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International audience; RNA molecules play integral roles in gene regulation, and understanding their structures gives us important insights into their biological functions. Despite recent developments in template-based and parameterized energy functions, the structure of RNA--in particular the nonhelical regions--is still difficult to predict. Knowledge-based potentials have proven efficient in protein structure prediction. In this work, we describe two differentiable knowledge-based potentials derived from a curated data set of RNA structures, with all-atom or coarse-grained representation, respectively. We focus on one aspect of the prediction problem: the identification of native-like RNA conformations from a set of near-native models. Using a variety of near-native RNA models generated from three independent methods, we show that our potential is able to distinguish the native structure and identify native-like conformations, even at the coarse-grained level. The all-atom version of our knowledge-based potential performs better and appears to be more effective at discriminating near-native RNA conformations than one of the most highly regarded parameterized potential. The fully differentiable form of our potentials will additionally likely be useful for structure refinement and/or molecular dynamics simulations.

The folding energy landscape of RNA is greatly affected by interactions between the RNA and counterions that neutralize the backbone negative charges and may also participate in tertiary contacts. Valence, size, coordination number, and electron shell structure can all contribute to the energetic stabilization of specific RNA conformations. Using single-molecule fluorescence resonance energy transfer (smFRET), we have examined the folding properties of the RNA transcript of human mitochondrial tRNA(Lys), which possesses two different folded states in addition to the unfolded one under conditions of thermodynamic equilibrium. We have quantitatively analyzed the degree of RNA tertiary structure stabilization for different types of cations based on a thermodynamic model that accounts for multiple conformational states and RNA-ion interactions within each state. We have observed that small monovalent ions stabilize the tRNA tertiary structure more efficiently than larger ones. More ions were found in close vicinity of compact RNA structures, independent of the type of ion. The largest conformation-dependent binding specificity of ions of the same charge was found for divalent ions, for which the ionic radii and coordination properties were responsible for shaping the folding free energy.

RNA often folds hierarchically, so that its sequence defines its secondary structure (helical base-paired regions connected by single-stranded junctions), which subsequently defines its tertiary fold. To preserve base-pairing and chain connectivity, the three-dimensional conformations that RNA can explore are strongly confined compared to when secondary structure constraints are not enforced. Using three examples, we studied how secondary structure confines and dictates an RNA’s preferred conformations. We made use of Macromolecular Conformations by SYMbolic programming (MC-Sym) fragment assembly to generate RNA conformations constrained by secondary structure. Then, to understand the correlations between different helix placements and orientations, we robustly clustered all RNA conformations by employing unique methods to remove outliers and estimate the best number of conformational clusters. We observed that the preferred conformation (as judged by largest cluster size) for each type of RNA junction molecule tested is consistent with its biological function. Further, the improved quality of models in our pruned datasets facilitates subsequent discrimination using scoring functions based either on statistical analysis (knowledge based) or experimental data.

Riboswitches regulate gene expression via ligand binding to an aptamer domain which induces conformational changes in a regulatory expression platform. By unfolding and refolding single add adenine riboswitch molecules in an optical trap, an integrated picture of the folding was developed and related to the regulatory mechanism. Force-extension curves (FECs) and constant-force folding trajectories measured on the aptamer alone revealed multiple partially-folded states, including several misfolded states not on the native folding pathway. All states were correlated to key structural components and interactions within hierarchical folding pathways. FECs of the full-length riboswitch revealed that the thermodynamically stable conformation switches upon ligand binding from a structure repressing translation to one permitting it. Along with rapid equilibration of the two structures in the absence of adenine, these results support a thermodynamically-controlled regulatory mechanism, in contrast with the kinetic control of the closely-related pbuE adenine riboswitch. Comparison of the folding of these riboswitches revealed many similarities arising from shared structural features but also essential differences related to their different regulatory mechanisms. © 2011 The Author(s).

The annealing of nucleic acids to (partly) complementary RNA or DNA strands is involved in important cellular processes. A variety of proteins have been shown to accelerate RNA/RNA annealing but their mode of action is still mainly uncertain. In order to study the mechanism of protein-facilitated acceleration of annealing we selected a short peptide, HIV-1 Tat(44-61), which accelerates the reaction efficiently. The activity of the peptide is strongly regulated by mono- and divalent cations which hints at the importance of electrostatic interactions between RNA and peptide. Mutagenesis of the peptide illustrated the dominant role of positively charged amino acids in RNA annealing--both the overall charge of the molecule and a precise distribution of basic amino acids within the peptide are important. Additionally, we found that Tat(44-61) drives the RNA annealing reaction via entropic rather than enthalpic terms. One-dimensional-NMR data suggest that the peptide changes the population distribution of possible RNA structures to favor an annealing-prone RNA conformation, thereby increasing the fraction of colliding RNA molecules that successfully anneal.

In vitro transcribed thymidylate synthase (TS) mRNA which is 100% substituted with 5-fluorouracil (FUra) was analyzed for changes in mRNA secondary structure, for alterations in translational efficiency, and for evidence of translational miscoding in vitro. FUra substitution in TS mRNA results in an altered migration pattern in non-denaturing RNA gels and in decreased hyperchromicity in RNA melting temperature studies, consistent with a change in mRNA secondary structure. However, no change in the translational efficiency of FUra-substituted TS mRNA is seen compared to control TS mRNA in either rabbit reticulocyte lysate or wheat germ extract in vitro translation systems. Analysis of the in vitro translation product of FUra-substituted TS mRNA by Western immunoblotting, isoelectric focusing, 5-fluoro-2‘-deoxyuridine 5‘-monophosphate binding, and TS catalytic activity experiments shows no difference compared to control TS mRNA. We conclude that the in vitro translation products of FUra-substituted and control TS mRNA are identical. Our findings do not support the hypothesis that changes in the mRNA template are responsible for the RNA-directed cytotoxicity of FUra.

