Coplanar and coaxial orientations of RNA bases and helices

Electrostatic interactions, base-pairing, and especially base-stacking dominate RNA three-dimensional structures. In an A-form RNA helix, base-stacking results in nearly perfect parallel orientations of all bases in the helix. Interestingly, when an RNA structure containing multiple helices is visualized at the atomic level, it is often possible to find an orientation such that only the edges of most bases are visible. This suggests that a general aspect of higher level RNA structure is a coplanar arrangement of base-normal vectors. We have analyzed all solved RNA crystal structures to determine the degree to which RNA base-normal vectors are globally coplanar. Using a statistical test based on the Watson-Girdle distribution, we determined that 330 out of 331 known RNA structures show statistically significant (p < 0.05; false discovery rate [FDR] = 0.05) coplanar normal vector orientations. Not surprisingly, 94% of the helices in RNA show bipolar arrangements of their base-normal vectors (p < 0.05). This allows us to compute a mean axis for each helix and compare their orientations within an RNA structure. This analysis revealed that 62% (208/331) of the RNA structures exhibit statistically significant coaxial packing of helices (p < 0.05, FDR = 0.08). Further analysis reveals that the bases in hairpin loops and junctions are also generally planar. This work demonstrates coplanar base orientation and coaxial helix packing as an emergent behavior of RNA structure and may be useful as a structural modeling constraint.     

Antisense oligonucleotide containing an internal, non-nucleotide-based linker promote site-specific cleavage of RNA.

We have designed and synthesized a series of novel antisense methylphosphonate oligonucleotide (MPO) cleaving agents that promote site-specific cleavage on a complementary RNA target. These MPOs contain a non- nucleotide-based linking moiety near the middle of the sequence in place of one of the nucleotide bases. The region surrounding the unpaired base on the RNA strand (i.e. the one directly opposite the non-nucleotide-linker) is sensitive to hydrolytic cleavage catalyzed by ethylenediamine hydrochloride. Furthermore, the regions of the RNA comprising hydrogen bonded domains are resistant to cleavage compared with single-stranded RNA alone. Several catalytic moieties capable of supporting acid/base hydrolysis were coupled to the non-nucleotide-based linker via simple aqueous coupling chemistries. When tethered to the MPO in this manner these moieties are shown to catalyze site-specific cleavage on the RNA target without any additional catalyst.     

RNA structure and dynamics: A base pairing perspective

RNA is now known to possess various structural, regulatory and enzymatic functions for survival of cellular organisms. Functional RNA structures are generally created by three-dimensional organization of small structural motifs, formed by base pairing between self-complementary sequences from different parts of the RNA chain. In addition to the canonical Watson–Crick or wobble base pairs, several non-canonical base pairs are found to be crucial to the structural organization of RNA molecules. They appear within different structural motifs and are found to stabilize the molecule through long-range intra-molecular interactions between basic structural motifs like double helices and loops. These base pairs also impart functional variation to the minor groove of A-form RNA helices, thus forming anchoring site for metabolites and ligands. Non-canonical base pairs are formed by edge-to-edge hydrogen bonding interactions between the bases. A large number of theoretical studies have been done to detect and analyze these non-canonical base pairs within crystal or NMR derived structures of different functional RNA. Theoretical studies of these isolated base pairs using ab initio quantum chemical methods as well as molecular dynamics simulations of larger fragments have also established that many of these non-canonical base pairs are as stable as the canonical Watson–Crick base pairs. This review focuses on the various structural aspects of non-canonical base pairs in the organization of RNA molecules and the possible applications of these base pairs in predicting RNA structures with more accuracy.     

A base-triple structural domain in RNA.

An oligonucleotide modeled on a proposed base-triple domain of the Tetrahymena group I intron has been characterized by NMR. The oligonucleotide contains two double-helix regions with adjacent single-stranded nucleotides. The NMR data show that the two helices stack coaxially, although the rotation between the two helices is approximately twice as large as the rotation between normal base pairs. The rotation between the two helices allows the single-stranded nucleotides to form U.U.G and A.G.C base triples in the minor groove. The A.G.C base triple contains a hydrogen bond between the adenine N1 and a 2'-hydroxyl in the minor groove of the G.C pair. A similar hydrogen bond between an adenine and a 2'-hydroxyl in transfer RNA suggests that this could be a recurring tertiary interaction in RNA.     

