.org/project/mdscan/). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

BitQT: a graph-based approach to the quality threshold clustering of molecular dynamics.

MOTIVATION: Classical Molecular Dynamics (MD) is a standard computational approach to model time-dependent processes at the atomic level. The inherent sparsity of increasingly huge generated trajectories demands clustering algorithms to reduce other post-simulation analysis complexity. The Quality Threshold (QT) variant is an appealing one from the vast number of available clustering methods. It guarantees that all members of a particular cluster will maintain a collective similarity established by a user-defined threshold. Unfortunately, its high computational cost for processing big data limits its application in the molecular simulation field. RESULTS: In this work, we propose a methodological parallel between QT clustering and another well-known algorithm in the field of Graph Theory, the Maximum Clique Problem. Molecular trajectories are represented as graphs whose nodes designate conformations, while unweighted edges indicate mutual similarity between nodes. The use of a binary-encoded RMSD matrix coupled to the exploitation of bitwise operations to extract clusters significantly contributes to reaching a very affordable algorithm compared to the few implementations of QT for MD available in the literature. Our alternative provides results in good agreement with the exact one while strictly preserving the collective similarity of clusters. AVAILABILITY AND IMPLEMENTATION: The source code and documentation of BitQT are free and publicly available on GitHub (https://github.com/LQCT/BitQT.git) and ReadTheDocs (https://bitqt.readthedocs.io/en/latest/), respectively. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Ribozyme Chemistry: To Be or Not To Be under High Pressure.

The use of high hydrostatic pressure to investigate structure-function relationships in biomacromolecules in solution provides precise information about conformational changes and variations of the interactions between these macromolecules and the solvent, as well as the volume changes associated with their activity. The complementary use of osmotic pressure reveals quantitatively the extent and direction of the water exchanges between the macromolecules and the solvent and the number of water molecules involved in these exchanges. In this review, the chemistry of ribozymes and the influence of pressure is described. In the case of the hairpin ribozyme, pressure slowed down the self-cleavage reaction on the basis that the formation of the transition state involves a positive ΔV⧧ of activation and the release of 78 ± 4 water molecules. The self-cleaving activity of the hammerhead ribozyme is also slowed down by pressure on the basis of kinetic parameters and ΔVs comparable to those of the hairpin ribozymes. However, it appears that the solution of the hammerhead ribozyme used in this study contains two populations of molecules which differ by the values of these parameters. The results obtained in the case of small self-cleaving ribozymes containing adenine bulges are consistent with the hypothesis that these small RNAs that bind amino acids or peptides could have appeared in prebiotic chemistry under extreme conditions in deep-sea vents or hydrothermal surface sites.

Revisiting the structure/function relationships of H/ACA(-like) RNAs: a unified model for Euryarchaea and Crenarchaea.

A structural and functional classification of H/ACA and H/ACA-like motifs is obtained from the analysis of the H/ACA guide RNAs which have been identified previously in the genomes of Euryarchaea (Pyrococcus) and Crenarchaea (Pyrobaculum). A unified structure/function model is proposed based on the common structural determinants shared by H/ACA and H/ACA-like motifs in both Euryarchaea and Crenarchaea. Using a computational approach, structural and energetic rules for the guide:target RNA-RNA interactions are derived from structural and functional data on the H/ACA RNP particles. H/ACA(-like) motifs found in Pyrococcus are evaluated through the classification and their biological relevance is discussed. Extra-ribosomal targets found in both Pyrococcus and Pyrobaculum might support the hypothesis of a gene regulation mediated by H/ACA(-like) guide RNAs in archaea.

RNA at 92 °C: the non-coding transcriptome of the hyperthermophilic archaeon Pyrococcus abyssi.

The non-coding transcriptome of the hyperthermophilic archaeon Pyrococcus abyssi is investigated using the RNA-seq technology. A dedicated computational pipeline analyzes RNA-seq reads and prior genome annotation to identify small RNAs, untranslated regions of mRNAs, and cis-encoded antisense transcripts. Unlike other archaea, such as Sulfolobus and Halobacteriales, P. abyssi produces few leaderless mRNA transcripts. Antisense transcription is widespread (215 transcripts) and targets protein-coding genes that are less conserved than average genes. We identify at least three novel H/ACA-like guide RNAs among the newly characterized non-coding RNAs. Long 5' UTRs in mRNAs of ribosomal proteins and amino-acid biosynthesis genes strongly suggest the presence of cis-regulatory leaders in these mRNAs. We selected a high-interest subset of non-coding RNAs based on their strong promoters, high GC-content, phylogenetic conservation, or abundance. Some of the novel small RNAs and long 5' UTRs display high GC contents, suggesting unknown structural RNA functions. However, we were surprised to observe that most of the high-interest RNAs are AU-rich, which suggests an absence of stable secondary structure in the high-temperature environment of P. abyssi. Yet, these transcripts display other hallmarks of functionality, such as high expression or high conservation, which leads us to consider possible RNA functions that do not require extensive secondary structure.

