Showing papers on "Nucleic acid secondary structure published in 2012"

Automated 3D structure composition for large RNAs

Abstract: Understanding the numerous functions that RNAs play in living cells depends critically on knowledge of their three-dimensional structure. Due to the difficulties in experimentally assessing structures of large RNAs, there is currently great demand for new high-resolution structure prediction methods. We present the novel method for the fully automated prediction of RNA 3D structures from a user-defined secondary structure. The concept is founded on the machine translation system. The translation engine operates on the RNA FRABASE database tailored to the dictionary relating the RNA secondary structure and tertiary structure elements. The translation algorithm is very fast. Initial 3D structure is composed in a range of seconds on a single processor. The method assures the prediction of large RNA 3D structures of high quality. Our approach needs neither structural templates nor RNA sequence alignment, required for comparative methods. This enables the building of unresolved yet native and artificial RNA structures. The method is implemented in a publicly available, user-friendly server RNAComposer. It works in an interactive mode and a batch mode. The batch mode is designed for large-scale modelling and accepts atomic distance restraints. Presently, the server is set to build RNA structures of up to 500 residues.

SSE: a nucleotide and amino acid sequence analysis platform

Abstract: There is an increasing need to develop bioinformatic tools to organise and analyse the rapidly growing amount of nucleotide and amino acid sequence data in organisms ranging from viruses to eukaryotes. A simple sequence editor (SSE) was developed to create an integrated environment where sequences can be aligned, annotated, classified and directly analysed by a number of built-in bioinformatic programs. SSE incorporates a sequence editor for the creation of sequence alignments, a process assisted by integrated CLUSTAL/MUSCLE alignment programs and automated removal of indels. Sequences can be fully annotated and classified into groups and annotated of sequences and sequence groups and access to analytical programs that analyse diversity, recombination and RNA secondary structure. Methods for analysing sequence diversity include measures of divergence and evolutionary distances, identity plots to detect regions of nucleotide or amino acid homology, reconstruction of sequence changes, mono-, di- and higher order nucleotide compositional biases and codon usage. Association Index calculations, GroupScans, Bootscanning and TreeOrder scans perform phylogenetic analyses that reconcile group membership with tree branching orders and provide powerful methods for examining segregation of alleles and detection of recombination events. Phylogeny changes across alignments and scoring of branching order differences between trees using the Robinson-Fould algorithm allow effective visualisation of the sites of recombination events. RNA secondary and tertiary structures play important roles in gene expression and RNA virus replication. For the latter, persistence of infection is additionally associated with pervasive RNA secondary structure throughout viral genomic RNA that modulates interactions with innate cell defences. SSE provides several programs to scan alignments for RNA secondary structure through folding energy thermodynamic calculations and phylogenetic methods (detection of co-variant changes, and structure conservation between divergent sequences). These analyses complement methods based on detection of sequence constraints, such as suppression of synonymous site variability. For each program, results can be plotted in real time during analysis through an integrated graphics package, providing publication quality graphs. Results can be also directed to tabulated datafiles for import into spreadsheet or database programs for further analysis. SSE combines sequence editor functions with analytical tools in a comprehensive and user-friendly package that assists considerably in bioinformatic and evolution research.

A rule of seven in Watson-Crick base-pairing of mismatched sequences

Abstract: Sequence recognition through base-pairing is essential for DNA repair and gene regulation, but the basic rules governing this process remain elusive. In particular, the kinetics of annealing between two imperfectly matched strands is not well characterized, despite its potential importance in nucleic acid-based biotechnologies and gene silencing. Here we use single-molecule fluorescence to visualize the multiple annealing and melting reactions of two untethered strands inside a porous vesicle, allowing us to precisely quantify the annealing and melting rates. The data as a function of mismatch position suggest that seven contiguous base pairs are needed for rapid annealing of DNA and RNA. This phenomenological rule of seven may underlie the requirement for seven nucleotides of complementarity to seed gene silencing by small noncoding RNA and may help guide performance improvement in DNA- and RNA-based bio- and nanotechnologies, in which off-target effects can be detrimental.

