Isostericity and tautomerism of base pairs in nucleic acids

The natural bases of nucleic acids form a great variety of base pairs with at least two hydrogen bonds between them. They are classified in twelve main families, with the Watson–Crick family being one of them. In a given family, some of the base pairs are isosteric between them, meaning that the positions and the distances between the C1′ carbon atoms are very similar. The isostericity of Watson–Crick pairs between the complementary bases forms the basis of RNA helices and of the resulting RNA secondary structure. Several defined suites of non-Watson–Crick base pairs assemble into RNA modules that form recurrent, rather regular, building blocks of the tertiary architecture of folded RNAs. RNA modules are intrinsic to RNA architecture are therefore disconnected from a biological function specifically attached to a RNA sequence. RNA modules occur in all kingdoms of life and in structured RNAs with diverse functions. Because of chemical and geometrical constraints, isostericity between non-Watson–Crick pairs is restricted and this leads to higher sequence conservation in RNA modules with, consequently, greater difficulties in extracting 3D information from sequence analysis. Nucleic acid helices have to be recognised in several biological processes like replication or translational decoding. In polymerases and the ribosomal decoding site, the recognition occurs on the minor groove sides of the helical fragments. With the use of alternative conformations, protonated or tautomeric forms of the bases, some base pairs with Watson–Crick-like geometries can form and be stabilized. Several of these pairs with Watson–Crick-like geometries extend the concept of isostericity beyond the number of isosteric pairs formed between complementary bases. These observations set therefore limits and constraints to geometric selection in molecular recognition of complementary Watson–Crick pairs for fidelity in replication and translation processes.     

Non-Watson Crick base pairs might stabilize RNA structural motifs in ribozymes — a comparative study of group-I intron structures

In recent decades studies on RNA structure and function have gained significance due to discoveries on diversified functions of RNA. A common element for RNA secondary structure formed by series of non-Watson/Watson Crick base pairs, internal loops and pseudoknots have been the highlighting feature of recent structural determination of RNAs. The recent crystal structure of group-I introns has demonstrated that these might constitute RNA structural motifs in ribozymes, playing a crucial role in their enzymatic activity. To understand the functional significance of these non-canonical base pairs in catalytic RNA, we analysed the sequences of group-I introns from nuclear genes. The results suggest that they might form the building blocks of folded RNA motifs which are crucial to the catalytic activity of the ribozyme. The conservation of these, as observed from divergent organisms, argues for the presence of non-canonical base pairs as an important requisite for the structure and enzymatic property of ribozymes by enabling them to carry out functions such as replication, polymerase activity etc. in primordial conditions in the absence of proteins.     

Stacking geometry for non‐canonical G:U wobble base pair containing dinucleotide sequences in RNA: dispersion‐corrected DFT‐D study

Emergence of thousands of crystal structures of noncoding RNA molecules indicates its structural and functional diversity. RNA function is based upon a large variety of structural elements which are specifically assembled in the folded molecules. Along with the canonical Watson‐Crick base pairs, different orientations of the bases to form hydrogen‐bonded non‐canonical base pairs have also been observed in the available RNA structures. Frequencies of occurrences of different non‐canonical base pairs in RNA indicate their important role to maintain overall structure and functions of RNA. There are several reports on geometry and energetic stabilities of these non‐canonical base pairs. However, their stacking geometry and stacking stability with the neighboring base pairs are not well studied. Among the different non‐canonical base pairs, the G:U wobble base pair (G:U W:WC) is most frequently observed in the RNA double helices. Using quantum chemical method and available experimental data set we have studied the stacking geometry of G:U W:WC base pair containing dinucleotide sequences in roll‐slide parameters hyperspace for different values of twist. This study indicates that the G:U W:WC base pair can stack well with the canonical base pairs giving rise to large interaction energy. The overall preferred stacking geometry in terms of roll, twist and slide for the eleven possible dinucleotide sequences is seen to be quite dependent on their sequences. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 328–338, 2015.     