Prediction of molecular interactions and physicochemical properties relevant for vasopressin V2 receptor antagonism.

We have developed two ligand- and receptor-based computational approaches to study the physicochemical properties relevant to the biological activity of vasopressin V2 receptor (V2R) antagonist and eventually to predict the expected binding mode to V2R. The obtained quantitative structure activity relationship (QSAR) model showed a correlation of the antagonist activity with the hydration energy (EH2O), the polarizability (P), and the calculated partial charge on atom N7 (q6) of the common substructure. The first two descriptors showed a positive contribution to antagonist activity, while the third one had a negative contribution. V2R was modeled and further relaxed on a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline (POPC) membrane by molecular dynamics simulations. The receptor antagonist complexes were guessed by molecular docking, and the stability of the most relevant structures was also evaluated by molecular dynamics simulations. As a result, amino acid residues Q96, W99, F105, K116, F178, A194, F307, and M311 were identified with the probably most relevant antagonist-receptor interactions on the studied complexes. The proposed QSAR model could explain the molecular properties relevant to the antagonist activity. The contributions to the antagonist-receptor interaction appeared also in agreement with the binding mode of the complexes obtained by molecular docking and molecular dynamics. These models will be used in further studies to look for new V2R potential antagonist molecules.

Deficiency of the tRNATyr:Psi 35-synthase aPus7 in Archaea of the Sulfolobales order might be rescued by the H/ACA sRNA-guided machinery.

Up to now, Psi formation in tRNAs was found to be catalysed by stand-alone enzymes. By computational analysis of archaeal genomes we detected putative H/ACA sRNAs, in four Sulfolobales species and in Aeropyrum pernix, that might guide Psi 35 formation in pre-tRNA(Tyr)(GUA). This modification is achieved by Pus7p in eukarya. The validity of the computational predictions was verified by in vitro reconstitution of H/ACA sRNPs using the identified Sulfolobus solfataricus H/ACA sRNA. Comparison of Pus7-like enzymes encoded by archaeal genomes revealed amino acid substitutions in motifs IIIa and II in Sulfolobales and A. pernix Pus7-like enzymes. These conserved RNA:Psi-synthase- motifs are essential for catalysis. As expected, the recombinant Pyrococcus abyssi aPus7 was fully active and acted at positions 35 and 13 and other positions in tRNAs, while the recombinant S. solfataricus aPus7 was only found to have a poor activity at position 13. We showed that the presence of an A residue 3' to the target U residue is required for P. abyssi aPus7 activity, and that this is not the case for the reconstituted S. solfataricus H/ACA sRNP. In agreement with the possible formation of Psi 35 in tRNA(Tyr)(GUA) by aPus7 in P. abyssi and by an H/ACA sRNP in S. solfataricus, the A36G mutation in the P. abyssi tRNA(Tyr)(GUA) abolished Psi 35 formation when using P. abyssi extract, whereas the A36G substitution in the S. solfataricus pre-tRNA(Tyr) did not affect Psi 35 formation in this RNA when using an S. solfataricus extract.

MCSS-Based Predictions of Binding Mode and Selectivity of Nucleotide Ligands.

Computational fragment-based approaches are widely used in drug design and discovery. One of their limitations is the lack of performance of docking methods, mainly the scoring functions. With the emergence of fragment-based approaches for single-stranded RNA ligands, we analyze the performance in docking and screening powers of an MCSS-based approach. The performance is evaluated on a benchmark of protein-nucleotide complexes where the four RNA residues are used as fragments. The screening power can be considered the major limiting factor for the fragment-based modeling or design of sequence-selective oligonucleotides. We show that the MCSS sampling is efficient even for such large and flexible fragments. Hybrid solvent models based on some partial explicit representations improve both the docking and screening powers. Clustering of the n best-ranked poses can also contribute to a lesser extent to better performance. A detailed analysis of molecular features suggests various ways to optimize the performance further.