Systematic discovery of structural elements governing stability of mammalian messenger RNAs

Abstract: Decoding post-transcriptional regulatory programs in RNA is a critical step towards the larger goal of developing predictive dynamical models of cellular behaviour. Despite recent efforts, the vast landscape of RNA regulatory elements remains largely uncharacterized. A long-standing obstacle is the contribution of local RNA secondary structure to the definition of interaction partners in a variety of regulatory contexts, including--but not limited to--transcript stability, alternative splicing and localization. There are many documented instances where the presence of a structural regulatory element dictates alternative splicing patterns (for example, human cardiac troponin T) or affects other aspects of RNA biology. Thus, a full characterization of post-transcriptional regulatory programs requires capturing information provided by both local secondary structures and the underlying sequence. Here we present a computational framework based on context-free grammars and mutual information that systematically explores the immense space of small structural elements and reveals motifs that are significantly informative of genome-wide measurements of RNA behaviour. By applying this framework to genome-wide human mRNA stability data, we reveal eight highly significant elements with substantial structural information, for the strongest of which we show a major role in global mRNA regulation. Through biochemistry, mass spectrometry and in vivo binding studies, we identified human HNRPA2B1 (heterogeneous nuclear ribonucleoprotein A2/B1, also known as HNRNPA2B1) as the key regulator that binds this element and stabilizes a large number of its target genes. We created a global post-transcriptional regulatory map based on the identity of the discovered linear and structural cis-regulatory elements, their regulatory interactions and their target pathways. This approach could also be used to reveal the structural elements that modulate other aspects of RNA behaviour.

Selective RNA Versus DNA G‐Quadruplex Targeting by In Situ Click Chemistry

Abstract: It all clicks into place: A potent telomere-targeting small molecule has been identified by using the copper-free 1,3-dipolar cycloaddition of a series of alkyne and azide building blocks catalyzed by a non-Watson-Crick DNA secondary structure (see picture). This method rapidly identifies, otherwise unanticipated, potent small-molecule probes to selectively target a given RNA or DNA.

R-chie: a web server and R package for visualizing RNA secondary structures

Abstract: Visually examining RNA structures can greatly aid in understanding their potential functional roles and in evaluating the performance of structure prediction algorithms. As many functional roles of RNA structures can already be studied given the secondary structure of the RNA, various methods have been devised for visualizing RNA secondary structures. Most of these methods depict a given RNA secondary structure as a planar graph consisting of base-paired stems interconnected by roundish loops. In this article, we present an alternative method of depicting RNA secondary structure as arc diagrams. This is well suited for structures that are difficult or impossible to represent as planar stem-loop diagrams. Arc diagrams can intuitively display pseudo-knotted structures, as well as transient and alternative structural features. In addition, they facilitate the comparison of known and predicted RNA secondary structures. An added benefit is that structure information can be displayed in conjunction with a corresponding multiple sequence alignments, thereby highlighting structure and primary sequence conservation and variation. We have implemented the visualization algorithm as a web server R-chie as well as a corresponding R package called R4RNA, which allows users to run the software locally and across a range of common operating systems.

Integrating chemical footprinting data into RNA secondary structure prediction.

Abstract: Chemical and enzymatic footprinting experiments, such as shape (selective 2'-hydroxyl acylation analyzed by primer extension), yield important information about RNA secondary structure. Indeed, since the [Formula: see text]-hydroxyl is reactive at flexible (loop) regions, but unreactive at base-paired regions, shape yields quantitative data about which RNA nucleotides are base-paired. Recently, low error rates in secondary structure prediction have been reported for three RNAs of moderate size, by including base stacking pseudo-energy terms derived from shape data into the computation of minimum free energy secondary structure. Here, we describe a novel method, RNAsc (RNA soft constraints), which includes pseudo-energy terms for each nucleotide position, rather than only for base stacking positions. We prove that RNAsc is self-consistent, in the sense that the nucleotide-specific probabilities of being unpaired in the low energy Boltzmann ensemble always become more closely correlated with the input shape data after application of RNAsc. From this mathematical perspective, the secondary structure predicted by RNAsc should be 'correct', in as much as the shape data is 'correct'. We benchmark RNAsc against the previously mentioned method for eight RNAs, for which both shape data and native structures are known, to find the same accuracy in 7 out of 8 cases, and an improvement of 25% in one case. Furthermore, we present what appears to be the first direct comparison of shape data and in-line probing data, by comparing yeast asp-tRNA shape data from the literature with data from in-line probing experiments we have recently performed. With respect to several criteria, we find that shape data appear to be more robust than in-line probing data, at least in the case of asp-tRNA.