A trans-acting hammerhead ribozyme (D0) was designed to cleave at the first GUC decreases X target sequence in Alzheimer amyloid precursor (beta APP) mRNA. A synthetic 26-base substrate analog of beta APP mRNA, labeled with either [alpha-32P]CTP or [alpha-35S]CTP underwent Mg(+2)-dependent, site-specific cleavage in vitro. Cleavage was found to occur at two sites. The major product of the reaction, a 13-base oligonucleotide, resulted from cleavage at the consensus cut site of hammerhead ribozymes. A minor 12-base product was formed by cleavage one base 5' to this site. When the beta APP substrate analog was labeled with [alpha-35S]ATP (Rp isomer) another cleavage site, amounting to 25-30% of the total product formed, was also activated. A series of ribozyme deletion mutants delimited the new cleavage site to the phosphate linkage 5' to the G residue of the hammerhead consensus cut site, GUC decreases X. We conclude that small changes in RNA conformation can dramatically alter ribozyme specificity in vitro. This may have implications for targeting hammerhead ribozymes in vivo.

The limited number of RNA structures determined by X-ray crystallography and NMR spectroscopy compels the use of experimental and theoretical methods that are less precise to obtain information on RNA conformation. RNA flexibility, a consequence of rotational freedom about seven intra- and internucleotide bonds, is unfortunately of such magnitude that these alternate techniques fall short of providing sufficient information to build robust tertiary structures. Various RNA modeling methods, described herein, permit the organization of this structural data to the form of three-dimensional structures. Interactive computer graphics techniques, for example, have generated several useful models. Also, conventional computer algorithms involving the minimization of empirical energy functions, previously limited to small molecules, are giving way to methods able to handle much larger molecules. Modified distance geometry and molecular mechanics algorithms, using simplified "pseudoatom" representations, can generate structures consistent with input data. A constraint satisfaction algorithm combined with discrete representations of nucleotide conformations systematically explores poorly defined regions of a molecule yielding all-atom representations, but requires enough structural constraints to avoid a computational explosion.

A series of coarse-grained models have been developed for study of the molecular dynamics of RNA nanostructures. The models in the series have one to three beads per nucleotide and include different amounts of detailed structural information. Such a treatment allows us to reach, for systems of thousands of nucleotides, a time scale of microseconds (i.e. by three orders of magnitude longer than in full atomistic modeling) and thus to enable simulations of large RNA polymers in the context of bionanotechnology. We find that the three-beads-per-nucleotide models, described by a set of just a few universal parameters, are able to describe different RNA conformations and are comparable in structural precision to the models where detailed values of the backbone P-C4′ dihedrals taken from a reference structure are included. These findings are discussed in the context of RNA conformation classes. © 2010 IOP Publishing Ltd.

We have used single-molecule fluorescence microscopy to study the folded state of human telomerase RNA (hTR). Here we show that hTR adopts a new conformation on binding to human telomerase reverse transcriptase (hTERT) and reconstitution of an active ribonucleoprotein complex. Our data are consistent with the formation of an RNA pseudoknot in active human telomerase.

Genome-wide association studies (GWAS) often identify disease-associated mutations in intergenic and non-coding regions of the genome. Given the high percentage of the human genome that is transcribed, we postulate that for some observed associations the disease phenotype is caused by a structural rearrangement in a regulatory region of the RNA transcript. To identify such mutations, we have performed a genome-wide analysis of all known disease-associated Single Nucleotide Polymorphisms (SNPs) from the Human Gene Mutation Database (HGMD) that map to the untranslated regions (UTRs) of a gene. Rather than using minimum free energy approaches (e.g. mFold), we use a partition function calculation that takes into consideration the ensemble of possible RNA conformations for a given sequence. We identified in the human genome disease-associated SNPs that significantly alter the global conformation of the UTR to which they map. For six disease-states (Hyperferritinemia Cataract Syndrome, beta-Thalassemia, Cartilage-Hair Hypoplasia, Retinoblastoma, Chronic Obstructive Pulmonary Disease (COPD), and Hypertension), we identified multiple SNPs in UTRs that alter the mRNA structural ensemble of the associated genes. Using a Boltzmann sampling procedure for sub-optimal RNA structures, we are able to characterize and visualize the nature of the conformational changes induced by the disease-associated mutations in the structural ensemble. We observe in several cases (specifically the 5' UTRs of FTL and RB1) SNP-induced conformational changes analogous to those observed in bacterial regulatory Riboswitches when specific ligands bind. We propose that the UTR and SNP combinations we identify constitute a "RiboSNitch," that is a regulatory RNA in which a specific SNP has a structural consequence that results in a disease phenotype. Our SNPfold algorithm can help identify RiboSNitches by leveraging GWAS data and an analysis of the mRNA structural ensemble.

Pulsed electron double resonance (PELDOR) spectroscopy reveals a prearranged tertiary structure of the 27 nucleotides long engineered neomycin-responsive riboswitch. Measured distances between spin labels at positions U4-U14, U4-U15, U14-U26, and U15-U26 were unchanged upon neomycin binding which implies that the global stem-loop architecture is preserved in the absence and presence of the ligand. On the basis of our results, we infer that low-temperature PELDOR data unambiguously demonstrate the existence of an enthalpically favorable set of RNA conformations ready to bind the ligand without major global rearrangement.