Combined in silico and experimental identification of the Pyrococcus abyssi H/ACA sRNAs and their target sites in ribosomal RNAs.

How far do H/ACA sRNPs contribute to rRNA pseudouridylation in Archaea was still an open question. Hence here, by computational search in three Pyrococcus genomes, we identified seven H/ACA sRNAs and predicted their target sites in rRNAs. In parallel, we experimentally identified 17 Psi residues in P. abyssi rRNAs. By in vitro reconstitution of H/ACA sRNPs, we assigned 15 out of the 17 Psi residues to the 7 identified H/ACA sRNAs: one H/ACA motif can guide up to three distinct pseudouridylations. Interestingly, by using a 23S rRNA fragment as the substrate, one of the two remaining Psi residues could be formed in vitro by the aCBF5/aNOP10/aGAR1 complex without guide sRNA. Our results shed light on structural constraints in archaeal H/ACA sRNPs: the length of helix H2 is of 5 or 6 bps, the distance between the ANA motif and the targeted U residue is of 14 or 15 nts, and the stability of the interaction formed by the substrate rRNA and the 3'-guide sequence is more important than that formed with the 5'-guide sequence. Surprisingly, we showed that a sRNA-rRNA interaction with the targeted uridine in a single-stranded 5'-UNN-3' trinucleotide instead of the canonical 5'-UN-3' dinucleotide is functional.

Hammerhead ribozymes: true metal or nucleobase catalysis? Where is the catalytic power from?

The hammerhead ribozyme was first considered as a metalloenzyme despite persistent inconsistencies between structural and functional data. In the last decade, metal ions were confirmed as catalysts in self-splicing ribozymes but displaced by nucleobases in self-cleaving ribozymes. However, a model of catalysis just relying on nucleobases as catalysts does not fully fit some recent data. Gathering and comparing data on metal ions in self-cleaving and self-splicing ribozymes, the roles of divalent metal ions and nucleobases are revisited. Hypothetical models based on cooperation between metal ions and nucleobases are proposed for the catalysis and evolution of this prototype in RNA catalysis.

RNA Back and Forth: Looking through Ribozyme and Viroid Motifs.

Current cellular facts allow us to follow the link from chemical to biochemical metabolites, from the ancient to the modern world. In this context, the "RNA world" hypothesis proposes that early in the evolution of life, the ribozyme was responsible for the storage and transfer of genetic information and for the catalysis of biochemical reactions. Accordingly, the hammerhead ribozyme (HHR) and the hairpin ribozyme belong to a family of endonucleolytic RNAs performing self-cleavage that might occur during replication. Furthermore, regarding the widespread occurrence of HHRs in several genomes of modern organisms (from mammals to small parasites and elsewhere), these small ribozymes have been regarded as living fossils of a primitive RNA world. They fold into 3D structures that generally require long-range intramolecular interactions to adopt the catalytically active conformation under specific physicochemical conditions. By studying viroids as plausible remains of ancient RNA, we recently demonstrated that they replicate in non-specific hosts, emphasizing their adaptability to different environments, which enhanced their survival probability over the ages. All these results exemplify ubiquitous features of life. Those are the structural and functional versatility of small RNAs, ribozymes, and viroids, as well as their diversity and adaptability to various extreme conditions. All these traits must have originated in early life to generate novel RNA populations.

A dedicated computational approach for the identification of archaeal H/ACA sRNAs.

Whereas dedicated computational approaches have been developed for the search of C/D sRNAs and snoRNAs, as yet no dedicated computational approach has been developed for the search of archaeal H/ACA sRNAs. Here we describe a computational approach allowing a fast and selective identification of H/ACA sRNAs in archaeal genomes. It is easy to use, even for biologists having no special expertise in computational biology. This approach is a stepwise knowledge-based approach, combining the search for common structural features of H/ACA motifs and the search for their putative target sequences. The first step is based on the ERPIN software. It depends on the establishment of a secondary structure-based "profile." We explain how this profile is built and how to use ERPIN to optimize the search for H/ACA motifs. Several examples of applications are given to illustrate how powerful the method is, its limits, and how the results can be evaluated. Then, the possible target rRNA sequences corresponding to the identified H/ACA motifs are searched by use of a descriptor-based method (RNAMOT). The principles and the practical aspects of this method are also explained, and several examples are given here as well to help users in the interpretation of the results.

Two-metal-ion mechanism for hammerhead-ribozyme catalysis.