The biased nucleotide composition of the HIV genome: a constant factor in a highly variable virus

Abstract: Viruses often deviate from their hosts in the nucleotide composition of their genomes. The RNA genome of the lentivirus family of retroviruses, including human immunodeficiency virus (HIV), contains e.g. an above average percentage of adenine (A) nucleotides, while being extremely poor in cytosine (C). Such a deviant base composition has implications for the amino acids that are encoded by the open reading frames (ORFs), both in the requirement of specific tRNA species and in the preference for amino acids encoded by e.g. A-rich codons. Nucleotide composition does obviously affect the secondary and tertiary structure of the RNA genome and its biological functions, but it does also influence phylogenetic analysis of viral genome sequences, and possibly the activity of the integrated DNA provirus. Over time, the nucleotide composition of the HIV-1 genome is exceptionally conserved, varying by less than 1% per base position per isolate within either group M, N, or O during 1983–2009. This extreme stability of the nucleotide composition may possibly be achieved by negative selection, perhaps conserving semi-stable RNA secondary structure as reverse transcription would be significantly affected for a less A-rich genome where secondary structures are expected to be more stable and thus more difficult to unfold. This review will discuss all aspects of the lentiviral genome composition, both of the RNA and of its derived double-stranded DNA genome, with a focus on HIV-1, the nucleotide composition over time, the effects of artificially humanized codons as well as contributions of immune system pressure on HIV nucleotide bias.

Input-dependent induction of oligonucleotide structural motifs for performing molecular logic

Abstract: The K+–H+-triggered structural conversion of multiple nucleic acid helices involving duplexes, triplexes, G-quadruplexes, and i-motifs is studied by gel electrophoresis, circular dichroism, and thermal denaturation. We employ the structural interconversions for perfoming molecular logic operations, as verified by fluorimetry and colorimetry. Short G-rich and C-rich cDNA and RNA single strands are hybridized to produce four A-form and B-form duplexes. Addition of K+ triggers the unwinding of the duplexes by inducing the folding of G-rich strands into DNA- or RNA G-quadruplex mono- and multimers, respectively. We found a decrease in pH to have different consequences on the resulting structural output, depending on whether the C-rich strand is DNA or RNA: while the protonated C-rich DNA strand folds into at least two isomers of a stable i-motif structure, the protonated C-rich RNA strand binds a DNA/RNA hybrid duplex to form a Y·RY parallel triplex. When using K+ and H+ as external stimuli, or inputs, and th...

Testing the Nearest Neighbor Model for Canonical RNA Base Pairs: Revision of GU Parameters

Abstract: Thermodynamic parameters for GU pairs are important for predicting the secondary structures of RNA and for finding genomic sequences that code for structured RNA. Optical melting curves were measured for 29 RNA duplexes with GU pairs to improve nearest neighbor parameters for predicting stabilities of helixes. The updated model eliminates a prior penalty assumed for terminal GU pairs. Six additional duplexes with the 5′GG/3′UU motif were added to the single representation in the previous database. This revises the ΔG°37 for the 5′GG/3′UU motif from an unfavorable 0.5 kcal/mol to a favorable −0.2 kcal/mol. Similarly, the ΔG°37 for the 5′UG/3′GU motif changes from 0.3 to −0.6 kcal/mol. The correlation coefficients between predicted and experimental ΔG°37, ΔH°, and ΔS° for the expanded database are 0.95, 0.89, and 0.87, respectively. The results should improve predictions of RNA secondary structure.

RNA unwinding by the Trf4/Air2/Mtr4 polyadenylation (TRAMP) complex.

Abstract: Many RNA-processing events in the cell nucleus involve the Trf4/Air2/Mtr4 polyadenylation (TRAMP) complex, which contains the poly(A) polymerase Trf4p, the Zn-knuckle protein Air2p, and the RNA helicase Mtr4p. TRAMP polyadenylates RNAs designated for processing by the nuclear exosome. In addition, TRAMP functions as an exosome cofactor during RNA degradation, and it has been speculated that this role involves disruption of RNA secondary structure. However, it is unknown whether TRAMP displays RNA unwinding activity. It is also not clear how unwinding would be coordinated with polyadenylation and the function of the RNA helicase Mtr4p in modulating poly(A) addition. Here, we show that TRAMP robustly unwinds RNA duplexes. The unwinding activity of Mtr4p is significantly stimulated by Trf4p/Air2p, but the stimulation of Mtr4p does not depend on ongoing polyadenylation. Nonetheless, polyadenylation enables TRAMP to unwind RNA substrates that it otherwise cannot separate. Moreover, TRAMP displays optimal unwinding activity on substrates with a minimal Mtr4p binding site comprised of adenylates. Our results suggest a model for coordination between unwinding and polyadenylation activities by TRAMP that reveals remarkable synergy between helicase and poly(A) polymerase.