The interaction networks of structured RNAs

All pairwise interactions occurring between bases which could be detected in three-dimensional structures of crystallized RNA molecules are annotated on new planar diagrams. The diagrams attempt to map the underlying complex networks of base–base interactions and, especially, they aim at conveying key relationships between helical domains: co-axial stacking, bending and all Watson–Crick as well as non-Watson–Crick base pairs. Although such wiring diagrams cannot replace full stereographic images for correct spatial understanding and representation, they reveal structural similarities as well as the conserved patterns and distances between motifs which are present within the interaction networks of folded RNAs of similar or unrelated functions. Finally, the diagrams could help devising methods for meaningfully transforming RNA structures into graphs amenable to network analysis.     

Context-sensitivity of isosteric substitutions of non-Watson–Crick basepairs in recurrent RNA 3D motifs

Sequence variation in a widespread, recurrent, structured RNA 3D motif, the Sarcin/Ricin (S/R), was studied to address three related questions: First, how do the stabilities of structured RNA 3D motifs, composed of non-Watson–Crick (non-WC) basepairs, compare to WC-paired helices of similar length and sequence? Second, what are the effects on the stabilities of such motifs of isosteric and non-isosteric base substitutions in the non-WC pairs? And third, is there selection for particular base combinations in non-WC basepairs, depending on the temperature regime to which an organism adapts? A survey of large and small subunit rRNAs from organisms adapted to different temperatures revealed the presence of systematic sequence variations at many non-WC paired sites of S/R motifs. UV melting analysis and enzymatic digestion assays of oligonucleotides containing the motif suggest that more stable motifs tend to be more rigid. We further found that the base substitutions at non-Watson–Crick pairing sites can significantly affect the thermodynamic stabilities of S/R motifs and these effects are highly context specific indicating the importance of base-stacking and base-phosphate interactions on motif stability. This study highlights the significance of non-canonical base pairs and their contributions to modulating the stability and flexibility of RNA molecules.     

RNAMotifScan: automatic identification of RNA structural motifs using secondary structural alignment

Recent studies have shown that RNA structural motifs play essential roles in RNA folding and interaction with other molecules. Computational identification and analysis of RNA structural motifs remains a challenging task. Existing motif identification methods based on 3D structure may not properly compare motifs with high structural variations. Other structural motif identification methods consider only nested canonical base-pairing structures and cannot be used to identify complex RNA structural motifs that often consist of various non-canonical base pairs due to uncommon hydrogen bond interactions. In this article, we present a novel RNA structural alignment method for RNA structural motif identification, RNAMotifScan, which takes into consideration the isosteric (both canonical and non-canonical) base pairs and multi-pairings in RNA structural motifs. The utility and accuracy of RNAMotifScan is demonstrated by searching for kink-turn, C-loop, sarcin-ricin, reverse kink-turn and E-loop motifs against a 23S rRNA (PDBid: 1S72), which is well characterized for the occurrences of these motifs. Finally, we search these motifs against the RNA structures in the entire Protein Data Bank and the abundances of them are estimated. RNAMotifScan is freely available at our supplementary website (http://genome.ucf.edu/RNAMotifScan).     

The UA_handle: a versatile submotif in stable RNA architectures

Stable RNAs are modular and hierarchical 3D architectures taking advantage of recurrent structural motifs to form extensive non-covalent tertiary interactions. Sequence and atomic structure analysis has revealed a novel submotif involving a minimal set of five nucleotides, termed the UA_handle motif (5′XU/AN_nX3′). It consists of a U:A Watson–Crick: Hoogsteen trans base pair stacked over a classic Watson–Crick base pair, and a bulge of one or more nucleotides that can act as a handle for making different types of long-range interactions. This motif is one of the most versatile building blocks identified in stable RNAs. It enters into the composition of numerous recurrent motifs of greater structural complexity such as the T-loop, the 11-nt receptor, the UAA/GAN and the G-ribo motifs. Several structural principles pertaining to RNA motifs are derived from our analysis. A limited set of basic submotifs can account for the formation of most structural motifs uncovered in ribosomal and stable RNAs. Structural motifs can act as structural scaffoldings and be functionally and topologically equivalent despite sequence and structural differences. The sequence network resulting from the structural relationships shared by these RNA motifs can be used as a proto-language for assisting prediction and rational design of RNA tertiary structures.     