The hammerhead ribozyme is one of the best studied ribozymes, but it still presents challenges for our understanding of RNA catalysis. It catalyzes a transesterification reaction that converts a 5',3' diester to a 2',3' cyclic phosphate diester via an S(N)2 mechanism. Thus, the overall reaction corresponds to that catalyzed by bovine pancreatic ribonuclease. However, an essential distinguishing aspect is that metal ions are not involved in RNase catalysis but appear to be important in ribozymes. Although various techniques have been used to assign specific functions to metals in the hammerhead ribozyme, their number and roles in catalysis is not clear. Two recent theoretical studies on RNA catalysis examined the reaction mechanism of a single-metal-ion model. A two-metal-ion model, which is supported by experiment and based on ab initio and density functional theory calculations, is described here. The proposed mechanism of the reaction has four chemical steps with three intermediates and four transition states along the reaction pathway. Reaction profiles are calculated in the gas phase and in solution. The early steps of the reaction are found to be fast (with low activation barriers), and the last step, corresponding to the departure of the leaving group, is rate limiting. This two-metal-ion model differs from the models proposed previously in that the two metal ions function not only as Lewis acids but also as general acids/bases. Comparison with experiment shows good agreement with thermodynamic and kinetic data. A detailed analysis based on natural bond orbitals (NBOs) and natural energy decomposition (NEDA) provides insights into the role of metal ions and other factors important for catalysis.

Nucleophilic attack on phosphate diesters: a density functional study of in-line reactivity in dianionic, monoanionic, and neutral systems.

A density functional study of the hydrolysis reaction of phosphodiesters with a series of attacking nucleophiles in the gas phase and in solution is presented. The nucleophiles HOH, HO-, CH3OH, and CH3O- were studied in reactions with ethylene phosphate, 2'3'-ribose cyclic phosphate and in their neutral (protonated) and monoanionic forms. Stationary-point geometries for the reactions were determined at the density functional B3LYP/6-31++G(d,p) level followed by energy refinement at the B3LYP/6-311++G(3df,2p) level. Solvation effects were estimated by using a dielectric approximation with the polarizable continuum model (PCM) at the gas-phase optimized geometries. This series of reactions characterizes factors that influence the intrinsic reactivity of the model phosphate compounds, including the effect of nucleophile, protonation state, cyclic structure, and solvent. The present study of the in-line mechanism for phosphodiester hydrolysis, a reaction of considerable biological importance, has implications for enzymatic mechanisms. The analysis generally supports the associative mechanism for phosphate ester hydrolysis. The results highlight the importance for the reaction barrier of charge neutralization resulting from the protonation of the nonbridging phosphoryl oxygens and the role of internal hydrogen transfer in the gas-phase mechanism. It also shows that solvent stabilization has a profound influence on the relative barrier heights for the dianionic, monoanionic, and neutral reactions. The calculations provide a comprehensive data set for the in-line hydrolysis mechanisms that can be used for the development of improved semiempirical quantum models for phosphate hydrolysis reactions.

The ERPIN server: an interface to profile-based RNA motif identification.

ERPIN is an RNA motif identification program that takes an RNA sequence alignment as an input and identifies related sequences using a profile-based dynamic programming algorithm. ERPIN differs from other RNA motif search programs in its ability to capture subtle biases in the training set and produce highly specific and sensitive searches, while keeping CPU requirements at a practical level. In its latest version, ERPIN also computes E-values, which tell biologists how likely they are to encounter a specific sequence match by chance-a useful indication of biological significance. We present here the ERPIN online search interface (http://tagc.univ-mrs.fr/erpin/). This web server automatically performs ERPIN searches for different RNA genes or motifs, using predefined training sets and search parameters. With a couple of clicks, users can analyze an entire bacterial genome or a genomic segment of up to 5Mb for the presence of tRNAs, 5S rRNAs, SRP RNA, C/D box snoRNAs, hammerhead motifs, miRNAs and other motifs. Search results are displayed with sequence, score, position, E-value and secondary structure graphics. An example of a complete genome scan is provided, as well as an evaluation of run times and specificity/sensitivity information for all available motifs.

Self-assembly Controls Self-cleavage of HHR from ASBVd (-): a Combined SANS and Modeling Study.