Effects of single-base bulges on intercalator binding to small RNA and DNA hairpins and a ribosomal RNA fragment

The way in which a single-base bulge might affect the structure of an RNA helix has been examined by preparing a series of six RNA hairpins, all with seven base pairs and a four-nucleotide loop. Five of the hairpins have single-base bulges at different positions. The intercalating cleavage reagent (methidiumpropyl)-EDTA-Fe(II) [MPE-Fe(II)] binds preferentially at a CpG sequence in the helix lacking a bulge and in four of the five hairpins with bulges. Hairpins with a bulge one or two bases to the 3' side of the CpG sequence bind ethidium 4-5-fold more strongly than the others. V1 RNase, which is sensitive to RNA backbone conformation in helices, detects a conformational change in all of the helices when ethidium binds; the most dramatic changes, involving the entire hairpin stem, are in one of the two hairpins with enhanced ethidium affinity. Only a slight conformational change is detected in the hairpin lacking a bulge. A bulge adjacent to a CpG sequence in a 100-nucleotide ribosomal RNA fragment enhances MPE-Fe(II) binding by an order of magnitude. These results extend our previous observations of bulges at a single position in an RNA hairpin [White, S. A., & Draper, D.E. (1987) Nucleic Acids Res. 15, 4049] and show that (1) a structural change in an RNA helix may be propagated for several base pairs, (2) bulges tend to increase the number of conformations available to a helix, and (3) the effects observed in small RNA hairpins are relevant to larger RNAs with more extensive structure. A bulge in a DNA hairpin identical in sequence with the RNA hairpins does not enhance MPE-Fe(II) binding affinity, relative to a control DNA hairpin. The effects of bulges on ethidium intercalation are evidently modulated by helix structure.     

Secondary structure model for 23S ribosomal RNA.

A secondary structure model for 23S ribosomal RNA has been constructed on the basis of comparative sequence data, including the complete sequences from E. coli. Bacillus stearothermophilis, human and mouse mitochondria and several partial sequences. The model has been tested extensively with single strand-specific chemical and enzymatic probes. Long range base-paired interactions organize the molecule into six major structural domains containing over 100 individual helices in all. Regions containing the sites of interaction with several ribosomal proteins and 5S RNA have been located. Segments of the 23S RNA structure corresponding to eucaryotic 5.8S and 25 RNA have been identified, and base paired interactions in the model suggest how they are attached to 28S RNA. Functionally important regions, including possible sites of contact with 30S ribosomal subunits, the peptidyl transferase center and locations of intervening sequences in various organisms are discussed. Models for molecular 'switching' of RNA molecules based on coaxial stacking of helices are presented, including a scheme for tRNA-23S RNA interaction.     

The hydrodynamic shape, conformation, and molecular model of Escherichia coli ribosomal 5 S RNA.

The structure of ribosomal 5 S RNA has been examined using several physical biochemical techniques. Hydrodynamic measurements yield a s020,omega and [eta] of 5.5 x 10(-13) x and 6.9 ml/g, respectively. Other parameters calculated from these values indicate the shape of 5 S RNA is consistent with that of a prolate ellipsoid 160 A in length and 32 A wide. Sedimentation equilibrium results show that 5 S RNA exists as a monomer in the reconstitution buffer with an apparent molecular weight of 44,000. Ultraviolet absorption difference spectra show that approximately 75% of the bases in 5 S RNA are involved in base pairing, and of these base pairs 70% are G-C and 30% are A-U. These results on the overall shape and secondary structure of 5 S RNA have been incorporated with the results of other investigators as to the possible location of single-stranded and double-stranded helical regions, and a molecular model for 5 S RNA is proposed. The molecular model consists of three double helices in the shape of a prolate ellipsoid, with two of the double helical regions at one end of the molecule. The structure is consistent with the available data on the structure and function of 5 S RNA and bears similarity to the molecular model proposed by Osterberg et al. ((1976) Eur. J. Biochem. 68, 481-487) based on small angle x-ray scattering results and the secondary structure proposed by Madison ((1968) Annu. Rev. Biochem. 37, 131-148).     

Secondary structure of 5S RNA: NMR experiments on RNA molecules partially labeled with nitrogen-15

A method has been found for reassembling fragment 1 of Escherichia coli 5S RNA from mixtures containing strand III (bases 69-87) and the complex consisting of strand II (bases 89-120) and strand IV (bases 1-11). The reassembled molecule is identical with unreconstituted fragment 1. With this technique, fragment 1 molecules have been constructed 15N-labeled either in strand III or in the strand II-strand IV complex. Spectroscopic data obtained with these partially labeled molecules show that the terminal helix of 5S RNA includes the GU and GC base pairs at positions 9 and 10 which the standard model for 5S secondary structure predicts [see Delihas, N., Anderson, J., & Singhal, R. P. (1984) Prog. Nucleic Acid Res. Mol. Biol. 31, 161-190] but that these base pairs are unstable both in the fragment and in native 5S RNA. The data also assign three resonances to the helix V region of the molecule (bases 70-77 and 99-106). None of these resonances has a "normal" chemical shift even though two of them correspond to AU or GU base pairs in the standard model. The implications of these findings for our understanding of the structure of 5S RNA and its complex with ribosomal protein L25 are discussed.     