Certain high copy-number mutants of the ColE1 plasmid produce a primer RNA that, unlike the wild-type, is resistant to inhibition by the plasmid-encoded replication inhibitor RNA I. We show that this resistance is associated with the ability of mutant primer RNA to hybridize to the DNA template strand more efficiently than does the wild-type transcript in vitro. We have isolated two second-site intramolecular suppressor mutations that partially restore wild-type copy number behavior to the high copy-number mutant in vivo. Each of these mutations alters a second base in primer RNA near the original mutation. We show that the primer RNA made by the pseudo-revertants regained wild-type-like sensitivity to RNA I in vitro. Also, the efficiency of RNA-DNA hybrid formation by the pseudo-revertant primer RNAs is restored to a level similar to that of wild-type primer. Using non-denaturing gel electrophoresis as an indication of RNA conformation, we identified two primer RNA conformers, each of 550 nucleotides, whose equilibrium distribution differs between wild-type and the mutant plasmid. The pseudo-revertant plasmids have a conformer distribution similar to that of wild-type, indicating that these primer sequence changes have long-range effects on primer conformation. An oligonucleotide complementary to the primer domain containing the mutation reduced hybrid formation when present during primer elongation. These results indicate that the copy-number behavior of these plasmids is a consequence of conformational alterations in primer RNA that alter its hybridization efficiency with the DNA template strand and its sensitivity to inhibition by RNA I.

The coexistence of multiple codes in messenger RNA molecules is investigated. A unique biological example of multiple coding is revealed by the rev responsive element (RRE) of lentiviruses. This element overlaps with the env gene and forms a specific, multibranched RNA conformation. An analysis of the similarity in the nucleotide sequences and the similarity in the secondary structures, as well as their relationship, is described. The data suggest a novel evolutionary pathway for the RRE structures: the shifting of base pairings in the secondary structure while preserving both a similar overall RNA conformation and, apparently, the activity of the protein encoded by the overlapping gene. This means that corresponding parts of the consensus secondary structure in different RRE species are formed by non-homologous nucleotides. This pattern has so far not been observed in comparative structural studies of other sets of functionally equivalent RNAs (like tRNAs and rRNAs). The multiple-coding problem is discussed with respect to the structure and function of the RRE and the high rate of viral evolution.

RNA molecules were found to separate into numerous metastable conformational forms upon non-denaturing gel electrophoresis. The equilibration of the conformations was accelerated by heating or mild denaturing conditions. Single-base substitutions in the sequence of the RNAs caused changes in the conformational patterns, including mobility shifts of major and minor conformations, appearance of new conformations and loss of other conformations. This sequence-dependent RNA conformational polymorphism was used to detect point mutations in p53 and, dihydrofolate reductase genes. Sense and anti-sense RNA strands corresponding to the same segment of the p53 gene gave entirely different conformational patterns. To generate the RNA, short regions of the target genes (up to about 250 bp) were amplified by the polymerase chain reaction and the resulting DNA segments transcribed to RNA by T7 RNA polymerase. The method is rapid, simple, amenable to non-radioactive visualization and was successful in several cases when DNA single-strand conformational polymorphism analysis (Orita et al. (1989) Genomics 5, 874-879) failed to detect the point mutation.

A comparative analysis of TAR RNA structures in human and simian immunodeficiency viruses reveals the conservation of certain structural features despite the divergence in sequence. Both the TAR elements of HIV-1 and SIV-chimpanzee can be folded into relatively simple one-stem hairpin structures. Chemical and RNAase probes were used to analyze the more complex structure of HIV-2 TAR RNA, which folds into a branched hairpin structure. A surprisingly similar RNA conformation can be proposed for SIV-mandrill, despite considerable divergence in nucleotide sequence. A third structural presentation of TAR sequences is seen for SIV-african green monkey. These results are generally consistent with the classification of HIV-SIV viruses in four subgroups based on sequence analyses (both nucleotide- and amino acid-sequences). However, some conserved TAR structures were detected for members of different virus subgroups. It is therefore proposed that RNA structure analysis might provide an additional tool for determining phylogenetic relationships among the HIV-SIV viruses.

Three nanobiocomposites (PRAg31, PRAg11, and PRAg13; the numbers indicate the weight ratios of poly(o-methoxyaniline) (POMA) and ribonucleic acid (RNA), respectively), produced from the same amount of POMA (P) and silver nitrate (AgNO(3)) with differing proportions of RNA (R) are prepared by aging the aqueous solutions of the mixture for 3 weeks at 30 degrees C. The scanning and transmission electron microscopy (SEM and TEM) indicate Ag nanoparticle formation on the hybrid fiber surface and in the PRAg31 system the hybrid fibrils become coated with metallic Ag, the phenomenon being termed as "nanojacketting". The circular dichroism (CD) spectra indicate a small distortion of RNA conformation from A helix toward B helix. FTIR and UV-vis spectra suggest that POMA (emeraldine base, EB) being doped by Ag(+) become oxidized to its pernigraniline base (PB) form and reduce Ag(+) to metallic Ag. The Ag nanoparticles thus produced become stabilized on the fibril surface by co-ordination through nitrogen atoms of POMA (PB) chains. The much slower red shift of pi band to polaron band transition peak in PRAg31 than that of the other two nanobiocomposites is indicative of difficulty in conformational transitions of POMA chain in the "nanojacketted" hybrid fibrils. The dc-conductivity values of the nanobiocomposites are two orders higher than that of the pure POMA-RNA hybrids. The PRAg31 system exhibits rectification property in the I-V characteristic curves and a probable explanation based on the feasibility of p-n junction formation arising from the transfer of lone pair of electrons of nitrogen of POMA (p-type) to the Ag nanoparticles (n-type) has been offered.

Few antimicrobial drugs function by directly targeting RNA. A small molecule that binds the hepatitis C viral genome by 'locking' in a particular RNA conformation to inhibit viral protein production suggests a new paradigm for drug design.