Triple-helical recognition of RNA using 2-aminopyridine-modified PNA at physiologically relevant conditions.

Abstract: Peptide nucleic acids containing thymidine and 2-aminopyridine (M) nucleobases form stable and sequence-selective triple helices with double-stranded RNA at physiologically relevant conditions. The M-modified PNA showed unique RNA selectivity by having two orders of magnitude higher affinity for the double-stranded RNAs than for the same DNA sequences.

Targeting a pre-mRNA structure with bipartite antisense molecules modulates tau alternative splicing

Abstract: Approximately 15% of human genetic diseases are estimated to involve dysregulation of alternative pre-mRNA splicing. Antisense molecules designed to alter these and other splicing events typically target continuous linear sequences of the message. Here, we show that a structural feature in a pre-mRNA can be targeted by bipartite antisense molecules designed to hybridize with the discontinuous elements that flank the structure and thereby alter splicing. We targeted a hairpin structure at the boundary between exon 10 and intron 10 of the pre-mRNA of tau. Mutations in this region that are associated with certain forms of frontotemporal dementia, destabilize the hairpin to cause increased inclusion of exon 10. Via electrophoretic mobility shift and RNase protection assays, we demonstrate that bipartite antisense molecules designed to simultaneously interact with the available sequences that immediately flank the tau pre-mRNA hairpin do indeed bind to this structured region. Moreover, these agents inhibit exon 10 splicing and reverse the effect of destabilizing disease-causing mutations, in both in vitro splicing assays and cell culture. This general bipartite antisense strategy could be employed to modulate other splicing events that are regulated by RNA secondary structure.

Dissecting the protein–RNA interface: the role of protein surface shapes and RNA secondary structures in protein–RNA recognition

Abstract: Protein–RNA interactions are essential for many biological processes. However, the structural mechanisms underlying these interactions are not fully understood. Here, we analyzed the protein surface shape (dented, intermediate or protruded) and the RNA base pairing properties (paired or unpaired nucleotides) at the interfaces of 91 protein–RNA complexes derived from the Protein Data Bank. Dented protein surfaces prefer unpaired nucleotides to paired ones at the interface, and hydrogen bonds frequently occur between the protein backbone and RNA bases. In contrast, protruded protein surfaces do not show such a preference, rather, electrostatic interactions initiate the formation of hydrogen bonds between positively charged amino acids and RNA phosphate groups. Interestingly, in many protein–RNA complexes that interact via an RNA loop, an aspartic acid is favored at the interface. Moreover, in most of these complexes, nucleotide bases in the RNA loop are flipped out and form hydrogen bonds with the protein, which suggests that aspartic acid is important for RNA loop recognition through a base-flipping process. This study provides fundamental insights into the role of the shape of the protein surface and RNA secondary structures in mediating protein–RNA interactions.

Evaluating our ability to predict the structural disruption of RNA by SNPs

Abstract: The structure of RiboNucleic Acid (RNA) has the potential to be altered by a Single Nucleotide Polymorphism (SNP). Disease-associated SNPs mapping to non-coding regions of the genome that are transcribed into RiboNucleic Acid (RNA) can potentially affect cellular regulation (and cause disease) by altering the structure of the transcript. We performed a large-scale meta-analysis of Selective 2'-Hydroxyl Acylation analyzed by Primer Extension (SHAPE) data, which probes the structure of RNA. We found that several single point mutations exist that significantly disrupt RNA secondary structure in the five transcripts we analyzed. Thus, every RNA that is transcribed has the potential to be a “RiboSNitch;” where a SNP causes a large conformational change that alters regulatory function. Predicting the SNPs that will have the largest effect on RNA structure remains a contemporary computational challenge. We therefore benchmarked the most popular RNA structure prediction algorithms for their ability to identify mutations that maximally affect structure. We also evaluated metrics for rank ordering the extent of the structural change. Although no single algorithm/metric combination dramatically outperformed the others, small differences in AUC (Area Under the Curve) values reveal that certain approaches do provide better agreement with experiment. The experimental data we analyzed nonetheless show that multiple single point mutations exist in all RNA transcripts that significantly disrupt structure in agreement with the predictions.

