Strong correlation between SHAPE chemistry and the generalized NMR order parameter (S2) in RNA

The functions of most RNA molecules are critically dependent on the distinct local dynamics that characterize secondary structure and tertiary interactions and on structural changes that occur upon binding by proteins and small molecule ligands. Measurements of RNA dynamics at nucleotide resolution set the foundation for understanding the roles of individual residues in folding, catalysis, and ligand recognition. In favorable cases, local order in small RNAs can be quantitatively analyzed by NMR in terms of a generalized order parameter, S2. Alternatively, SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) chemistry measures local nucleotide flexibility in RNAs of any size using structure-sensitive reagents that acylate the 2'-hydroxyl position. In this work, we compare per-residue RNA dynamics, analyzed by both S2 and SHAPE, for three RNAs: the HIV-1 TAR element, the U1A protein binding site, and the Tetrahymena telomerase stem loop 4. We find a very strong correlation between the two measurements: nucleotides with high SHAPE reactivities consistently have low S2 values. We conclude that SHAPE chemistry quantitatively reports local nucleotide dynamics and can be used with confidence to analyze dynamics in large RNAs, RNA-protein complexes, and RNAs in vivo.     

Time-resolved RNA SHAPE chemistry

Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry yields quantitative RNA secondary and tertiary structure information at single nucleotide resolution. SHAPE takes advantage of the discovery that the nucleophilic reactivity of the ribose 2'-hydroxyl group is modulated by local nucleotide flexibility in the RNA backbone. Flexible nucleotides are reactive toward hydroxyl-selective electrophiles, whereas constrained nucleotides are unreactive. Initial versions of SHAPE chemistry, which employ isatoic anhydride derivatives that react on the minute time scale, are emerging as the ideal technology for monitoring equilibrium structures of RNA in a wide variety of biological environments. Here, we extend SHAPE chemistry to a benzoyl cyanide scaffold to make possible facile time-resolved kinetic studies of RNA in approximately 1 s snapshots. We then use SHAPE chemistry to follow the time-dependent folding of an RNase P specificity domain RNA. Tertiary interactions form in two distinct steps with local tertiary contacts forming an order of magnitude faster than long-range interactions. Rate-determining tertiary folding requires minutes despite that no non-native interactions must be disrupted to form the native structure. Instead, overall folding is limited by simultaneous formation of interactions approximately 55 A distant in the RNA. Time-resolved SHAPE holds broad potential for understanding structural biogenesis and the conformational interconversions essential to the functions of complex RNA molecules at single nucleotide resolution.     

The mechanisms of RNA SHAPE chemistry

The biological functions of RNA are ultimately governed by the local environment at each nucleotide. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry is a powerful approach for measuring nucleotide structure and dynamics in diverse biological environments. SHAPE reagents acylate the 2'-hydroxyl group at flexible nucleotides because unconstrained nucleotides preferentially sample rare conformations that enhance the nucleophilicity of the 2'-hydroxyl. The critical corollary is that some constrained nucleotides must be poised for efficient reaction at the 2'-hydroxyl group. To identify such nucleotides, we performed SHAPE on intact crystals of the Escherichia coli ribosome, monitored the reactivity of 1490 nucleotides in 16S rRNA, and examined those nucleotides that were hyper-reactive toward SHAPE and had well-defined crystallographic conformations. Analysis of these conformations revealed that 2'-hydroxyl reactivity is broadly facilitated by general base catalysis involving multiple RNA functional groups and by two specific orientations of the bridging 3'-phosphate group. Nucleotide analog studies confirmed the contributions of these mechanisms to SHAPE reactivity. These results provide a strong mechanistic explanation for the relationship between SHAPE reactivity and local RNA dynamics and will facilitate interpretation of SHAPE information in the many technologies that make use of this chemistry.     

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.     

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.     

Femtomole SHAPE reveals regulatory structures in the authentic XMRV RNA genome

Higher-order structure influences critical functions in nearly all noncoding and coding RNAs. Most single-nucleotide resolution RNA structure determination technologies cannot be used to analyze RNA from scarce biological samples, like viral genomes. To make quantitative RNA structure analysis applicable to a much wider array of RNA structure-function problems, we developed and applied high-sensitivity selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) to structural analysis of authentic genomic RNA of the xenotropic murine leukemia virus-related virus (XMRV). For analysis of fluorescently labeled cDNAs generated in high-sensitivity SHAPE experiments, we developed a two-color capillary electrophoresis approach with zeptomole molecular detection limits and subfemtomole sensitivity for complete SHAPE experiments involving hundreds of individual RNA structure measurements. High-sensitivity SHAPE data correlated closely (R = 0.89) with data obtained by conventional capillary electrophoresis. Using high-sensitivity SHAPE, we determined the dimeric structure of the XMRV packaging domain, examined dynamic interactions between the packaging domain RNA and viral nucleocapsid protein inside virion particles, and identified the packaging signal for this virus. Despite extensive sequence differences between XMRV and the intensively studied Moloney murine leukemia virus, architectures of the regulatory domains are similar and reveal common principles of gammaretrovirus RNA genome packaging.     

