Fingerprinting Noncanonical and Tertiary RNA Structures by Differential SHAPE Reactivity

Many RNA structures are composed of simple secondary structure elements linked by a few critical tertiary interactions. SHAPE chemistry has made interrogation of RNA dynamics at single-nucleotide resolution straightforward. However, de novo identification of nucleotides involved in tertiary interactions remains a challenge. Here we show that nucleotides that form noncanonical or tertiary contacts can be detected by comparing information obtained using two SHAPE reagents, N-methylisatoic anhydride (NMIA) and 1-methyl-6-nitroisatoic anhydride (1M6). Nucleotides that react preferentially with NMIA exhibit slow local nucleotide dynamics and usually adopt the less common C2'-endo ribose conformation. Experiments and first-principles calculations show that 1M6 reacts preferentially with nucleotides in which one face of the nucleobase allows an unhindered stacking interaction with the reagent. Differential SHAPE reactivities were used to detect noncanonical and tertiary interactions in four RNAs with diverse structures and to identify preformed noncanonical interactions in partially folded RNAs. Differential SHAPE reactivity analysis will enable experimentally concise, large-scale identification of tertiary structure elements and ligand binding sites in complex RNAs and in diverse biological environments.     

Novel features for identifying A-minors in three-dimensional RNA molecules

RNA tertiary interactions or tertiary motifs are conserved structural patterns formed by pairwise interactions between nucleotides. They include base-pairing, base-stacking, and base-phosphate interactions. A-minor motifs are the most common tertiary interactions in the large ribosomal subunit. The A-minor motif is a nucleotide triple in which minor groove edges of an adenine base are inserted into the minor groove of neighboring helices, leading to interaction with a stabilizing base pair. We propose here novel features for identifying and predicting A-minor motifs in a given three-dimensional RNA molecule. By utilizing the features together with machine learning algorithms including random forests and support vector machines, we show experimentally that our approach is capable of predicting A-minor motifs in the given RNA molecule effectively, demonstrating the usefulness of the proposed approach. The techniques developed from this work will be useful for molecular biologists and biochemists to analyze RNA tertiary motifs, specifically A-minor interactions.     

Slow conformational dynamics at C2'-endo nucleotides in RNA

RNA molecules undergo local conformational dynamics on timescales spanning picoseconds to minutes. Slower local motions have the greater potential to govern RNA folding, ligand recognition, and ribonucleoprotein assembly reactions but are difficult to detect in large RNAs with complex structures. RNA SHAPE chemistry employs acylation of the ribose 2'-hydroxyl position to measure local nucleotide flexibility in RNA and is well-characterized by a mechanism in which each nucleotide samples unreactive (closed) and reactive (open) states. We monitor RNA conformational dynamics over distinct time domains by varying the electrophilicity of the acylating reagent. Select C2'-endo nucleotides are nonreactive toward fast reagents but reactive toward slower SHAPE reagents in both model RNAs and in a large RNA with a tertiary fold. We conclude, first, that the C2'-endo conformation by itself does not govern SHAPE reactivity. However, some C2'-endo nucleotides undergo extraordinarily slow conformational changes, on the order of 10(-4) s(-1). Due to their distinctive local dynamics, C2'-endo nucleotides have the potential to function as rate-determining molecular switches and are likely to play central, currently unexplored, roles in RNA folding and function.     

SAR by MS

RNAs have recently emerged as an exciting new target for small molecule therapeutics. Conventional HTS discovery strategies measuring disruption of RNAprotein interactions have proven unsuccessful. We describe a ligand-based drug discovery strategy that addresses the inherent difficulties RNA targets. The strategy is based on: 1) using a MS spectrometry (MS)-based assay to measure the affinity of compounds for a target; 2) performing competitive binding experiments and molecular modeling with the motifs to determine the binding site(s) of the ligands; 3) design and synthesis of derivatives of interesting binders to establish the linking sites; 4) identifying the appropriate linker group using MS; 5) fusing motifs into a more complex structure to afford higher affinity compounds. Example of applying this strategy to identify new classes of lead molecules with affinity and specificity for ribosomal RNA targets will be presented.     