Ribosomal proteins stabilize the folded structure of the ribosomal RNA and enable the recruitment of further proteins to the complex. Quantitative hydroxyl radical footprinting was used to measure the extent to which three different primary assembly proteins, S4, S17, and S20, stabilize the three-dimensional structure of the Escherichia coli 16S 5' domain. The stability of the complexes was perturbed by varying the concentration of MgCl(2). Each protein influences the stability of the ribosomal RNA tertiary interactions beyond its immediate binding site. S4 and S17 stabilize the entire 5' domain, while S20 has a more local effect. Multistage folding of individual helices within the 5' domain shows that each protein stabilizes a different ensemble of structural intermediates that include nonnative interactions at low Mg(2+) concentration. We propose that the combined interactions of S4, S17, and S20 with different helical junctions bias the free-energy landscape toward a few RNA conformations that are competent to add the secondary assembly protein S16 in the next step of assembly.

A broad structural landscape often needs to be characterized in order to fully understand how regulatory RNAs perform their biological functions at the atomic level. We present a protocol for visualizing thermally accessible RNA conformations at atomic-resolution and with timescales extending up to milliseconds. The protocol combines molecular dynamics (MD) simulations with experimental residual dipolar couplings (RDCs) measured in partially aligned (13)C/(15)N isotopically enriched elongated RNA samples. The structural ensembles generated in this manner provide insights into RNA dynamics and its role in functionally important transitions.

Telomerase, a ribonucleoprotein (RNP) expressed in all highly proliferating cells, is comprised of telomerase RNA, telomerase reverse transcriptase (TERT) and other protein cofactors. The complex compensates for the chromosomal shortening that arises with each round of DNA replication. Since mutations altering telomerase expression, assembly and regulation cause multiple human diseases, it is of utmost importance to understand telomerase at the structural level. However, while the structure of TERT in the absence of RNA has been recently published, the full RNP structure remains unknown.Using a combination of single-molecule FRET and ensemble footprinting, we are probing the functional structure of telomerase RNP. In particular, we have determined the active conformation adopted by two conserved regions of telomerase RNA - the stemloop IV and the pseudoknot - upon assembly with TERT and other protein cofactors.The pseudoknot, unformed in naked telomerase RNA, was folded in active RNPs. Proper pseudoknot folding was required for catalysis, as demonstrated by mutations that abolished telomerase activity when formation of either pseudoknot stem was disallowed. Conversely, compensatory mutations that reinstated basepairing in the pseudoknot region also restored telomerase activity.Binding of TERT, in addition to allowing pseudoknot formation, brought loop IV into the proximity of the pseudoknot without affecting its conformation. Mutations in the loop IV region that abolished TERT binding and telomerase activity resulted in an extended RNA conformation with a substantially larger loop IV-pseudoknot distance, as well as protection of the pseudoknot by RNP proteins. Interestingly, the telomerase holoenzyme protein p65 could compensate for these effects of loop IV mutations, restoring the loop IV-TERT interaction, the folded conformation of stemloop IV and telomerase activity.These results suggest that proper conformations of the pseudoknot and stemloop IV of telomerase RNA are critical for enzyme activity.

Riboswitches are functional RNA molecules that control gene expression through conformational changes in response to small-molecule ligand binding. In addition, riboswitch 3D structure, like that of other RNA molecules, is dependent on cation-RNA interactions as the RNA backbone is highly negatively charged. Here, we show how small-angle X-ray scattering (SAXS) can be used to probe RNA conformations as a function of ligand and ion concentration. In a recent study of a glycine-binding tandem aptamer from Vibrio cholerae, we have used SAXS data and thermodynamic modeling to investigate how Mg(2+)-dependent folding and glycine binding are energetically coupled. In addition, we have employed ab initio shape reconstruction algorithms to obtain low-resolution models of the riboswitch structure from SAXS data under different solution conditions.

The antibiotic thiostrepton, a thiazole-containing peptide, inhibits translation and ribosomal GTPase activity by binding directly to a limited and highly conserved region of the large subunit ribosomal RNA termed the GTPase center. We have previously used a filter binding assay to examine the binding of ribosomal protein L11 to a set of ribosomal RNA fragments encompassing the Escherichia coli GTPase center sequence. We show here that thiostrepton binding to the same RNA fragments can also be detected in a filter binding assay. Binding is relatively independent of monovalent salt concentration and temperature but requires a minimum Mg2+ concentration of about 0.5 mM. To help determine the RNA features recognized by L11 and thiostrepton, a set of over 40 RNA sequence variants was prepared which, taken together, change every nucleotide within the 1051 to 1108 recognition domain while preserving the known secondary structure of the RNA. Binding constants for L11 and thiostrepton interaction with these RNAs were measured. Only a small number of sequence variants had more than fivefold effects on L11 binding affinities, and most of these were clustered around a junction of helical segments. These same mutants had similar effects on thiostrepton binding, but more than half of the other sequence changes substantially reduced thiostrepton binding. On the basis of these data and chemical modification studies of this RNA domain in the literature, we propose that L11 makes few, if any, contacts with RNA bases, but recognizes the three-dimensional conformation of the RNA backbone. We also argue from the data that thiostrepton is probably sensitive to small changes in RNA conformation. The results are discussed in terms of a model in which conformational flexibility of the GTPase center RNA is functionally important during the ribosome elongation cycle.

DNA/RNA chromatography presents a versatile platform for the analysis of nucleic acids. Although the mechanism of separation of double stranded (ds) DNA fragments is largely understood, the mechanism by which RNA is separated appears more complicated. To further understand the separation mechanisms of RNA using ion pair reverse phase liquid chromatography, we have analysed a number of dsRNA and single stranded (ss) RNA fragments. The high-resolution separation of dsRNA was observed, in a similar manner to dsDNA under non-denaturing conditions. Moreover, the high-resolution separation of ssRNA was observed at high temperatures (75 degrees C) in contrast to ssDNA. It is proposed that the presence of duplex regions/secondary structures within the RNA remain at such temperatures, resulting in high-resolution RNA separations. The retention time of the nucleic acids reflects the relative hydrophobicity, through contributions of the nucleic sequence and the degree of secondary structure present. In addition, the analysis of RNA using such approaches was extended to enable the discrimination of bacterial 16S rRNA fragments and as an aid to conformational analysis of RNA. RNA:RNA interactions of the human telomerase RNA component (hTR) were analysed in conjunction with the incorporation of Mg2+ during chromatography. This novel chromatographic procedure permits analysis of the temperature dependent formation of dimeric RNA species.