Intrinsic contribution of the 2'-hydroxyl to RNA conformational heterogeneity

Canonical duplex RNA assumes only the A-form conformation at the secondary structure level while, in contrast, a wide range of noncanonical, tertiary conformations of RNA occur. Here, we show how the 2'-hydroxyl controls RNA conformational properties. Quantum mechanical calculations reveal that the orientation of the 2'-hydroxyl significantly alters the intrinsic flexibility of the phosphodiester backbone, favoring the A-form in duplex RNA when it is in the base orientation and facilitating sampling of a wide range of noncanonical, tertiary structures when it is in the O3' orientation. Influencing the orientation of the 2'-hydroxyl are interactions with the environment, as evidenced by crystallographic survey data, indicating the 2'-hydroxyl to sample more of the O3' orientation in noncanonical RNA structures. These results indicate that the 2'-hydroxyl acts as a "switch", both limiting the conformation of RNA to the A-form at the secondary structure level and allowing RNA to sample a wide range of noncanonical tertiary conformations.     

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.     

Atomistic analysis of pseudoknotted RNA unfolding

As important functional structures, RNA pseudoknots provide excellent models for studying the interplay between secondary and tertiary structures and the roles of triplexes, noncanonical interactions, and coaxial stacking in the folding/unfolding process. Here we report a first atomistic and statistical analysis of the unfolding of the pseudoknot within gene 32 mRNA of bacteriophage T2. Multiple unfolding pathways, diverse transition states, and various intermediate structures were observed. Water molecules were found to be coupled with the unfolding process via the expulsion or concurrent mechanism.     

Noncanonical hydrogen bonding in nucleic acids. Benchmark evaluation of key base-phosphate interactions in folded RNA molecules using quantum-chemical calculations and molecular dynamics simulations

RNA molecules are stabilized by a wide range of noncanonical interactions that are not present in DNA. Among them, the recently classified base-phosphate (BPh) interactions belong to the most important ones. Twelve percent of nucleotides in the ribosomal crystal structures are involved in BPh interactions. BPh interactions are highly conserved and provide major constraints on RNA sequence evolution. Here we provide assessment of the energetics of BPh interactions using MP2 computations extrapolated to the complete basis set of atomic orbitals and corrected for higher-order electron correlation effects. The reference computations are compared with DFT-D and DFT-D3 approaches, the SAPT method, and the molecular mechanics force field. The computations, besides providing the basic benchmark for the BPh interactions, allow some refinements of the original classification, including identification of some potential doubly bonded BPh patterns. The reference computations are followed by analysis of some larger RNA fragments that consider the context of the BPh interactions. The computations demonstrate the complexity of interaction patterns utilizing the BPh interactions in real RNA structures. The BPh interactions are often involved in intricate interaction networks. We studied BPh interactions of protonated adenine that can contribute to catalysis of hairpin ribozyme, the key BPh interaction in the S-turn motif of the sarcin-ricin loop, which may predetermine the S-turn topology and complex BPh patterns from the glmS riboswitch. Finally, the structural stability of BPh interactions in explicit solvent molecular dynamics simulations is assessed. The simulations well preserve key BPh interactions and allow dissection of structurally/functionally important water-meditated BPh bridges, which could not be considered in earlier bioinformatics classification of BPh interactions.     

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.     

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.     

Screening of Minimalist Noncanonical Sites in Duplex DNA and RNA Reveals Context and Motif-Selective Binding by Fluorogenic Base Probes

We hypothesize that programmable hybridization to noncanonical nucleic acid motifs may be achieved by macromolecular display of binders to individual noncanonical pairs (NCPs). As each recognition element may individually have weak binding to an NCP, we developed a semi-rational approach to detect low affinity interactions between selected nitrogenous bases and noncanonical sites in duplex DNA and RNA. A set of fluorogenic probes was synthesized by coupling abiotic (triazines, pyrimidines) and native RNA bases to thiazole orange (TO) dye. This probe library was screened against duplex nucleic acid substrates bearing single abasic, single NCP, and tandem NCP sites. Probe engagement with NCP sites was reported by 100–1000× fluorescence enhancement over background. Binding is strongly context-dependent, reflective of both molecular recognition and stability: less stable motifs are more likely to bind a synthetic probe. Further, DNA and RNA substrates exhibit entirely different abasic and single NCP binding profiles. While probe binding in the abasic and single NCP screens was monotonous, much richer binding profiles were observed with the screen of tandem NCP sites in RNA, in part due to increased steric accessibility. In addition to known binding interactions between the triazine melamine (M) and T/U sites, the NCP screens identified new targeting elements for pyrimidine-rich motifs in single NCPs and 2×2 internal bulges. We anticipate that semi-rational approaches of this type will lead to programmable noncanonical hybridization strategies at the macromolecular level.     