Strategies for the Design of Drugs Targeting RNA and RNA-Protein Complexes

In many steps of gene replication and expression, RNA molecules participate as key players, which renders them attractive targets for therapeutic intervention. While the function of nucleic acids as carriers of genetic material is based on their sequence, a number of important RNAs are involved in processes that depend on the defined three-dimensional structures of these molecules. As for proteins, numerous complex folds of RNA exist. The development of drugs that bind specifically to RNA folds opens exciting new ways to expand greatly the existing repertoire of protein-targeted therapeutics. Most functions of RNAs involve interactions with proteins that contain RNA-binding domains. Effector molecules targeted at RNA may either alter the functional three-dimensional structure of the nucleic acid, so the interaction with proteins is thereby inhibited or enhanced, or, as interface inhibitors, they may directly prevent the formation of competent RNA-protein complexes. While the same tools used for the design of protein-targeted drugs may be considered for studying effectors binding to nucleic acids, the differences between proteins and RNAs in the forces which dominate their three-dimensional folding call for novel drug design strategies. In the present review, I will outline how our rapidly expanding knowledge of RNA three-dimensional structure and function facilitates rational approaches to develop RNA-binding compounds. Putative RNA targets for therapeutic intervention will be discussed along with recent advances in understanding RNA-small molecule and RNA-protein interactions.     

The shape-shifting quasispecies of RNA: one sequence, many functional folds

E Unus pluribum, or "Of One, Many", may be at the root of decoding the RNA sequence-structure-function relationship. RNAs embody the large majority of genes in higher eukaryotes and fold in a sequence-directed fashion into three-dimensional structures that perform functions conserved across all cellular life forms, ranging from regulating to executing gene expression. While it is the most important determinant of the RNA structure, the nucleotide sequence is generally not sufficient to specify a unique set of secondary and tertiary interactions due to the highly frustrated nature of RNA folding. This frustration results in folding heterogeneity, a common phenomenon wherein a chemically homogeneous population of RNA molecules folds into multiple stable structures. Often, these alternative conformations constitute misfolds, lacking the biological activity of the natively folded RNA. Intriguingly, a number of RNAs have recently been described as capable of adopting multiple distinct conformations that all perform, or contribute to, the same function. Characteristically, these conformations interconvert slowly on the experimental timescale, suggesting that they should be regarded as distinct native states. We discuss how rugged folding free energy landscapes give rise to multiple native states in the Tetrahymena Group I intron ribozyme, hairpin ribozyme, sarcin-ricin loop, ribosome, and an in vitro selected aptamer. We further describe the varying degrees to which folding heterogeneity impacts function in these RNAs, and compare and contrast this impact with that of heterogeneities found in protein folding. Embracing that one sequence can give rise to multiple native folds, we hypothesize that this phenomenon imparts adaptive advantages on any functionally evolving RNA quasispecies.     

A powerful approach for the selection of 2-aminopurine substitution sites to investigate RNA folding

A precise tertiary structure must be adopted to allow the function of many RNAs in cells. Accordingly, increasing resources have been devoted to the elucidation of RNA structures and the folding of RNAs. 2-Aminopurine (2AP), a fluorescent nucleobase analogue, can be substituted in strategic positions of DNA or RNA molecules to act as site-specific probe to monitor folding and folding dynamics of nucleic acids. Recent studies further demonstrated the potential of 2AP modifications in the assessment of folding kinetics during ligand-induced secondary and tertiary RNA structure rearrangements. However, an efficient way to unambiguously identify reliable positions for 2AP sensors is as yet unavailable and would represent a major asset, especially in the absence of crystallographic or NMR structural data for a target molecule. We report evidence of a novel and direct correlation between the 2'-OH flexibility of nucleotides, observed by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) probing and the fluorescence response following nucleotide substitutions by 2AP. This correlation leads to a straightforward method, using SHAPE probing with benzoyl cyanide, to select appropriate nucleotide sites for 2AP substitution. This clear correlation is presented for three model RNAs of biological significance: the SAM-II, adenine (addA), and preQ(1) class II (preQ(1)cII) riboswitches.     