Novel RNA base pair with higher specificity using single selenium atom

Abstract: Specificity of nucleobase pairing provides essential foundation for genetic information storage, replication, transcription and translation in all living organisms. However, the wobble base pairs, where U in RNA (or T in DNA) pairs with G instead of A, might compromise the high specificity of the base pairing. The U/G wobble pairing is ubiquitous in RNA, especially in non-coding RNA. In order to increase U/A pairing specificity, we have hypothesized to discriminate against U/G wobble pair by tailoring the steric and electronic effects at the 2-exo position of uridine and replacing the 2-exo oxygen with a selenium atom. We report here the first synthesis of the 2-Se-U-RNAs as well as the 2-Se-uridine ((Se)U) phosphoramidite. Our biophysical and structural studies of the (Se)U-RNAs indicate that this single atom replacement can indeed create a novel U/A base pair with higher specificity than the natural one. We reveal that the (Se)U/A pair maintains a structure virtually identical to the native U/A base pair, while discriminating against U/G wobble pair. This oxygen replacement with selenium offers a unique chemical strategy to enhance the base pairing specificity at the atomic level.

RNA secondary structure mediates alternative 3'ss selection in Saccharomyces cerevisiae.

Abstract: Alternative splicing is the mechanism by which different combinations of exons in the pre-mRNA give rise to distinct mature mRNAs. This process is mediated by splicing factors that bind the pre-mRNA and affect the recognition of its splicing signals. Saccharomyces species lack many of the regulatory factors present in metazoans. Accordingly, it is generally assumed that the amount of alternative splicing is limited. However, there is recent compelling evidence that yeast have functional alternative splicing, mainly in response to environmental conditions. We have previously shown that sequence and structure properties of the pre-mRNA could explain the selection of 39 splice sites (ss) in Saccharomyces cerevisiae. In this work, we extend our previous observations to build a computational classifier that explains most of the annotated 39ss in the CDS and 59 UTR of this organism. Moreover, we show that the same rules can explain the selection of alternative 39ss. Experimental validation of a number of predicted alternative 39ss shows that their usage is low compared to annotated 39ss. The majority of these alternative 39ss introduce premature termination codons (PTCs), suggesting a role in expression regulation. Furthermore, a genome-wide analysis of the effect of temperature, followed by experimental validation, yields only a small number of changes, indicating that this type of regulation is not widespread. Our results are consistent with the presence of alternative 39ss selection in yeast mediated by the pre-mRNA structure, which can be responsive to external cues, like temperature, and is possibly related to the control of gene expression.

GTfold: enabling parallel RNA secondary structure prediction on multi-core desktops.

Abstract: Accurate and efficient RNA secondary structure prediction remains an important open problem in computational molecular biology. Historically, advances in computing technology have enabled faster and more accurate RNA secondary structure predictions. Previous parallelized prediction programs achieved significant improvements in runtime, but their implementations were not portable from niche high-performance computers or easily accessible to most RNA researchers. With the increasing prevalence of multi-core desktop machines, a new parallel prediction program is needed to take full advantage of today’s computing technology. We present here the first implementation of RNA secondary structure prediction by thermodynamic optimization for modern multi-core computers. We show that GTfold predicts secondary structure in less time than UNAfold and RNAfold, without sacrificing accuracy, on machines with four or more cores. GTfold supports advances in RNA structural biology by reducing the timescales for secondary structure prediction. The difference will be particularly valuable to researchers working with lengthy RNA sequences, such as RNA viral genomes.