Unusually short RNA sequences: design of a 13-mer RNA that selectively binds and recognizes theophylline

RNA plays critical roles in numerous biological processes and constitutes valuable therapeutic targets. RNA is significant not only for its roles in transmitting the genetic code but also for its enzymatic functions in ribozymes and in peptide bond formation in ribosomes. Recent studies have shown that RNAs containing as few as 22 nucleotides can be key elements in cellular functions. This suggests the possibility of using short RNAs as regulatory elements. Here, we show that ligand recognition and selectivity by RNA molecules can occur with only the presence of a binding pocket and as few as six additional scaffolding nucleotides holding the binding pocket in place. A 13-mer RNA truncation of a 33-mer aptamer for theophylline preserves the ability to bind to theophylline and to discriminate against the structurally similar compound caffeine. The truncated aptamer retains nearly all of the same structural elements in its binding site as those present in the original aptamer. This is the first demonstration of selective ligand binding by a 13-mer RNA.     

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.     

Deoxyribozymes with 2'-5' RNA ligase activity

In vitro selection was used to identify deoxyribozymes that ligate two RNA substrates. In the ligation reaction, a 2'-5' RNA phosphodiester linkage is created from a 2',3'-cyclic phosphate and a 5'-hydroxyl group. The new Mg(2+)-dependent deoxyribozymes provide 50-60% yield of ligated RNA in overnight incubations at pH 7.5 and 37 degrees C, and they afford 40-50% yield in 1 h at pH 9.0 and 37 degrees C. Various RNA substrate sequences may be joined by simple Watson-Crick covaration of the DNA binding arms that interact with the two RNA substrates. The current deoxyribozymes have some RNA substrate sequence requirements at the nucleotides immediately surrounding the ligation junction (either UAUA GGAA or UAUN GGAA, where the arrow denotes the ligation site and N equals any nucleotide). One of the new deoxyribozymes was used to prepare by ligation the Tetrahymena group I intron RNA P4-P6 domain, a representative structured RNA. Nondenaturing gel electrophoresis revealed that a 2'-5' linkage between nucleotides A233 and G234 of P4-P6 does not disrupt its Mg(2+)-dependent folding (DeltaDeltaG degrees ' < 0.2 kcal/mol). This demonstrates that a 2'-5' linkage does not necessarily interfere with structure in a folded RNA. Therefore, these non-native linkages may be acceptable in modified RNAs when structure/function relationships are investigated. Deoxyribozymes that ligate RNA should be particularly useful for preparing site-specifically modified RNAs for studies of RNA structure, folding, and catalysis.     

Synthesis, gene silencing, and molecular modeling studies of 4'-C-aminomethyl-2'-O-methyl modified small interfering RNAs

The linear syntheses of 4'-C-aminomethyl-2'-O-methyl uridine and cytidine nucleoside phosphoramidites were achieved using glucose as the starting material. The modified RNA building blocks were incorporated into small interfering RNAs (siRNAs) by employing solid phase RNA synthesis. Thermal melting studies showed that the modified siRNA duplexes exhibited slightly lower T(m) (～1 °C/modification) compared to the unmodified duplex. Molecular dynamics simulations revealed that the 4'-C-aminomethyl-2'-O-methyl modified nucleotides adopt South-type conformation in a siRNA duplex, thereby altering the stacking and hydrogen-bonding interactions. These modified siRNAs were also evaluated for their gene silencing efficiency in HeLa cells using a luciferase-based reporter assay. The results indicate that the modifications are well tolerated in various positions of the passenger strand and at the 3' end of the guide strand but are less tolerated in the seed region of the guide strand. The modified siRNAs exhibited prolonged stability in human serum compared to unmodified siRNA. This work has implications for the use of 4'-C-aminomethyl-2'-O-methyl modified nucleotides to overcome some of the challenges associated with the therapeutic utilities of siRNAs.     

Folding of aromatic oligoimides of trans-1,2-diaminocyclohexane

Chiral oligomeric diimides prepared from pyromellitic dianhydride, (R,R)-1,2-diaminocyclohexane and phthalic anhydride fold into M or P helical conformers; trimer 1 folds into the P conformer in the crystal but the M conformer dominates in solution; longer chain oligomers 2 and 3 form preferentially P conformers in solution, as a result of intermolecular interactions.