New loop-loop tertiary interactions in self-splicing introns of subgroup IC and ID: a complete 3D model of the Tetrahymena thermophila ribozyme

Background: Group I introns self-splice via two consecutive trans-esterification reactions in the presence of guanosine cofactor and magnesium ions. Comparative sequence analysis has established that a catalytic core of about 120 nucleotides is conserved in all known group I introns. This core is generally not sufficient for activity, however, and most self-splicing group I introns require non-nonserved peripheral elements to stabilize the complete three-dimensional (3D) structure. The physico-chemical properties of group I introns make them excellent systems for unraveling the structural basis of the RNA-RNA interactions responsible for promoting the self-assembly of complex RNAs.Results: We present phylogenetic and experimental evidence for the existence of three additional tertiary base pairings between hairpin loops within peripheral components of subgroup IC1 and ID introns. Each of these new long range interactions, called P13, P14 and P16, involves a terminal loop located in domain 2. Although domains 2 of IC and ID introns share very strong sequence similarity, their terminal loops interact with domains 5 and 9 (subgroup IC1) and domain 6 (subgroup ID). Based on these tertiary contacts, comparative sequence analysis, and published experimental results such as Fe(II)-EDTA protection patterns, we propose 3D models for two entire group I introns, the subgroup IC1 intron in the large ribosomal precursor RNA of Tetrahymena thermophila and the SdCob.1 subgroup ID intron found in the cytochrome b gene of Saccharomyces douglasii.Conclusions: Three-dimensional models of group I introns belonging to four different subgroups are now available. They all emphasize the modular and hierarchical organization of the architecture of group I introns and the widespread use of base-pairings between terminal hairpin loops for stabilizing the folded and active structures of large and complex RNA molecules.     

Reductive Charge Transfer through an RNA Aptamer

The transfer of charges through double helical DNA is a very well investigated bioelectric phenomenon. RNA, on the contrary, has been less studied in this regard. The few available data report on charge transfer through RNA duplex structures mainly composed of homonucleotide sequences. In the light of the RNA world scenarios, it is an interesting question, if charge transfer can be coupled with RNA function. Functional RNAs however, contain versatile structural motifs. Therefore, electron transport also through non-Watson–Crick base-paired regions might be required. We here demonstrate distance-dependent reductive charge transfer through RNA duplexes and through the non-Watson–Crick base-paired region of an RNA aptamer.     

Aminoglycoside Conjugation for RNA Targeting: Antimicrobials and Beyond

Natural aminoglycosides are therapeutically useful antibiotics and very efficient RNA ligands. They are oligosaccharides that contain several ammonium groups able to interfere with the translation process in prokaryotes upon binding to bacterial ribosomal RNA (rRNA), and thus, impairing protein synthesis. Even if aminoglycosides are commonly used in therapy, these RNA binders lack selectivity and are able to bind to a wide number of RNA sequences/structures. This is one of the reasons for their toxicity and limited applications in therapy. At the same time, the ability of aminoglycosides to bind to various RNAs renders them a great source of inspiration for the synthesis of new binders with improved affinity and specificity toward several therapeutically relevant RNA targets. Thus, a number of studies have been performed on these complex and highly functionalized compounds, leading to the development of various synthetic methodologies toward the synthesis of conjugated aminoglycosides. The aim of this review is to highlight recent progress in the field of aminoglycoside conjugation, paying particular attention to modifications performed toward the improvement of affinity and especially to the selectivity of the resulting compounds. This will help readers to understand how to introduce a desired chemical modification for future developments of RNA ligands as antibiotics, antiviral, and anticancer compounds.     

The size of RNA as an ideal branched polymer

Because of the branching arising from partial self-complementarity, long single-stranded (ss) RNA molecules are significantly more compact than linear arrangements (e.g., denatured states) of the same sequence of monomers. To elucidate the dependence of compactness on the nature and extent of branching, we represent ssRNA secondary structures as tree graphs which we treat as ideal branched polymers, and use a theorem of Kramers for evaluating their root-mean-square radius of gyration, ?R(g)=√R(g)(2). We consider two sets of sequences--random and viral--with nucleotide sequence lengths (N) ranging from 100 to 10,000. The RNAs of icosahedral viruses are shown to be more compact (i.e., to have smaller ?R(g)) than the random RNAs. For the random sequences we find that ?R(g) varies as N(1/3). These results are contrasted with the scaling of ?R(g) for ideal randomly branched polymers (N(1/4)), and with that from recent modeling of (relatively short, N ≤ 161) RNA tertiary structures (N(2/5)).     