Solution conformation of a bulged adenosine base in an RNA duplex by relaxation matrix refinement

Bulges are common structural motifs in RNA secondary structure and are thought to play important roles in RNA-protein and RNA-drug interactions. Adenosine bases are the most commonly occurring unpaired base in double helical RNA secondary structures. The solution conformation and dynamics of a 25-nucleotide RNA duplex containing an unpaired adenosine, r(GGCAGAGUGCCGC): r(GCGGCACCUGCC) have been studied by NMR spectroscopy and MORASS iterative relaxation matrix structural refinement. The results show that the bulged adenosine residue stacks into the RNA duplex with little perturbation around the bulged region. Most of the bases in the RNA duplex adopt C(3)'-endo conformation, exhibiting the N-type sugar pucker as found in the A form helices. The sugars of the bulged residue and the 5' flanking residue to it are found to exhibit C(2)'-endo conformation. None of the residues are in syn conformation.     

DbpA is a region‐specific RNA helicase

DbpA is a DEAD‐box RNA helicase implicated in RNA structural rearrangements in the peptidyl transferase center. DbpA contains an RNA binding domain, responsible for tight binding of DbpA to hairpin 92 of 23S ribosomal RNA, and a RecA‐like catalytic core responsible for double‐helix unwinding. It is not known if DbpA unwinds only the RNA helices that are part of a specific RNA structure, or if DbpA unwinds any RNA helices within the catalytic core's grasp. In other words, it is not known if DbpA is a site‐specific enzyme or region‐specific enzyme. In this study, we used protein and RNA engineering to investigate if DbpA is a region‐specific or a site‐specific enzyme. Our data suggest that DbpA is a region‐specific enzyme. This conclusion has an important implication for the physiological role of DbpA. It suggests that during ribosome assembly, DbpA could bind with its C‐terminal RNA binding domain to hairpin 92, while its catalytic core may unwind any double‐helices in its vicinity. The only requirement for a double‐helix to serve as a DbpA substrate is for the double‐helix to be positioned within the catalytic core's grasp.     

Differential helix stabilities and sites pre-organized for tertiary interactions revealed by monitoring local nucleotide flexibility in the bI5 group I intron RNA

The local environment at adenosine residues in the bI5 group I intron RNA was monitored as a function of Mg(2+) using both the traditional method of dimethyl sulfate (DMS) N1 methylation and a new approach, selective acylation of 2'-amine substituted nucleotides. These probes yield complementary structural information because N1 methylation reports accessibility at the base pairing face, whereas 2'-amine acylation scores overall residue flexibility. 2'-Amine acylation robustly detects RNA secondary structure and is sensitive to higher order interactions not monitored by DMS. Disruption of RNA structure due to the 2'-amine substitution is rare and can be compensated by stabilizing folding conditions. Peripheral helices that do not interact with other parts of the RNA are more stable than both base paired helices and tertiary interactions in the conserved catalytic core. The equilibrium state of the bI5 intron RNA, prior to assembly with its protein cofactor, thus features a relatively loosely packed core anchored by more stable external stem-loop structures. Adenosine residues in J4/5 and P9.0 form structures in which the nucleotide is constrained but the N1 position is accessible, consistent with pre-organization to form long-range interactions with the 5' and 3' splice sites.     

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.     

Structural study of an RNA aptamer for a Tat protein complexed with ligands.

An RNA aptamer for an HIV Tat protein has been isolated by the in vitro SELEX method. The RNA aptamer binds to the Tat protein 50-100 times more strongly than native TAR RNA does. Here, we have investigated the structure of the RNA aptamer complexed with ligands, partial peptide fragments of the Tat protein or argininamide, by multidimensional 1H/13C/15N NMR. It is strongly suggested that two U:A:U base triples are formed in the RNA aptamer upon binding of ligands. Specific hydrogen bonds between arginine side chains of ligands and guanine bases located adjacent to the base triples are identified. On the basis of many intramolecular and intermolecular NOEs, a structural model of the complex has been constructed.     