Structures of Non-canonical Tandem Base Pairs in RNA Helices: Review

Abstract

The structures of tandem non-canonical base pairs, a frequently recurring motif in RNA molecules, are reviewed and analysed. The tandem non-canonical base pair motifs can be roughly divided in three groups, containing seven subgroups based on their base pairing patterns and local geometries. Structural details and helical parameters that can be used to numerically distinguish between the subgroups are tabulated. Remarkably, while the individual helical twists of the tandem and adjacent base pair steps can be substantially smaller or larger than the typical A-form value of 32.7°, the average value is close to A-form. This and other striking regularities resulting from compensating geometrical adjustments, important for understanding and predicting the configurations of non-canonical base pairs geometries are discussed.     

Hybrid simulation approach incorporating microscopic interaction along with rigid body degrees of freedom for stacking between base pairs

Stacking interaction between the aromatic heterocyclic bases plays an important role in the double helical structures of nucleic acids. Considering the base as rigid body, there are total of 18 degrees of freedom of a dinucleotide step. Some of these parameters show sequence preferences, indicating that the detailed atomic interactions are important in the stacking. Large variants of non‐canonical base pairs have been seen in the crystallographic structures of RNA. However, their stacking preferences are not thoroughly deciphered yet from experimental results. The current theoretical approaches use either the rigid body degrees of freedom where the atomic information are lost or computationally expensive all atom simulations. We have used a hybrid simulation approach incorporating Monte‐Carlo Metropolis sampling in the hyperspace of 18 stacking parameters where the interaction energies using AMBER‐parm99bsc0 and CHARMM‐36 force‐fields were calculated from atomic positions. We have also performed stacking energy calculations for structures from Monte‐Carlo ensemble by Dispersion corrected density functional theory. The available experimental data with Watson–Crick base pairs are compared to establish the validity of the method. Stacking interaction involving A:U and G:C base pairs with non‐canonical G:U base pairs also were calculated and showed that these structures were also sequence dependent. This approach could be useful to generate multiscale modeling of nucleic acids in terms of coarse‐grained parameters where the atomic interactions are preserved. This method would also be useful to predict structure and dynamics of different base pair steps containing non Watson–Crick base pairs, as found often in the non‐coding RNA structures. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 212–226, 2016.     

Structural characterization of naturally occurring RNA single mismatches

RNA is known to be involved in several cellular processes; however, it is only active when it is folded into its correct 3D conformation. The folding, bending and twisting of an RNA molecule is dependent upon the multitude of canonical and non-canonical secondary structure motifs. These motifs contribute to the structural complexity of RNA but also serve important integral biological functions, such as serving as recognition and binding sites for other biomolecules or small ligands. One of the most prevalent types of RNA secondary structure motifs are single mismatches, which occur when two canonical pairs are separated by a single non-canonical pair. To determine sequence–structure relationships and to identify structural patterns, we have systematically located, annotated and compared all available occurrences of the 30 most frequently occurring single mismatch-nearest neighbor sequence combinations found in experimentally determined 3D structures of RNA-containing molecules deposited into the Protein Data Bank. Hydrogen bonding, stacking and interaction of nucleotide edges for the mismatched and nearest neighbor base pairs are described and compared, allowing for the identification of several structural patterns. Such a database and comparison will allow researchers to gain insight into the structural features of unstudied sequences and to quickly look-up studied sequences.     