Sugar edge/sugar edge base pairs in RNA: stabilities and structures from quantum chemical calculations

Cis and trans sugar edge/sugar edge (SE/SE) binding patterns are essential building units of RNAs. For example, SE/SE interactions form the A-minor motifs, the most important tertiary interaction type in functional RNAs. This study provides an in-depth structure and stability analysis for these two base pair families. Gas-phase-optimized geometries are reported for 12 cis and 7 trans SE/SE base pairs and contrasted to their X-ray counterparts. Interaction energies are computed at the RIMP2 level of theory using the density-functional-theory-optimized geometries. There is a good overall agreement between the optimized and X-ray geometries of the cis SE/SE base pairs. In contrast, only three of the seven trans SE/SE binding patterns could be optimized without a significant distortion of the X-ray geometry. Note, however, that many SE/SE base pairs participate in broader networks of interactions; thus it is not surprising to see some of them to deviate from the X-ray geometry in a complete isolation. Computed interaction energies reveal that all 12 known cis SE/SE binding patterns are very stable. Among the trans SE/SE binding patterns, only the rG/rG, rG/rC, and rA/rG base pairs are sufficiently stable in the crystal geometry. Prediction has been made for some structures not yet detected by crystallography, namely, cis rC/rC, rG/rC, rG/rU, and rU/rU and trans rG/rA base pairs. Interestingly, the new cis SE/SE binding patterns are not necessarily isosteric with the remaining 12 members of this family. The trans rG/rA base pair represents a viable option for base pairing in RNA to be identified by future X-ray studies. In a complete lack of structural information, prediction of other unknown members of the trans SE/SE family was not attempted. Analysis of the interaction energies shows a very large electron correlation component of the interaction energy, pointing at the elevated role of dispersion energy as compared to other types of base pairs. This likely is profitable for stabilization of SE/SE binding patterns in polar environments and could be one of the reasons why the A-minor motif is the leading type of tertiary interactions in RNAs.     

Generating new specific RNA interaction interfaces using C-loops

New RNA interaction interfaces are reported for designing RNA modules for directional supramolecular self-assembly. The new interfaces are generated from existing ones by inserting C-loops between the interaction motifs that mediate supramolecular assembly. C-Loops are new modular motifs recently identified in crystal structures that increase the helical twist of RNA helices in which they are inserted and thus reduce the distance between pairs of loop or loop-receptor motifs from 11 to 9 base-stacking layers while maintaining correct orientation for binding to cognate interaction interfaces. Binding specificities of C-loop-containing molecules for cognate molecules that also have inserted C-loops were found to range up to 20-fold. Binding affinities for most C-loop-containing molecules were generally equal or higher than those for the parent molecules lacking C-loops.     

Tertiary interactions determine the accuracy of RNA folding

RNAs must fold into unique three-dimensional structures to function in the cell, but how each polynucleotide finds its native structure is not understood. To investigate whether the stability of the tertiary structure determines the speed and accuracy of RNA folding, docking of a tetraloop with its receptor in a bacterial group I ribozyme was perturbed by site-directed mutagenesis. Disruption of the tetraloop or its receptor destabilizes tertiary interactions throughout the ribozyme by 2-3 kcal/mol, demonstrating that tertiary interactions form cooperatively in the transition from a native-like intermediate to the native state. Nondenaturing PAGE and RNase T1 digestion showed that base pairs form less homogeneously in the mutant RNAs during the transition from the unfolded state to the intermediate. Thus, tertiary interactions between helices bias the ensemble of secondary structures toward native-like conformations. Time-resolved hydroxyl radical footprinting showed that the wild-type ribozyme folds completely within 5-20 ms. By contrast, only 40-60% of a tetraloop mutant ribozyme folds in 30-40 ms, with the remainder folding in 30-200 s via nonnative intermediates. Therefore, destabilization of tetraloop-receptor docking introduces an alternate folding pathway in the otherwise smooth energy landscape of the wild-type ribozyme. Our results show that stable tertiary structure increases the flux through folding pathways that lead directly and rapidly to the native structure.     