Identification and assignment of base pairs in three helical stems of Torulopsis utilis ribosomal 5S RNA and its RNase T1 and RNase T2 cleaved fragments via 500-MHz proton homonuclear overhauser enhancements

Imino proton resonances in the downfield region (10-14 ppm) of the 500-MHz 1H NMR spectrum of Torulopsis utilis 5S RNA are identified (A X U, G X C, or G X U) and assigned to base pairs in helices I, IV, and V via analysis of homonuclear Overhauser enhancements (NOE) from intact T. utilis 5S RNA, its RNase T1 and RNase T2 digested fragments, and a second yeast (Saccharomyces cerevisiae) 5S RNA whose nucleotide sequence differs at only six residues from that of T. utilis 5S RNA. The near-identical chemical shifts and NOE behavior of most of the common peaks from these four RNAs strongly suggest that helices I, IV, and V retain the same conformation after RNase digestion and that both T. utilis and S. cerevisiae 5S RNAs share a common secondary and tertiary structure. Of the four G X U base pairs identified in the intact 5S RNA, two are assigned to the terminal stem (helix I) and the other two to helices IV and V. Seven of the nine base pairs of the terminal stem have been assigned. Our experimental demonstration of a G X U base pair in helix V supports the 5S RNA secondary structural model of Luehrsen and Fox [Luehrsen, K. R., & Fox, G.E. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2150-2154]. Finally, the base-pair proton peak assigned to the terminal G X U in helix V of the RNase T2 cleaved fragment is shifted downfield from that in the intact 5S RNA, suggesting that helices I and V may be coaxial in intact T. utilis 5S RNA.     

Determination of secondary structure in rabbit globin messenger RNA by thermal denaturation.

The secondary structure of highly purified globin messenger RNA has been investigated by alkaline hydrolysis, nuclease digestion, and thermal denaturation. The thermal denaturation properties of globin messenger have been compared to poly(U), poly (A), and a synthetic random sequence RNA copolymer. From these studies it is concluded that globin mRNA contains considerable secondary structure and that the amount of helical structure is greater than that which occurs with a random sequence polyribonucleotide. Globin mRNA contains, by comparison to the secondary structures of native DNA, tRNAs, or 18S rRNA, helices with involve 55-62% of the bases or 58-68% if a correction is made for the 3'-terminal poly(A) segment. The helices of globin mRNA appear to be unique as differences in the NaCl stabilization of this RNA have been noted when compared to other naturally ooccurring and synthetic RNAs. Comparison of the hyperchromicity maxima, obtained at 260 and 280 nm for globin mRNA and 18S rRNA, indicates that the helices of the two RNAs contain similar numbers of G-C base pairs. Differential analysis of NaCl stabilization curves indicate three discrete thermally denaturable helix types in globin mRNA.     

Analysis of RNA motifs

RNA motifs are directed and ordered stacked arrays of non-Watson-Crick base pairs forming distinctive foldings of the phosphodiester backbones of the interacting RNA strands. They correspond to the 'loops' - hairpin, internal and junction - that intersperse the Watson-Crick two-dimensional helices as seen in two-dimensional representations of RNA structure. RNA motifs mediate the specific interactions that induce the compact folding of complex RNAs. RNA motifs also constitute specific protein or ligand binding sites. A given motif is characterized by all the sequences that fold into essentially identical three-dimensional structures with the same ordered array of isosteric non-Watson-Crick base pairs. It is therefore crucial, when analyzing a three-dimensional RNA structure in order to identify and compare motifs, to first classify its non-Watson-Crick base pairs geometrically.     

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.     

Hydration of transfer RNA molecules: a crystallographic study

Four crystal structures of transfer RNA molecules were refined at 3 A resolution with the inclusion of the solvent molecules found in the difference maps: yeast tRNA-phe in the orthorhombic form, yeast tRNA-phe in the monoclinic form and yeast tRNA-asp in the A and B forms. Over 100 solvent molecules were located in each tRNA crystal. Several hydration schemes are found repeatedly in the 4 crystals. The tertiary interactions in the corner of the L-shaped molecule attract numerous solvent molecules which bridge the ribose hydroxyl O(2') atoms, base exocyclic atoms and phosphate anionic oxygen atoms. Conservation of bases leads to conservative localized hydration patterns. Several solvent molecules are found stabilizing unusual base pairs like the G-U pairs and those involving the pseudouridine base. Water bridges between the O(2') and the exocyclic atom O2 of pyrimidines or the N3 atom of purines are common. Water bridges occur frequently between successive anionic oxygen atoms of each strand as well as between N7 or other exocyclic atoms of successive bases in the major groove. Magnesium ions or spermine molecules are found to bind in the major groove of tRNA helices without specific interactions.