ClaRNA: a classifier of contacts in RNA 3D structures based on a comparative analysis of various classification schemes

The understanding of folding and function of RNA molecules depends on the identification and classification of interactions between ribonucleotide residues. We developed a new method named ClaRNA for computational classification of contacts in RNA 3D structures. Unique features of the program are the ability to identify imperfect contacts and to process coarse-grained models. Each doublet of spatially close ribonucleotide residues in a query structure is compared to clusters of reference doublets obtained by analysis of a large number of experimentally determined RNA structures, and assigned a score that describes its similarity to one or more known types of contacts, including pairing, stacking, base–phosphate and base–ribose interactions. The accuracy of ClaRNA is 0.997 for canonical base pairs, 0.983 for non-canonical pairs and 0.961 for stacking interactions. The generalized squared correlation coefficient (GC²) for ClaRNA is 0.969 for canonical base pairs, 0.638 for non-canonical pairs and 0.824 for stacking interactions. The classifier can be easily extended to include new types of spatial relationships between pairs or larger assemblies of nucleotide residues. ClaRNA is freely available via a web server that includes an extensive set of tools for processing and visualizing structural information about RNA molecules.     

Studies of translational misreading in vivo show that the ribosome very efficiently discriminates against most potential errors

Protein synthesis must rapidly and repeatedly discriminate between a single correct and many incorrect aminoacyl-tRNAs. We have attempted to measure the frequencies of all possible missense errors by tRNA, tRNA and tRNA. The most frequent errors involve three types of mismatched nucleotide pairs, U•U, U•C, or U•G, all of which can form a noncanonical base pair with geometry similar to that of the canonical U•A or C•G Watson–Crick pairs. Our system is sensitive enough to measure errors at other potential mismatches that occur at frequencies as low as 1 in 500,000 codons. The ribosome appears to discriminate this efficiently against any pair with non-Watson–Crick geometry. This extreme accuracy may be necessary to allow discrimination against the errors involving near Watson–Crick pairing.     

NCIR: a database of non-canonical interactions in known RNA structures

The secondary and tertiary structure of an RNA molecule typically includes a number of non-canonical base–base interactions. The known occurrences of these interactions are tabulated in the NCIR database, which can be accessed from http://prion.bchs.uh.edu/bp_type/. The number of examples is now over 1400, which is an increase of >700% since the database was first published. This dramatic increase reflects the addition of data from the recently published crystal structures of the 50S (2.4 Å) and 30S (3.0 Å) ribosomal subunits. In addition, non-canonical interactions observed in published crystal and NMR structures of tRNAs, group I introns, ribozymes, RNA aptamers and synthetic oligonucleotides are included. Properties associated with these interactions, such as sequence context, sugar pucker conformation, glycosidic angle conformation, melting temperature, chemical shift and free energy, are also reported when available. Out of the 29 anticipated pairs with at least two hydrogen bonds, 28 have been observed to date. In addition, several novel examples, not generally predicted, have also been encountered, bringing the total of such pairs to 36. Added to this list are a variety of single, bifurcated, triple and quadruple interactions. The most common non-canonical pairs are the sheared GA, GA imino, AU reverse Hoogsteen, and the GU and AC wobble pairs. The most frequent triple interaction connects N3 of an A with the amino of a G that is also involved in a standard Watson–Crick pair.     

Probing the structural hierarchy and energy landscape of an RNA T-loop hairpin

The T-loop motif is an important recurrent RNA structural building block consisting of a U-turn sub-motif and a UA trans Watson–Crick/Hoogsteen base pair. In the presence of a hairpin stem, the UA non-canonical base pair becomes part of the UA-handle motif. To probe the hierarchical organization and energy landscape of the T-loop, we performed replica exchange molecular dynamics (REMD) simulations of the T-loop in isolation and as part of a hairpin. Our simulations reveal that the isolated T-loop adopts coil conformers stabilized by base stacking. The T-loop hairpin shows a highly rugged energy landscape featuring multiple local minima with a transition state for folding consisting of partially zipped states. The U-turn displays a high conformational flexibility both when the T-loop is in isolation and as part of a hairpin. On the other hand, the stability of the UA non-canonical base pair is enhanced in the presence of the UA-handle. This motif is apparently a key component for stabilizing the T-loop, while the U-turn is mostly involved in long-range interaction. Our results suggest that the stability and folding of small RNA motifs are highly dependent on local context.     