Learning how native RNA conformations can be stabilized relative to unfolded states is an important objective, for both understanding natural RNAs and improving the design of artificial functional RNAs. Here we show that covalently attached double-stranded DNA constraints (ca. 14 base pairs in length) can significantly stabilize the native conformation of an RNA molecule. Using the P4-P6 domain of the Tetrahymena group I intron as the test system, we identified pairs of RNA sites where attaching a DNA duplex is predicted to be structurally compatible with only the folded state of the RNA. The DNA-constrained RNAs were synthesized and shown by nondenaturing polyacrylamide gel electrophoresis (native PAGE) to have substantial decreases in their Mg2+ midpoints ([Mg2+]1/2 values). These changes are equivalent to free energy stabilizations as large as DeltaDeltaGdegrees = -2.5 kcal/mol, which is approximately 14% of the total tertiary folding energy. For comparison, the sole modification of P4-P6 previously reported to stabilize this RNA is a single-nucleotide deletion (DeltaC209) that provides only 1.1 kcal/mol of stabilization. Our findings indicate that nature has not completely optimized P4-P6 RNA folding. Furthermore, the DNA constraints are designed not to interact directly and extensively with the RNA, but rather more indirectly to modulate the relative stabilities of folded and unfolded RNA states. The successful implementation of this strategy to further stabilize a natively folded RNA conformation suggests an important element of modularity in stabilization of RNA structure, with implications for how nature might use other molecules such as proteins to stabilize specific RNA conformations.

We report the use of light for two distinct kinds of investigation of the conformational changes of, respectively, DNA and RNA. First, 'Deoxyribosensors' are a class of DNA constructs that incorporate aptamers, and which report the binding of a ligand to the aptamer by attenuating the conformational/stacking relationship of two constituent DNA double helices within the sensor. Such attenuations can be monitored as electrical outputs via a light irradiation protocol. Second, RNA aptamers were selected for the specific binding of one but not the other isomer of different photochromic switch compounds. One such RNA aptamer, specific for binding the 'closed' isomer of a dihydropyrene compound, was used to create a highly effective, light-sensitive, RNA-cleaving ribozyme. Such ribozymes and riboswitches should find broad utility for the light-mediated control of gene expression within living cells and organisms.

Summary: Three-dimensional RNA structure prediction and folding is of significant interest in the biological research community. Here, we present iFoldRNA, a novel web-based methodology for RNA structure prediction with near atomic resolution accuracy and analysis of RNA folding thermodynamics. iFoldRNA rapidly explores RNA conformations using discrete molecular dynamics simulations of input RNA sequences. Starting from simplified linear-chain conformations, RNA molecules ( Availability: http://iFoldRNA.dokhlab.org. Contact: dokh@med.unc.edu Supplementary information: Supplementary data are available at Bioinformatics online.

RNA conformation is both highly dependent on and sensitive to the presence of charged ligands. Mono- and divalent ions stabilize the native fold of RNA, whereas other polyvalent cationic ligands can act to both stabilize or disrupt native RNA structure. In this work, we analyze the effects of two ligands, Mg (2+) and tobramycin, on the folding of S. cerevisiae tRNA (Asp) transcripts using single nucleotide resolution SHAPE chemistry. Surprisingly, reducing the Mg (2+) concentration favors a structural rearrangement in which the D- and variable loops pair. The tobramycin polycation binds to loops in tRNA (Asp) and induces RNA unfolding in two distinct transitions: the loss of tertiary interactions between the T- and D-loops followed by complete unfolding of the D-stem. Although Mg (2+) and tobramycin are relatively simple ligands, both modulate tRNA (Asp) folding in unanticipatedly complex ways, neither of which is consistent with simple hierarchical folding or unfolding of this RNA. Monitoring the structural consequences of ligand binding to RNA at single nucleotide resolution makes it possible to define intermediate structures that contribute to the complex energy landscapes often observed for RNA folding processes and lays the groundwork for a significantly improved understanding of the interactions between RNA and its solution environment.

A primary cause of failure of the highly active antiretroviral therapy (HAART) used to control the infection of human immunodeficiency virus type 1 (HIV-1) is the rapid emergence of strains that are resistant to one or more of the active agents used in typical multidrug regimens. Mutations of genes coding for inhibitor targets, such as protease, reverse transcriptase, and gp41, result in a reduced response to treatment from patients infected by resistant strains. The magnitude reached by the AIDS pandemic and the daunting problem of drug resistance elevate the need for new therapeutic agents with alternative mechanisms of action. In the life cycle of HIV-1, the processes of genome recognition, dimerization, and packaging provide very attractive opportunities for the development of novel anti-retroviral strategies. These essential functions are mediated by the interactions between the nucleocapsid (NC) domain of the Gag polyprotein and a highly conserved region of genomic RNA, known as the packaging signal, or Psi-RNA. Located in the 5'-untranslated leader (5'-UTR), the ~120 nucleotides comprising Psi-RNA have been shown to fold into relatively stable secondary structures identified as stem-loop 1 through 4 (SL1-4), which serve as possible binding sites for NC during viral replication. The characteristics and structural determinants of these protein-RNA interactions have been extensively investigated to elucidate the role played by the different elements and enable the identification of viable targets. Different strategies have been proposed to disrupt the mechanism of genome recognition, dimerization, and packaging by targeting key structures involved in these processes. We have employed an approach based on electrospray ionization (ESI) and Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) to investigate ligand-RNA interactions and evaluate the stability of NC-hairpin complexes in the presence of ligands. Furthermore, we have explored the possibility of employing tandem mass spectrometry to map the actual binding sites of small molecule ligands on individual stemloop domains of Psi-RNA. This approach was also extended to determine the effects of these ligands on the chaperone activity presented by NC. Finally, we have taken advantage of the high mass limits and resolution of ESI-FTICRMS to study the binding interactions of a peptide library with the complete Psi-RNA. These biophysical studies have provided an ideal platform to screen possible therapeutics, as well as identifying new viral targets, which could lead to the development of antiretroviral agents that are capable of interfering with these key steps in the viral life cycle.