Efficient Oligomerization of Aromatic Amino Acids Induced by Gaps in Four-Helix Bundles of DNA or RNA

The formation of peptides from amino acids is one of the processes associated with life. Because of the dominant role of translation in extant biology, peptide-forming processes that are RNA induced are of particular interest. We have previously reported the formation of phosphoramidate-linked peptido RNAs as the products of spontaneous condensation reactions between ribonucleotides and free amino acids in aqueous solution. We now asked whether four-helix bundle (4HB) DNA or RNA folding motifs with a single- or double-nucleotide gap next to a 5’-phosphate can act as reaction sites for phosphoramidate formation. For glycine, this was found to be the case, whereas phenylalanine and tryptophan showed accelerated formation of peptides without a covalent link to the nucleic acid. Free peptides with up to 11 tryptophan or phenylalanine residues were found in precipitates forming in the presence of gap-containing DNA or RNA 4HBs. Control experiments using motifs with just a nick or primer alone did not have the same effect. Because folded structures with a gap in a double helix are likely products of hybridization of strands formed in statistically controlled oligomerization reactions, our results are interesting in the context of prebiotic scenarios. Independent of a putative role in evolution, our findings suggest that for some aromatic amino acids an RNA-induced pathway for oligomerization exists that does not have a discernable link to translation.     

Site-specific platinum(II) cross-linking in a ribozyme active site

The function of RNA depends on its ability to adopt complex and dynamic structures, and the incorporation of site-specific cross-linking probes is a powerful method for providing distance constraints that are valuable in RNA structural biology. Here we describe a new RNA-RNA cross-linking strategy based on Pt(II) targeting of specific phosphorothioate substitutions. In this strategy cis-diammine Pt(II) complexes are kinetically recruited and anchored to a phosphorothioate substitution embedded within a structured RNA. Substitution of the remaining exchangeable Pt(II) ligand with a nucleophile supplied by a nearby RNA nucleobase results in metal-mediated cross-links that are stable during isolation. This type of cross-linking strategy was explored within the catalytic core of the Hammerhead ribozyme (HHRz). When a phosphorothioate substitution is installed at the scissile bond normally cleaved by the HHRz, Pt(II) cross-linking takes place to nucleotides G8 and G10 in the ribozyme active site. Both of these positions are predicted to be within ~8 ? of a phosphorothioate-bound Pt(II) metal center. Cross-linking depends on Mg(2+) ion concentration, reaching yields as high as 30%, with rates that indicate cation competition within the RNA three-helix junction. Cross-linking efficiency depends on accurate formation of the HHRz tertiary structure, and cross-links are not observed for RNA helices. Combined, these results show promise for using kinetically inert Pt(II) complexes as new site-specific cross-linking tools for exploring RNA structure and dynamics.     

Automated RNA structure prediction uncovers a kink-turn linker in double glycine riboswitches

The tertiary structures of functional RNA molecules remain difficult to decipher. A new generation of automated RNA structure prediction methods may help address these challenges but have not yet been experimentally validated. Here we apply four prediction tools to a class of double glycine riboswitches that can bind two ligands cooperatively. A novel method (BPPalign), RMdetect, JAR3D, and Rosetta 3D modeling give consistent predictions for a new stem P0 and a kink-turn motif. These elements structure the linker between the RNAs' double aptamers. Chemical mapping on the Fusobacterium nucleatum riboswitch with N-methylisatoic anhydride, dimethyl sulfate and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate probing, mutate-and-map studies, and mutation/rescue experiments all provide strong evidence for the structured linker. Under solution conditions that permit rigorous thermodynamic analysis, disrupting this helix-junction-helix structure gives 120- and 6-30-fold poorer dissociation constants for the RNA's two glycine-binding transitions, corresponding to an overall energetic impact of 4.3 ± 0.5 kcal/mol. Prior biochemical and crystallography studies did not include this critical element due to over-truncation of the RNA. We speculate that several further undiscovered elements are likely to exist in the flanking regions of this and other functional RNAs, and automated prediction tools can play a useful role in their detection and dissection.