In the Avocado Sunblotch Viroid (ASBVd: 249-nt) from the Avsunviroidae family, a symmetric rolling-circle replication operates through an autocatalytic mechanism mediated by hammerhead ribozymes (HHR) embedded in both polarity strands. The concatenated multimeric ASBVd (+) and ASBVd (-) RNAs thus generated are processed by cleavage to unit-length where ASBVd (-) self-cleaves with more efficiency. Absolute scale small angle neutron scattering (SANS) revealed a temperature-dependent dimer association in both ASBVd (-) and its derived 79-nt HHR (-). A joint thermodynamic analysis of SANS and catalytic data indicates the rate-determining step corresponds to the dimer/monomer transition. 2D and 3D models of monomeric and dimeric HHR (-) suggest that the inter-molecular contacts stabilizing the dimer (between HI and HII domains) compete with the intra-molecular ones stabilizing the active conformation of the full-length HHR required for an efficient self-cleavage. Similar competing intra- and inter-molecular contacts are proposed in ASBVd (-) though with a remoter region from an extension of the HI domain.

Drawing and editing the secondary structure(s) of RNA.

Secondary structure diagrams are essential, in RNA biology, to communicate functional hypotheses and summarize structural data, and communicate them visually as drafts or finalized publication-ready figures. While many tools are currently available to automate the production of such diagrams, their capacities are usually partial, making it hard for a user to decide which to use in a given context. In this chapter, we guide the reader through the steps involved in the production of expressive publication-quality illustrations featuring the RNA secondary structure. We present major existing representations and layouts, and give precise instructions to produce them using available free software, including jViz.RNA, the PseudoViewer, RILogo, R-chie, RNAplot, R2R, and VARNA. We describe the file formats and structural descriptions accepted by popular RNA visualization tools. We also provide command lines and Python scripts to ease the user's access to advanced features. Finally, we discuss and illustrate alternative approaches to visualize the secondary structure in the presence of probing data, pseudoknots, RNA-RNA interactions, and comparative data.

Effective atom volumes for implicit solvent models: comparison between Voronoi volumes and minimum fluctuation volumes.

An essential element of implicit solvent models, such as the generalized Born method, is a knowledge of the volume associated with the individual atoms of the solute. Two approaches for determining atomic volumes for the generalized Born model are described; one is based on Voronoi polyhedra and the other, on minimizing the fluctuations in the overall volume of the solute. Volumes to be used with various parameter sets for protein and nucleic acids in the CHARMM force field are determined from a large set of known structures. The volumes resulting from the two different approaches are compared with respect to various parameters, including the size and solvent accessibility of the structures from which they are determined. The question of whether to include hydrogens in the atomic representation of the solute volume is examined. Copyright 2001 John Wiley & Sons, Inc. J Comput Chem 22: 1857-1879, 2001

Comparative study of two box H/ACA ribonucleoprotein pseudouridine-synthases: relation between conformational dynamics of the guide RNA, enzyme assembly and activity.

Multiple RNA-guided pseudouridine synthases, H/ACA ribonucleoprotein particles (RNPs) which contain a guide RNA and four proteins, catalyze site-specific post-transcriptional isomerization of uridines into pseudouridines in substrate RNAs. In archaeal particles, the guide small RNA (sRNA) is anchored by the pseudouridine synthase aCBF5 and the ribosomal protein L7Ae. Protein aNOP10 interacts with both aCBF5 and L7Ae. The fourth protein, aGAR1, interacts with aCBF5 and enhances catalytic efficiency. Here, we compared the features of two H/ACA sRNAs, Pab21 and Pab91, from Pyrococcus abyssi. We found that aCBF5 binds much more weakly to Pab91 than to Pab21. Surprisingly, the Pab91 sRNP exhibits a higher catalytic efficiency than the Pab21 sRNP. We thus investigated the molecular basis of the differential efficiencies observed for the assembly and catalytic activity of the two enzymes. For this, we compared profiles of the extent of lead-induced cleavages in these sRNAs during a stepwise reconstitution of the sRNPs, and analyzed the impact of the absence of the aNOP10-L7Ae interaction. Such probing experiments indicated that the sRNAs undergo a series of conformational changes upon RNP assembly. These changes were also evaluated directly by circular dichroism (CD) spectroscopy, a tool highly adapted to analyzing RNA conformational dynamics. In addition, our results reveal that the conformation of helix P1 formed at the base of the H/ACA sRNAs is optimized in Pab21 for efficient aCBF5 binding and RNP assembly. Moreover, P1 swapping improved the assembly of the Pab91 sRNP. Nonetheless, efficient aCBF5 binding probably also relies on the pseudouridylation pocket which is not optimized for high activity in the case of Pab21.