RNA thermometers are thermosensors that regulate gene expression by temperature-induced changes in RNA conformation. Naturally occurring RNA thermometers exhibit complex secondary structures which are believed to undergo a series of gradual structural changes in response to temperature shifts. Here, we report the de novo design of considerably simpler RNA thermometers that provide useful RNA-only tools to regulate bacterial gene expression by a shift in the growth temperature. We show that a single small stem-loop structure containing the ribosome binding site is sufficient to construct synthetic RNA thermometers that work efficiently at physiological temperatures. Our data suggest that the thermometers function by a simple melting mechanism and thus provide minimum size on/off switches to experimentally induce or repress gene expression by temperature.

A synthetic gene of yeast aspartic acid tRNA with a promoter for phage T7 RNA polymerase was cloned in Escherichia coli. The in vitro transcribed tRNA(Asp) molecules are deprived of modified nucleotides and retain their aspartylation capacity. The solution conformation of these molecules was mapped with chemical structural probes and compared to that of fully modified molecules. Significant differences in reactivities were observed in Pb2+ cleavage of the RNAs and in modification of the bases with dimethyl sulphate. The most striking result concerns C56, which becomes reactive in unmodified tRNA(Asp), indicating the disruption of the C56-G19 base pair involved in the D- and T-loop interaction. The chemical data indicate that unmodified tRNA(Asp) transcripts possess a relaxed conformation compared to that of the native tRNA. This conclusion is confirmed by thermal melting experiments. Thus it can be proposed that post-transcriptional modifications of nucleotides in tRNA stabilize the biologically active conformations in these molecules.

Nanospray-FT-ICR has been employed to investigate the processes of genome dimerization, selection, and packaging in human immunodifficiency virus type 1, which are mediated by specific interactions between the nucleocapsid protein (NC) and the structural elements formed by the genome's packaging signal [ψ-ribonucleic acid (RNA)]. This analytical platform allowed for the unambiguous characterization of all the non-covalent complexes formed in vitro by simultaneous RNA–RNA and protein–RNA binding equilibria. Competitive binding experiments involving the isolated RNA elements were completed to evaluate their ability to sustain specific protein interactions. In similar fashion, ad hoc RNA mutants were used to locate two distinct binding sites on the apical loop and stem-bulge of the monomeric stemloop 1 (SL1) domain, which is responsible for initiating the dimerization process. The stem-bulge motifs provided viable binding sites in both the kissing-loop (KL) and the extended duplex forms of dimeric SL1, whereas the latter included additional sites corresponding to the A-bulge motifs that flank the annealed palindromes. A cross-linking approach using pre-derivatized, photo-cross-linkable NC demonstrated that the SL3 domain was the preferred site for protein binding in the context of full-length ψ-RNA. This concerted strategy is expected to provide new valuable insight into the effects induced by the global folding of ψ-RNA on its ability to interact with the NC protein during genome dimerization, selection and packaging.

We present a protocol for determining the relative orientation and dynamics of A-form helices in 13C/15N isotopically enriched RNA samples using NMR residual dipolar couplings (RDCs). Non-terminal Watson–Crick base pairs in helical stems are experimentally identified using NOE and trans-hydrogen bond connectivity and modeled using the idealized A-form helix geometry. RDCs measured in the partially aligned RNA are used to compute order tensors describing average alignment of each helix relative to the applied magnetic field. The order tensors are translated into Euler angles defining the average relative orientation of helices and order parameters describing the amplitude and asymmetry of interhelix motions. The protocol does not require complete resonance assignments and therefore can be implemented rapidly to RNAs much larger than those for which complete high-resolution NMR structure determination is feasible. The protocol is particularly valuable for exploring adaptive changes in RNA conformation that occur in response to biologically relevant signals. Following resonance assignments, the procedure is expected to take no more than 2 weeks of acquisition and data analysis time.

A key step in the rational design of new RNA binding small molecules necessitates a complete elucidation of the molecular aspects of the binding of existing molecules to RNA structures. This work focuses towards the understanding of the interaction of a DNA intercalator, quinacrine and a minor groove binder 4',6-diamidino-2-phenylindole (DAPI) with the right handed Watson-Crick base paired A-form and the left-handed Hoogsteen base paired H(L)-form of poly(rC).poly(rG) evaluated by multifaceted spectroscopic and viscometric techniques. The energetics of their interaction has also been elucidated by isothermal titration calorimetry. Results of this study converge to suggest that (i) quinacrine intercalates to both A-form and H(L)-form of poly(rC).poly(rG); (ii) DAPI shows both intercalative and groove-binding modes to the A-form of the RNA but binds by intercalative mode to the H(L)-form. Isothermal calorimetric patterns of quinacrine binding to both the forms of RNA and of DAPI binding to the H(L)-form are indicative of single binding while the binding of DAPI to the A-form reveals two kinds of binding. The binding of both the drugs to both conformations of RNA is exothermic; while the binding of quinacrine to both conformations and DAPI to the A-form (first site) is entropy driven, the binding of DAPI to the second site of A-form and H(L)-conformation is enthalpy driven. Temperature dependence of the binding enthalpy revealed that the RNA-ligand interaction reactions are accompanied by small heat capacity changes that are nonetheless significant. We conclude that the binding affinity characteristics and energetics of interaction of these DNA binding molecules to the RNA conformations are significantly different and may serve as data for the development of effective structure selective RNA-based antiviral drugs.