New insights into the fundamental role of topological constraints as a determinant of two-way junction conformation

Abstract: Recent studies have shown that topological constraints encoded at the RNA secondary structure level involving basic steric and stereochemical forces can significantly restrict the orientations sampled by helices across two-way RNA junctions. Here, we formulate these topological constraints in greater quantitative detail and use this topological framework to rationalize long-standing but poorly understood observations regarding the basic behavior of RNA two-way junctions. Notably, we show that the asymmetric nature of the A-form helix and the finite length of a bulge provide a physical basis for the experimentally observed directionality and bulge-length amplitude dependence of bulge induced inter-helical bends. We also find that the topologically allowed space can be modulated by variations in sequence, particularly with the addition of non-canonical GU base pairs at the junction, and, surprisingly, by the length of the 5′ and 3′ helices. A survey of two-way RNA junctions in the protein data bank confirms that junction residues have a strong preference to adopt looped-in, non-canonically base-paired conformations, providing a route for extending our bulge-directed framework to internal loop motifs and implying a simplified link between secondary and tertiary structure. Finally, our results uncover a new simple mechanism for coupling junction-induced topological constraints with tertiary interactions.

RNA CoSSMos: Characterization of Secondary Structure Motifs—a searchable database of secondary structure motifs in RNA three-dimensional structures

Abstract: RNA secondary structure is important for designing therapeutics, understanding protein-RNA binding and predicting tertiary structure of RNA. Several databases and downloadable programs exist that specialize in the three-dimensional (3D) structure of RNA, but none focus specifically on secondary structural motifs such as internal, bulge and hairpin loops. The RNA Characterization of Secondary Structure Motifs (RNA CoSSMos) database is a freely accessible and searchable online database and website of 3D characteristics of secondary structure motifs. To create the RNA CoSSMos database, 2156 Protein Data Bank (PDB) files were searched for internal, bulge and hairpin loops, and each loop's structural information, including sugar pucker, glycosidic linkage, hydrogen bonding patterns and stacking interactions, was included in the database. False positives were defined, identified and reclassified or omitted from the database to ensure the most accurate results possible. Users can search via general PDB information, experimental parameters, sequence and specific motif and by specific structural parameters in the subquery page after the initial search. Returned results for each search can be viewed individually or a complete set can be downloaded into a spreadsheet to allow for easy comparison. The RNA CoSSMos database is automatically updated weekly and is available at http://cossmos.slu.edu.

Evolutionarily conserved A-to-I editing increases protein stability of the alternative splicing factor Nova1.

Abstract: The structural complexity of the vertebrate brain is mirrored by its unparalleled transcriptome complexity. In particular, two post-transcriptional processes, alternative splicing and RNA editing, greatly diversify brain transcriptomes. Here we report a close connection between these two processes: we show A-to-I RNA editing in Nova1, a key brain-specific regulator of alternative splicing. Nova1 editing levels increase during embryonic development in mouse and chicken brains and show significant variation across postnatal brain regions. Evolutionary conservation of both editing and editing-associated RNA secondary structure of the Nova1 mRNA for 300 million years attests to the functional importance of Nova1 editing. Using a combination of different assays in human HEK293T cell lines, we report a novel post-translational role for this RNA editing. Whereas functional assays showed no effect of RNA editing on the regulatory splicing activity of the encoded proteins, we found evidence that edited forms exhibit reduced proteasome targeting and increased protein half-life. In addition, we found evidence for similar regulation of protein half-life by an evolutionarily conserved alternative splicing event in Nova1. These results open new venues of research on the multi-level integration of gene expression by: (1) revealing the novel role of RNA editing in regulating protein stability, and (2) establishing protein stability as a new target of multifaceted regulation.

Structural Map of a MicroRNA-122:Hepatitis C Virus Complex

Abstract: MicroRNA-122 (miR-122) enhances hepatitis C virus (HCV) fitness via targeting two sites in the 5′-untranslated region (UTR) of HCV. We used selective 2′-hydroxyl acylation analyzed by primer extension to resolve the HCV 5′-UTR's RNA secondary structure in the presence of miR-122. Nearly all nucleotides in miR-122 are involved in targeting the second site, beyond classic seed base pairings. These additional interactions enhance HCV replication in cell culture. To our knowledge, this is the first biophysical study of this complex to reveal the importance of ‘tail’ miR-122 nucleotide interactions.

Structural constraints identified with covariation analysis in ribosomal RNA.