New information content in RNA base pairing deduced from quantitative analysis of high-resolution structures

Non-canonical base pairs play important roles in organizing the complex three-dimensional folding of RNA. Here, we outline methodology developed both to analyze the spatial patterns of interacting base pairs in known RNA structures and to reconstruct models from the collective experimental information. We focus attention on the structural context and deformability of the seven pairing patterns found in greatest abundance in the helical segments in a set of well-resolved crystal structures, including (i–ii) the canonical A·U and G·C Watson–Crick base pairs, (iii) the G·U wobble pair, (iv) the sheared G·A pair, (v) the A·U Hoogsteen pair, (vi) the U·U wobble pair, and (vii) the G·A Watson–Crick-like pair. The non-canonical pairs stand out from the canonical associations in terms of apparent deformability, spanning a broader range of conformational states as measured by the six rigid-body parameters used to describe the spatial arrangements of the interacting bases, the root-mean-square deviations of the base-pair atoms, and the fluctuations in hydrogen-bonding geometry. The deformabilties, the modes of base-pair deformation, and the preferred sites of occurrence depend on sequence. We also characterize the positioning and overlap of the base pairs with respect to the base pairs that stack immediately above and below them in double-helical fragments. We incorporate the observed positions of the bases, base pairs, and intervening phosphorus atoms in models to predict the effects of the non-canonical interactions on overall helical structure.     

NMR structures of double loops of an RNA aptamer against mammalian initiation factor 4A

A high affinity RNA aptamer (APT58, 58 nt long) against mammalian initiation factor 4A (eIF4A) requires nearly its entire nucleotide sequence for efficient binding. Since splitting either APT58 or eIF4A into two domains diminishes the affinity for each other, it is suggested that multiple interactions or a global interaction between the two molecules accounts for the high affinity. To understand the structural basis of APT58's global recognition of eIF4A, we determined the solution structure of two essential nucleotide loops (AUCGCA and ACAUAGA) within the aptamer using NMR spectroscopy. The AUCGCA loop is stabilized by a U-turn motif and contains a non-canonical A:A base pair (the single hydrogen bond mismatch: Hoogsteen/Sugar-edge). On the other hand, the ACAUAGA loop is stabilized by an AUA tri-nucleotide loop motif and contains the other type of A:A base pair (single hydrogen bond mismatch: Watson–Crick/Watson–Crick). Considering the known structural and functional properties of APT58, we propose that the AUCGCA loop is directly involved in the interaction with eIF4A, while the flexibility of the ACAUAGA loop is important to support this interaction. The Watson–Crick edges of C7 and C9 in the AUCGCA loop may directly interact with eIF4A.     

Effect of single-residue bulges on RNA double-helical structures: crystallographic database analysis and molecular dynamics simulation studies

Asymmetric bulge loop motifs are widely dispersed in all types of functional RNAs. They are frequently occurring structural motifs in folded RNA structures and appear commonly in pre-microRNA and ribosomes, where they are involved in specific RNA–RNA and RNA–protein interactions. It is therefore necessary to understand such motifs from a structural point of view. We analyzed all available RNA structures and identified quite a few fragments of double helices that contain bulges. We found that these discontinuities often introduce kinks into the double helices, which also affects the stacking overlap between the base pairs across the irregularity. In order to understand the influence of these bulges on stability and flexibility, we carried out molecular dynamics simulations of three different single-residue bulge-containing RNA helices using the CHARMM36 force field. The structural variability at the junctions of RNA bulges is expected to differ from that in continuous double-helical stretches. The structural features of the junction region were observed to vary noticeably depending on the orientation of the bulge residue. When the base of the bulge residue is looped out, the RNA stretch behaves like a standard long A-form RNA double helix, whereas the entire RNA behaves differently when the base of the bulge residue is intercalated between base pairs inside the RNA stem. Such single-base intercalation was found to introduce a permanent kink into the composite double helix, which could be a recognition element for Dicer during the maturation of miRNA.