GrAfSS: a webserver for substructure similarity searching and comparisons in the structures of proteins and RNA.

The GrAfSS (Graph theoretical Applications for Substructure Searching) webserver is a platform to search for three-dimensional substructures of: (i) amino acid side chains in protein structures; and (ii) base arrangements in RNA structures. The webserver interfaces the functions of five different graph theoretical algorithms - ASSAM, SPRITE, IMAAAGINE, NASSAM and COGNAC - into a single substructure searching suite. Users will be able to identify whether a three-dimensional (3D) arrangement of interest, such as a ligand binding site or 3D motif, observed in a protein or RNA structure can be found in other structures available in the Protein Data Bank (PDB). The webserver also allows users to determine whether a protein or RNA structure of interest contains substructural arrangements that are similar to known motifs or 3D arrangements. These capabilities allow for the functional annotation of new structures that were either experimentally determined or computationally generated (such as the coordinates generated by AlphaFold2) and can provide further insights into the diversity or conservation of functional mechanisms of structures in the PDB. The computed substructural superpositions are visualized using integrated NGL viewers. The GrAfSS server is available at http://mfrlab.org/grafss/.

Regulation of Glycine Cleavage and Detoxification by a Highly Conserved Glycine Riboswitch in Burkholderia spp.

The glycine riboswitch is a known regulatory element that is unique in having two aptamers that are joined by a linker region. In this study, we investigated a glycine riboswitch located in the 5' untranslated region of a glycine cleavage system homolog (gcvTHP) in Burkholderia spp. Structure prediction using the sequence generated a model with a glycine binding pocket composed of base-triple interactions (G62-A64-A86 and G65-U84-C85) that are supported by A/G minor interactions (A17-C60-G88 and G16-C61-G87, respectively) and two ribose-zipper motifs (C11-G12 interacting with A248-A247 and C153-U154 interacting with A79-A78) which had not been previously reported. The capacity of the riboswitch to bind to glycine was experimentally validated by native gel assays and the crucial role of interactions that make up the glycine binding pocket were proven by mutations of A17U and G16C which resulted in conformational differences that may lead to dysfunction. Using glycine supplemented minimal media, we were able to prove that the expression of the gcvTHP genes found downstream of the riboswitch responded to the glycine concentrations introduced thus confirming the role of this highly conserved Burkholderia riboswitch and its associated genes as a putative glycine detoxification system in Burkholderia spp.

Drug ReposER: a web server for predicting similar amino acid arrangements to known drug binding interfaces for potential drug repositioning.

A common drug repositioning strategy is the re-application of an existing drug to address alternative targets. A crucial aspect to enable such repurposing is that the drug's binding site on the original target is similar to that on the alternative target. Based on the assumption that proteins with similar binding sites may bind to similar drugs, the 3D substructure similarity data can be used to identify similar sites in other proteins that are not known targets. The Drug ReposER (DRug REPOSitioning Exploration Resource) web server is designed to identify potential targets for drug repurposing based on sub-structural similarity to the binding interfaces of known drug binding sites. The application has pre-computed amino acid arrangements from protein structures in the Protein Data Bank that are similar to the 3D arrangements of known drug binding sites thus allowing users to explore them as alternative targets. Users can annotate new structures for sites that are similarly arranged to the residues found in known drug binding interfaces. The search results are presented as mappings of matched sidechain superpositions. The results of the searches can be visualized using an integrated NGL viewer. The Drug ReposER server has no access restrictions and is available at http://mfrlab.org/drugreposer/.

Self-assembly programming of DNA polyominoes.

Fabrication of functional DNA nanostructures operating at a cellular level has been accomplished through molecular programming techniques such as DNA origami and single-stranded tiles (SST). During implementation, restrictive and constraint dependent designs are enforced to ensure conformity is attainable. We propose a concept of DNA polyominoes that promotes flexibility in molecular programming. The fabrication of complex structures is achieved through self-assembly of distinct heterogeneous shapes (i.e., self-organised optimisation among competing DNA basic shapes) with total flexibility during the design and assembly phases. In this study, the plausibility of the approach is validated using the formation of multiple 3×4 DNA network fabricated from five basic DNA shapes with distinct configurations (monomino, tromino and tetrominoes). Computational tools to aid the design of compatible DNA shapes and the structure assembly assessment are presented. The formations of the desired structures were validated using Atomic Force Microscopy (AFM) imagery. Five 3×4 DNA networks were successfully constructed using combinatorics of these five distinct DNA heterogeneous shapes. Our findings revealed that the construction of DNA supra-structures could be achieved using a more natural-like orchestration as compared to the rigid and restrictive conventional approaches adopted previously.

InterRNA: a database of base interactions in RNA structures.