Abstract: Covariation analysis is used to identify those positions with similar patterns of sequence variation in an alignment of RNA sequences. These constraints on the evolution of two positions are usually associated with a base pair in a helix. While mutual information (MI) has been used to accurately predict an RNA secondary structure and a few of its tertiary interactions, early studies revealed that phylogenetic event counting methods are more sensitive and provide extra confidence in the prediction of base pairs. We developed a novel and powerful phylogenetic events counting method (PEC) for quantifying positional covariation with the Gutell lab’s new RNA Comparative Analysis Database (rCAD). The PEC and MI-based methods each identify unique base pairs, and jointly identify many other base pairs. In total, both methods in combination with an N-best and helix-extension strategy identify the maximal number of base pairs. While covariation methods have effectively and accurately predicted RNAs secondary structure, only a few tertiary structure base pairs have been identified. Analysis presented herein and at the Gutell lab’s Comparative RNA Web (CRW) Site reveal that the majority of these latter base pairs do not covary with one another. However, covariation analysis does reveal a weaker although significant covariation between sets of nucleotides that are in proximity in the three-dimensional RNA structure. This reveals that covariation analysis identifies other types of structural constraints beyond the two nucleotides that form a base pair.

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

Abstract: 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 noncan...

Stable stem enabled Shannon entropies distinguish non-coding RNAs from random backgrounds

Abstract: The computational identification of RNAs in genomic sequences requires the identification of signals of RNA sequences. Shannon base pairing entropy is an indicator for RNA secondary structure fold certainty in detection of structural, non-coding RNAs (ncRNAs). Under the Boltzmann ensemble of secondary structures, the probability of a base pair is estimated from its frequency across all the alternative equilibrium structures. However, such an entropy has yet to deliver the desired performance for distinguishing ncRNAs from random sequences. Developing novel methods to improve the entropy measure performance may result in more effective ncRNA gene finding based on structure detection. This paper shows that the measuring performance of base pairing entropy can be significantly improved with a constrained secondary structure ensemble in which only canonical base pairs are assumed to occur in energetically stable stems in a fold. This constraint actually reduces the space of the secondary structure and may lower the probabilities of base pairs unfavorable to the native fold. Indeed, base pairing entropies computed with this constrained model demonstrate substantially narrowed gaps of Z-scores between ncRNAs, as well as drastic increases in the Z-score for all 13 tested ncRNA sets, compared to shuffled sequences. These results suggest the viability of developing effective structure-based ncRNA gene finding methods by investigating secondary structure ensembles of ncRNAs.

Structure–Activity Studies on the Fluorescent Indicator in a Displacement Assay for the Screening of Small Molecules Binding to RNA

Abstract: A series of xanthone and thioxanthone derivatives with aminoalkoxy substituents were synthesized as fluorescent indicators for a displacement assay in the study of small-molecule-RNA interactions. The RNA-binding properties of these molecules were investigated in terms of the improved binding selectivity to the loop region in the RNA secondary structure relative to 2,7-bis(2-aminoethoxy)xanthone (X2S) by fluorimetric titration and displacement assay. An 11-mer double-stranded RNA and a hairpin RNA mimicking the stem loop IIB of Rev response element (RRE) RNA of HIV-1 mRNA were used. The X2S derivatives with longer aminoalkyl substituents showed a higher affinity to the double-stranded RNA than the parent molecule. Introduction of a methyl group on the aminoethoxy moiety of X2S effectively modulated the selectivity to the RNA secondary structure. Methyl group substitution at the C1' position suppressed the binding to the loop regions. Substitution with two methyl groups on the amino nitrogen atom resulted in reducing the affinity to the double-stranded region by a factor of 40%. The effect of methyl substitution on the amino nitrogen atom was also observed for a thioxanthone derivative. Titration experiments, however, suggested that thioxanthone derivatives showed a more prominent tendency of multiple binding to RNA than xanthone derivatives. The selectivity index calculated from the affinity to the double-stranded and loop regions suggested that the N,N-dimethyl derivative of X2S would be suitable for the screening of small molecules binding to RRE.

Dynamics in Sequence Space for RNA Secondary Structure Design.

Abstract: We have implemented a method for the design of RNA sequences that should fold to arbitrary secondary structures. A popular energy model allows one to take the derivative with respect to composition, which can then be interpreted as a force and used for Newtonian dynamics in sequence space. Combined with a negative design term, one can rapidly sample sequences which are compatible with a desired secondary structure via simulated annealing. Results for 360 structures were compared with those from another nucleic acid design program using measures such as the probability of the target structure and an ensemble-weighted distance to the target structure.