Replication of viroids, small non-protein-coding plant pathogenic RNAs, entails reiterative transcription of their incoming single-stranded circular genomes, to which the (þ) polarity is arbitrarily assigned, cleavage of the oligomeric strands of one or both polarities to unit-length, and ligation to circular RNAs. While cleavage in chloroplastic viroids (family Avsunviroidae) is mediated by hammerhead ribozymes, where and how cleavage of oligomeric (þ) RNAs of nuclear viroids (family Pospiviroidae) occurs in vivo remains controversial. Previous in vitro data indicated that a hairpin capped by a GAAA tetraloop is the RNA motif directing cleavage and a loop E motif ligation. Here we have reexamined this question in vivo, taking advantage of earlier findings showing that dimeric viroid (þ) RNAs of the family Pospiviroidae transgenically expressed in Arabidopsis thaliana are processed correctly. Using this methodology, we have mapped the processing site of three members of this family at equivalent positions of the hairpin I/doublestranded structure that the upper strand and flanking nucleotides of the central conserved region (CCR) can form. More specifically, from the effects of 16 mutations on Citrus exocortis viroid expressed transgenically in A. thaliana, we conclude that the substrate for in vivo cleavage is the conserved double-stranded structure, with hairpin I potentially facilitating the adoption of this structure, whereas ligation is determined by loop E and flanking nucleotides of the two CCR strands. These results have deep implications on the underlying mechanism of both processing reactions, which are most likely catalyzed by enzymes different from those generally assumed: cleavage by a member of the RNase III family, and ligation by an RNA ligase distinct from the only one characterized so far in plants, thus predicting the existence of at least a second plant RNA ligase., Work in JAD and RF laboratories was supported by grants BMC2002–03694 (MCyT), BFU2005–06808/BMC and AGL2004–06311-C02–01 (MEC), and ACOMP07/268 (GV). MEG received predoctoral fellowships from MCyT and Fundacio´n Bancaja.

The specific binding of HIV-1 nucleocapsid (NC) to the hinge region of the kissing-loop (KL) dimer formed by stemloop 1 (SL1) can have significant consequences on its ability to isomerize into the corresponding extended duplex (ED) form. The binding determinants and the effects on the isomerization process were investigated in vitro by a concerted strategy involving ad hoc RNA mutants and electrospray ionization-Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry, which enabled us to characterize the stoichiometry and conformational state of all possible protein-RNA and RNA-RNA assemblies present simultaneously in solution. For the first time, NC-hinge interactions were observed in constructs including at least one unpaired guanine at the 5'-end of the loop-loop duplex, whereas no interactions were detected when the unpaired guanine was placed at its 3'-end. This binding mode is supported by the presence of a grip-like motif described by recent crystal structures, which is formed by the 5'-purines of both hairpins held together by mutual stacking interactions. Using tandem mass spectrometry, hinge interactions were clearly shown to reduce the efficiency of KL/ED isomerization without inducing its complete block. This outcome is consistent with the partial stabilization of the extra-helical grip by the bound protein, which can hamper the purine components from parting ways and initiate the strand exchange process. These findings confirm that the broad binding and chaperone activities of NC induce unique effects that are clearly dependent on the structural context of the cognate nucleic acid substrate. For this reason, the presence of multiple binding sites on the different forms assumed by SL1 can produce seemingly contrasting effects that contribute to a fine modulation of the two-step process of RNA dimerization and isomerization.

The long non-coding RNA (lncRNA) Xist is a master regulator of X-chromosome inactivation in mammalian cells. Models for how Xist and other lncRNAs function depend on thermodynamically stable secondary and higher-order structures that RNAs can form in the context of a cell. Probing accessible RNA bases can provide data to build models of RNA conformation that provide insight into RNA function, molecular evolution, and modularity. To study the structure of Xist in cells, we built upon recent advances in RNA secondary structure mapping and modeling to develop Targeted Structure-Seq, which combines chemical probing of RNA structure in cells with target-specific massively parallel sequencing. By enriching for signals from the RNA of interest, Targeted Structure-Seq achieves high coverage of the target RNA with relatively few sequencing reads, thus providing a targeted and scalable approach to analyze RNA conformation in cells. We use this approach to probe the full-length Xist lncRNA to develop new models for functional elements within Xist, including the repeat A element in the 5’-end of Xist. This analysis also identified new structural elements in Xist that are evolutionarily conserved, including a new element proximal to the C repeats that is important for Xist function., Author Summary To do their jobs, many RNAs need to fold into structures (through base-paring). We were interested in the conformation of a specific mammalian RNA, Xist, when it is inside a cell. Xist is a very large non-coding RNA (lncRNA), that is >17,000 nt long. Xist is particularly important because it is one of the first lncRNAs to be discovered, and turns genes off across an entire chromosome. To figure out how Xist RNA is folded in mouse cells, we developed a new approach, Targeted Structure-Seq, to examine the conformation of large RNAs like Xist. Using computer modeling, we identified parts of Xist that are base paired into RNA duplexes. We also determined which parts of the Xist RNA are likely to be structured. This work provides a new tool for studying the secondary structure of any large RNA, and helps us understand what the important pieces of Xist look like while Xist does its work in the cell.

Splicing factors belonging to the DEAD-box superfamily can affect RNA conformation in vitro. These proteins may control the dynamic changes in RNA base-pairing interactions during spliceosome assembly.

