Knotted artifacts in predicted 3D RNA structures.

Unlike proteins, RNAs deposited in the Protein Data Bank do not contain topological knots. Recently, admittedly, the first trefoil knot and some lasso-type conformations have been found in experimental RNA structures, but these are still exceptional cases. Meanwhile, algorithms predicting 3D RNA models have happened to form knotted structures not so rarely. Interestingly, machine learning-based predictors seem to be more prone to generate knotted RNA folds than traditional methods. A similar situation is observed for the entanglements of structural elements. In this paper, we analyze all models submitted to the CASP15 competition in the 3D RNA structure prediction category. We show what types of topological knots and structure element entanglements appear in the submitted models and highlight what methods are behind the generation of such conformations. We also study the structural aspect of susceptibility to entanglement. We suggest that predictors take care of an evaluation of RNA models to avoid publishing structures with artifacts, such as unusual entanglements, that result from hallucinations of predictive algorithms.

When will RNA get its AlphaFold moment?

The protein structure prediction problem has been solved for many types of proteins by AlphaFold. Recently, there has been considerable excitement to build off the success of AlphaFold and predict the 3D structures of RNAs. RNA prediction methods use a variety of techniques, from physics-based to machine learning approaches. We believe that there are challenges preventing the successful development of deep learning-based methods like AlphaFold for RNA in the short term. Broadly speaking, the challenges are the limited number of structures and alignments making data-hungry deep learning methods unlikely to succeed. Additionally, there are several issues with the existing structure and sequence data, as they are often of insufficient quality, highly biased and missing key information. Here, we discuss these challenges in detail and suggest some steps to remedy the situation. We believe that it is possible to create an accurate RNA structure prediction method, but it will require solving several data quality and volume issues, usage of data beyond simple sequence alignments, or the development of new less data-hungry machine learning methods.

New prediction categories in CASP15.

Prediction categories in the Critical Assessment of Structure Prediction (CASP) experiments change with the need to address specific problems in structure modeling. In CASP15, four new prediction categories were introduced: RNA structure, ligand-protein complexes, accuracy of oligomeric structures and their interfaces, and ensembles of alternative conformations. This paper lists technical specifications for these categories and describes their integration in the CASP data management system.

WebTetrado: a webserver to explore quadruplexes in nucleic acid 3D structures.

Quadruplexes are four-stranded DNA/RNA motifs of high functional significance that fold into complex shapes. They are widely recognized as important regulators of genomic processes and are among the most frequently investigated potential drug targets. Despite interest in quadruplexes, few studies focus on automatic tools that help to understand the many unique features of their 3D folds. In this paper, we introduce WebTetrado, a web server for analyzing 3D structures of quadruplex structures. It has a user-friendly interface and offers many advanced features, including automatic identification, annotation, classification, and visualization of the motif. The program applies to the experimental or in silico generated 3D models provided in the PDB and PDBx/mmCIF files. It supports canonical G-quadruplexes as well as non-G-based quartets. It can process unimolecular, bimolecular, and tetramolecular quadruplexes. WebTetrado is implemented as a publicly available web server with an intuitive interface and can be freely accessed at https://webtetrado.cs.put.poznan.pl/.

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.

RNAloops: a database of RNA multiloops.

MOTIVATION: Knowledge of the 3D structure of RNA supports discovering its functions and is crucial for designing drugs and modern therapeutic solutions. Thus, much attention is devoted to experimental determination and computational prediction targeting the global fold of RNA and its local substructures. The latter include multi-branched loops-functionally significant elements that highly affect the spatial shape of the entire molecule. Unfortunately, their computational modeling constitutes a weak point of structural bioinformatics. A remedy for this is in collecting these motifs and analyzing their features. RESULTS: RNAloops is a self-updating database that stores multi-branched loops identified in the PDB-deposited RNA structures. A description of each loop includes angular data-planar and Euler angles computed between pairs of adjacent helices to allow studying their mutual arrangement in space. The system enables search and analysis of multiloops, presents their structure details numerically and visually, and computes data statistics. AVAILABILITY AND IMPLEMENTATION: RNAloops is freely accessible at https://rnaloops.cs.put.poznan.pl. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Characterizing domain-specific open educational resources by linking ISCB Communities of Special Interest to Wikipedia.

MOTIVATION: Wikipedia is one of the most important channels for the public communication of science and is frequently accessed as an educational resource in computational biology. Joint efforts between the International Society for Computational Biology (ISCB) and the Computational Biology taskforce of WikiProject Molecular Biology (a group of expert Wikipedia editors) have considerably improved computational biology representation on Wikipedia in recent years. However, there is still an urgent need for further improvement in quality, especially when compared to related scientific fields such as genetics and medicine. Facilitating involvement of members from ISCB Communities of Special Interest (COSIs) would improve a vital open education resource in computational biology, additionally allowing COSIs to provide a quality educational resource highly specific to their subfield. RESULTS: We generate a list of around 1500 English Wikipedia articles relating to computational biology and describe the development of a binary COSI-Article matrix, linking COSIs to relevant articles and thereby defining domain-specific open educational resources. Our analysis of the COSI-Article matrix data provides a quantitative assessment of computational biology representation on Wikipedia against other fields and at a COSI-specific level. Furthermore, we conducted similarity analysis and subsequent clustering of COSI-Article data to provide insight into potential relationships between COSIs. Finally, based on our analysis, we suggest courses of action to improve the quality of computational biology representation on Wikipedia.