A major component of RNA structure stabilization are the hydrogen bonded interactions between the base residues. The importance and biological relevance for large clusters of base interactions can be much more easily investigated when their occurrences have been systematically detected, catalogued and compared. In this paper, we describe the database InterRNA (INTERactions in RNA structures database-http://mfrlab.org/interrna/) that contains records of known RNA 3D motifs as well as records for clusters of bases that are interconnected by hydrogen bonds. The contents of the database were compiled from RNA structural annotations carried out by the NASSAM (http://mfrlab.org/grafss/nassam) and COGNAC (http://mfrlab.org/grafss/cognac) computer programs. An analysis of the database content and comparisons with the existing corpus of knowledge regarding RNA 3D motifs clearly show that InterRNA is able to provide an extension of the annotations for known motifs as well as able to provide novel interactions for further investigations.

DNA tetrominoes: the construction of DNA nanostructures using self-organised heterogeneous deoxyribonucleic acids shapes.

The unique programmability of nucleic acids offers alternative in constructing excitable and functional nanostructures. This work introduces an autonomous protocol to construct DNA Tetris shapes (L-Shape, B-Shape, T-Shape and I-Shape) using modular DNA blocks. The protocol exploits the rich number of sequence combinations available from the nucleic acid alphabets, thus allowing for diversity to be applied in designing various DNA nanostructures. Instead of a deterministic set of sequences corresponding to a particular design, the protocol promotes a large pool of DNA shapes that can assemble to conform to any desired structures. By utilising evolutionary programming in the design stage, DNA blocks are subjected to processes such as sequence insertion, deletion and base shifting in order to enrich the diversity of the resulting shapes based on a set of cascading filters. The optimisation algorithm allows mutation to be exerted indefinitely on the candidate sequences until these sequences complied with all the four fitness criteria. Generated candidates from the protocol are in agreement with the filter cascades and thermodynamic simulation. Further validation using gel electrophoresis indicated the formation of the designed shapes. Thus, supporting the plausibility of constructing DNA nanostructures in a more hierarchical, modular, and interchangeable manner.

COGNAC: a web server for searching and annotating hydrogen-bonded base interactions in RNA three-dimensional structures.

Hydrogen bonds are crucial factors that stabilize a complex ribonucleic acid (RNA) molecule's three-dimensional (3D) structure. Minute conformational changes can result in variations in the hydrogen bond interactions in a particular structure. Furthermore, networks of hydrogen bonds, especially those found in tight clusters, may be important elements in structure stabilization or function and can therefore be regarded as potential tertiary motifs. In this paper, we describe a graph theoretical algorithm implemented as a web server that is able to search for unbroken networks of hydrogen-bonded base interactions and thus provide an accounting of such interactions in RNA 3D structures. This server, COGNAC (COnnection tables Graphs for Nucleic ACids), is also able to compare the hydrogen bond networks between two structures and from such annotations enable the mapping of atomic level differences that may have resulted from conformational changes due to mutations or binding events. The COGNAC server can be accessed at http://mfrlab.org/grafss/cognac.

Comparative sequence and structure analysis reveals the conservation and diversity of nucleotide positions and their associated tertiary interactions in the riboswitches.

The tertiary motifs in complex RNA molecules play vital roles to either stabilize the formation of RNA 3D structure or to provide important biological functionality to the molecule. In order to better understand the roles of these tertiary motifs in riboswitches, we examined 11 representative riboswitch PDB structures for potential agreement of both motif occurrences and conservations. A total of 61 unique tertiary interactions were found in the reference structures. In addition to the expected common A-minor motifs and base-triples mainly involved in linking distant regions the riboswitch structures three highly conserved variants of A-minor interactions called G-minors were found in the SAM-I and FMN riboswitches where they appear to be involved in the recognition of the respective ligand's functional groups. From our structural survey as well as corresponding structure and sequence alignments, the agreement between motif occurrences and conservations are very prominent across the representative riboswitches. Our analysis provide evidence that some of these tertiary interactions are essential components to form the structure where their sequence positions are conserved despite a high degree of diversity in other parts of the respective riboswitches sequences. This is indicative of a vital role for these tertiary interactions in determining the specific biological function of riboswitch.

In-silico design of computational nucleic acids for molecular information processing.

Within recent years nucleic acids have become a focus of interest for prototype implementations of molecular computing concepts. During the same period the importance of ribonucleic acids as components of the regulatory networks within living cells has increasingly been revealed. Molecular computers are attractive due to their ability to function within a biological system; an application area extraneous to the present information technology paradigm. The existence of natural information processing architectures (predominately exemplified by protein) demonstrates that computing based on physical substrates that are radically different from silicon is feasible. Two key principles underlie molecular level information processing in organisms: conformational dynamics of macromolecules and self-assembly of macromolecules. Nucleic acids support both principles, and moreover computational design of these molecules is practicable. This study demonstrates the simplicity with which one can construct a set of nucleic acid computing units using a new computational protocol. With the new protocol, diverse classes of nucleic acids imitating the complete set of boolean logical operators were constructed. These nucleic acid classes display favourable thermodynamic properties and are significantly similar to the approximation of successful candidates implemented in the laboratory. This new protocol would enable the construction of a network of interconnecting nucleic acids (as a circuit) for molecular information processing.