RNAs in biological processes often interconvert between defined structures. These RNA structure conversions are assisted by proteins and are frequently coupled to ATP hydrolysis. It is not well understood how proteins coordinate RNA structure conversions and which role ATP hydrolysis has in these processes. Here, we have investigated in vitro how the DEAD-box ATPase Ded1 facilitates RNA structure conversions in a simple model system. We find that Ded1 assists RNA structure conversions via two distinct pathways. One pathway requires ATP hydrolysis and involves the complete disassembly of the RNA strands. This pathway represents a kinetically controlled steady state between the RNA structures, which allows formation of less stable from more stable RNA conformations and thus RNA structure conversion against thermodynamic equilibrium values. The other pathway is ATP-independent and proceeds via multipartite intermediates that are stabilized by Ded1. Our results provide a basic mechanistic framework for protein-assisted RNA structure conversions that illuminates the role of ATP hydrolysis and reveal an unexpected diversity of pathways.

Local conformation is an important determinant of RNA catalysis and binding. The analysis of RNA conformation is particularly difficult due to the large number of degrees of freedom (torsion angles) per residue. Proteins, by comparison, have many fewer degrees of freedom per residue. In this work, we use and extend classical tools from statistics and signal processing to search for clusters in RNA conformational space. Results are reported both for scalar analysis, where each torsion angle is separately studied, and for vectorial analysis, where several angles are simultaneously clustered. Adapting techniques from vector quantization and clustering to the RNA structure, we find torsion angle clusters and RNA conformational motifs. We validate the technique using well-known conformational motifs, showing that the simultaneous study of the total torsion angle space leads to results consistent with known motifs reported in the literature and also to the finding of new ones.

Determining RNA secondary structure computationally, rather than manually, has the advantage of being cheaper and quicker. This paper introduces a new set-based particle swarm optimization algorithm to optimize the structure of an RNA molecule, using an advanced thermodynamic model. Results show that it is possible to use this SetPSO algorithm to optimise RNA secondary structure and produce candidate RNA conformations.

Results from optical melting studies of Watson-Crick complementary heteroduplexes formed between 2'-O-methyl RNA and RNA oligonucleotides are used to determine nearest neighbor thermodynamic parameters for predicting the stabilities of such duplexes. The results are consistent with the physical model assumed by the individual nearest neighbor-hydrogen bonding model, which contains terms for helix initiation, base pair stacking and base pair composition. The sequence dependence is similar to that for Watson-Crick complementary RNA/RNA duplexes, which suggests that the sequence dependence may also be similar to that for other backbones that favor A-form RNA conformations.

Using a domain elongation strategy, we decoupled internal motions in RNA from overall rotational diffusion. This allowed us to site-specifically resolve a manifold of motional modes in two regulatory RNAs from HIV-1 with the use of nuclear magnetic resonance spin relaxation methods. Base and sugar librations vary on a picosecond time scale and occur within helical domains that move collectively at diffusion-limited nanosecond time scales. Pivot points are short, functionally important, and highly mobile internal loops. These spontaneous changes in RNA conformation correlate quantitatively with those that follow adaptive recognition of diverse targets. Thus, ligands may stabilize existing RNA conformations rather than inducing new ones.

All retroviruses encapsidate their genome as a dimer of homologous single-stranded RNAs. The dimerization initiation site (DIS) of human immunodeficiency virus type 1 (HIV-1) is located in the 5'-untranslated region of the viral genome and consists of a hairpin with a 6 nt self-complementary loop sequence. Genomic RNA dimerization, a crucial step for virion infectivity, is promoted by the formation of a loop-loop complex (or kissing complex) between two DIS hairpins. Crystal structures for the subtypes A, B and F of the HIV-1 DIS kissing complex have now been solved at 2.3 A, 1.9 A and 1.6 A, respectively. They revealed a polymorphism of bulged-out residues showing clearly that their conformation is not a mere consequence of crystal packing. They also provide more insights into ion binding, hydration, and RNA conformation and flexibility. In particular, we observed the binding of spermine to the loop-loop helix, which displaced a magnesium cation important for subtype A DIS dimerization. The excellent agreement between X-ray structures and the results of chemical probing and interference data on larger viral RNA fragments shows that the crystal structures are relevant for the DIS kissing complex present in solution and in viral particles. Accordingly, these structures will be helpful for designing new drugs derived from aminoglycoside antibiotics and targeted against the RNA dimerization step of the viral life-cycle.

Specific RNA-binding proteins regulate RNA conformations in protein-switched ribozymes. Ribozyme activity can be regulated with RNA-binding proteins, as in the case of natural RNP ribozymes like RNase P that consist of an RNA subunit and an RNA-binding protein. Recent studies have shown that both natural and artificial catalytic RNAs are convertible to allosterically regulated ribozymes by molecular design at the secondary structure or three-dimensional levels and also by performing selections from combinatorial libraries. In protein-switched ribozymes, the specific binding of a protein stabilizes a particular conformation of RNA in active or inactive form such that ribozyme activity is regulated by the presence or absence of the RNA-binding protein. In applications, a protein-regulated ribozyme can be utilized as a biosensor that detects the concentration of the binding proteins in vitro and in vivo. Further advances in the design and construction of protein-regulated ribozymes are expected because of rapid progress in structural biology. Future studies will also shed new light on the molecular evolution pathways of ribozymes.

We report that double-helical DNA constraints can be used to control the conformation of another molecule, RNA. When a covalently attached DNA constraint is structurally incompatible with the native Mg2+-dependent RNA conformation, RNA folding is disrupted, as revealed by nondenaturing gel electrophoresis and independently by chemical probing. Our approach is distinct from other efforts in DNA nanotechnology, which have prepared DNA objects by self-assembly, built static DNA lattices for assembly of other objects, and created nanomachines made solely of DNA. In contrast, our dynamic use of DNA to control the conformations of other macromolecules should have wide impact in nanotechnology applications ranging from materials science to biology.