DrawTetrado to create layer diagrams of G4 structures.

MOTIVATION: Quadruplexes are specific 3D structures found in nucleic acids. Due to the exceptional properties of these motifs, their exploration with the general-purpose bioinformatics methods can be problematic or insufficient. The same applies to visualizing their structure. A hand-drawn layer diagram is the most common way to represent the quadruplex anatomy. No molecular visualization software generates such a structural model based on atomic coordinates. RESULTS: DrawTetrado is an open-source Python program for automated visualization targeting the structures of quadruplexes and G4-helices. It generates static layer diagrams that represent structural data in a pseudo-3D perspective. The possibility to set color schemes, nucleotide labels, inter-element distances or angle of view allows for easy customization of the output drawing. AVAILABILITY AND IMPLEMENTATION: The program is available under the MIT license at https://github.com/RNApolis/drawtetrado.

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/.

ONQUADRO: a database of experimentally determined quadruplex structures.

ONQUADRO is an advanced database system that supports the study of the structures of canonical and non-canonical quadruplexes. It combines a relational database that collects comprehensive information on tetrads, quadruplexes, and G4-helices; programs to compute structure parameters and visualise the data; scripts for statistical analysis; automatic updates and newsletter modules; and a web application that provides a user interface. The database is a self-updating resource, with new information arriving once a week. The preliminary data are downloaded from the Protein Data Bank, processed, annotated, and completed. As of August 2021, ONQUADRO contains 1,661 tetrads, 518 quadruplexes, and 30 G4-helices found in 467 experimentally determined 3D structures of nucleic acids. Users can view and download their description: sequence, secondary structure (dot-bracket, classical diagram, arc diagram), tertiary structure (ball-and-stick, surface or vdw-ball model, layer diagram), planarity, twist, rise, chi angle (value and type), loop characteristics, strand directionality, metal ions, ONZ, and Webba da Silva classification (the latter by loop topology and tetrad combination), origin structure ID, assembly ID, experimental method, and molecule type. The database is freely available at https://onquadro.cs.put.poznan.pl/. It can be used on both desktop computers and mobile devices.

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.

BioCommons: a robust java library for RNA structural bioinformatics.

MOTIVATION: Biomolecular structures come in multiple representations and diverse data formats. Their incompatibility with the requirements of data analysis programs significantly hinders the analytics and the creation of new structure-oriented bioinformatic tools. Therefore, the need for robust libraries of data processing functions is still growing. RESULTS: BioCommons is an open-source, Java library for structural bioinformatics. It contains many functions working with the 2D and 3D structures of biomolecules, with a particular emphasis on RNA. AVAILABILITY AND IMPLEMENTATION: The library is available in Maven Central Repository and its source code is hosted on GitHub: https://github.com/tzok/BioCommons. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

RNAthor - fast, accurate normalization, visualization and statistical analysis of RNA probing data resolved by capillary electrophoresis.

RNAs adopt specific structures to perform their functions, which are critical to fundamental cellular processes. For decades, these structures have been determined and modeled with strong support from computational methods. Still, the accuracy of the latter ones depends on the availability of experimental data, for example, chemical probing information that can define pseudo-energy constraints for RNA folding algorithms. At the same time, diverse computational tools have been developed to facilitate analysis and visualization of data from RNA structure probing experiments followed by capillary electrophoresis or next-generation sequencing. RNAthor, a new software tool for the fully automated normalization of SHAPE and DMS probing data resolved by capillary electrophoresis, has recently joined this collection. RNAthor automatically identifies unreliable probing data. It normalizes the reactivity information to a uniform scale and uses it in the RNA secondary structure prediction. Our web server also provides tools for fast and easy RNA probing data visualization and statistical analysis that facilitates the comparison of multiple data sets. RNAthor is freely available at http://rnathor.cs.put.poznan.pl/.

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.

ElTetrado: a tool for identification and classification of tetrads and quadruplexes.

BACKGROUND: Quadruplexes are specific structure motifs occurring, e.g., in telomeres and transcriptional regulatory regions. Recent discoveries confirmed their importance in biomedicine and led to an intensified examination of their properties. So far, the study of these motifs has focused mainly on the sequence and the tertiary structure, and concerned canonical structures only. Whereas, more and more non-canonical quadruplex motifs are being discovered. RESULTS: Here, we present ElTetrado, a software that identifies quadruplexes (composed of guanine- and other nucleobase-containing tetrads) in nucleic acid structures and classifies them according to the recently introduced ONZ taxonomy. The categorization is based on the secondary structure topology of quadruplexes and their component tetrads. It supports the analysis of canonical and non-canonical motifs. Besides the class recognition, ElTetrado prepares a dot-bracket and graphical representations of the secondary structure, which reflect the specificity of the quadruplex's structure topology. It is implemented as a freely available, standalone application, available at https://github.com/tzok/eltetrado. CONCLUSIONS: The proposed software tool allows to identify and classify tetrads and quadruplexes based on the topology of their secondary structures. It complements existing approaches focusing on the sequence and 3D structure.

RNA-Puzzles toolkit: a computational resource of RNA 3D structure benchmark datasets, structure manipulation, and evaluation tools.

Significant improvements have been made in the efficiency and accuracy of RNA 3D structure prediction methods during the succeeding challenges of RNA-Puzzles, a community-wide effort on the assessment of blind prediction of RNA tertiary structures. The RNA-Puzzles contest has shown, among others, that the development and validation of computational methods for RNA fold prediction strongly depend on the benchmark datasets and the structure comparison algorithms. Yet, there has been no systematic benchmark set or decoy structures available for the 3D structure prediction of RNA, hindering the standardization of comparative tests in the modeling of RNA structure. Furthermore, there has not been a unified set of tools that allows deep and complete RNA structure analysis, and at the same time, that is easy to use. Here, we present RNA-Puzzles toolkit, a computational resource including (i) decoy sets generated by different RNA 3D structure prediction methods (raw, for-evaluation and standardized datasets), (ii) 3D structure normalization, analysis, manipulation, visualization tools (RNA_format, RNA_normalizer, rna-tools) and (iii) 3D structure comparison metric tools (RNAQUA, MCQ4Structures). This resource provides a full list of computational tools as well as a standard RNA 3D structure prediction assessment protocol for the community.

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.

RNAvista: a webserver to assess RNA secondary structures with non-canonical base pairs.

Motivation: In the study of 3D RNA structure, information about non-canonical interactions between nucleobases is increasingly important. Specialized databases support investigation of this issue based on experimental data, and several programs can annotate non-canonical base pairs in the RNA 3D structure. However, predicting the extended RNA secondary structure which describes both canonical and non-canonical interactions remains difficult. Results: Here, we present RNAvista that allows predicting an extended RNA secondary structure from sequence or from the list enumerating canonical base pairs only. RNAvista is implemented as a publicly available webserver with user-friendly interface. It runs on all major web browsers. Availability and implementation: http://rnavista.cs.put.poznan.pl.

RNAfitme: a webserver for modeling nucleobase and nucleoside residue conformation in fixed-backbone RNA structures.

BACKGROUND: Computational RNA 3D structure prediction and modeling are rising as complementary approaches to high-resolution experimental techniques for structure determination. They often apply to substitute or complement them. Recently, researchers' interests have directed towards in silico methods to fit, remodel and refine RNA tertiary structure models. Their power lies in a problem-specific exploration of RNA conformational space and efficient optimization procedures. The aim is to improve the accuracy of models obtained either computationally or experimentally. RESULTS: Here, we present RNAfitme, a versatile webserver tool for remodeling of nucleobase- and nucleoside residue conformations in the fixed-backbone RNA 3D structures. Our approach makes use of dedicated libraries that define RNA conformational space. They have been built upon torsional angle characteristics of PDB-deposited RNA structures. RNAfitme can be applied to reconstruct full-atom model of RNA from its backbone; remodel user-selected nucleobase/nucleoside residues in a given RNA structure; predict RNA 3D structure based on the sequence and the template of a homologous molecule of the same size; refine RNA 3D model by reducing steric clashes indicated during structure quality assessment. RNAfitme is a publicly available tool with an intuitive interface. It is freely accessible at http://rnafitme.cs.put.poznan.pl/ CONCLUSIONS: RNAfitme has been applied in various RNA 3D remodeling scenarios for several types of input data. Computational experiments proved its efficiency, accuracy, and usefulness in the processing of RNAs of any size. Fidelity of RNAfitme predictions has been thoroughly tested for RNA 3D structures determined experimentally and modeled in silico.

RNApdbee 2.0: multifunctional tool for RNA structure annotation.

In the field of RNA structural biology and bioinformatics, an access to correctly annotated RNA structure is of crucial importance, especially in the secondary and 3D structure predictions. RNApdbee webserver, introduced in 2014, primarily aimed to address the problem of RNA secondary structure extraction from the PDB files. Its new version, RNApdbee 2.0, is a highly advanced multifunctional tool for RNA structure annotation, revealing the relationship between RNA secondary and 3D structure given in the PDB or PDBx/mmCIF format. The upgraded version incorporates new algorithms for recognition and classification of high-ordered pseudoknots in large RNA structures. It allows analysis of isolated base pairs impact on RNA structure. It can visualize RNA secondary structures-including that of quadruplexes-with depiction of non-canonical interactions. It also annotates motifs to ease identification of stems, loops and single-stranded fragments in the input RNA structure. RNApdbee 2.0 is implemented as a publicly available webserver with an intuitive interface and can be freely accessed at http://rnapdbee.cs.put.poznan.pl/.