Nearest neighbor rules for RNA helix folding thermodynamics: improved end effects.

Nearest neighbor parameters for estimating the folding stability of RNA secondary structures are in widespread use. For helices, current parameters penalize terminal AU base pairs relative to terminal GC base pairs. We curated an expanded database of helix stabilities determined by optical melting experiments. Analysis of the updated database shows that terminal penalties depend on the sequence identity of the adjacent penultimate base pair. New nearest neighbor parameters that include this additional sequence dependence accurately predict the measured values of 271 helices in an updated database with a correlation coefficient of 0.982. This refined understanding of helix ends facilitates fitting terms for base pair stacks with GU pairs. Prior parameter sets treated 5'GGUC3' paired to 3'CUGG5' separately from other 5'GU3'/3'UG5' stacks. The improved understanding of helix end stability, however, makes the separate treatment unnecessary. Introduction of the additional terms was tested with three optical melting experiments. The average absolute difference between measured and predicted free energy changes at 37°C for these three duplexes containing terminal adjacent AU and GU pairs improved from 1.38 to 0.27 kcal/mol. This confirms the need for the additional sequence dependence in the model.

Perspectives on Viral RNA Genomes and the RNA Folding Problem.

Viral RNA genomes change shape as virus particles disassemble, form replication complexes, attach to ribosomes for translation, evade host defense mechanisms, and assemble new virus particles. These structurally dynamic RNA shapeshifters present a challenging RNA folding problem, because the RNA sequence adopts multiple structures and may sometimes contain regions of partial disorder. Recent advances in high resolution asymmetric cryoelectron microscopy and chemical probing provide new ways to probe the degree of structure and disorder, and have identified more than one conformation in dynamic equilibrium in viral RNA. Chemical probing and the Detection of RNA Folding Ensembles using Expectation Maximization (DREEM) algorithm has been applied to studies of the dynamic equilibrium conformations in HIV RNA in vitro, in virio, and in vivo. This new type of data provides insight into important questions about virus assembly mechanisms and the fundamental physical forces driving virus particle assembly.

Challenges and approaches to predicting RNA with multiple functional structures.

The revolution in sequencing technology demands new tools to interpret the genetic code. As in vivo transcriptome-wide chemical probing techniques advance, new challenges emerge in the RNA folding problem. The emphasis on one sequence folding into a single minimum free energy structure is fading as a new focus develops on generating RNA structural ensembles and identifying functional structural features in ensembles. This review describes an efficient combinatorially complete method and three free energy minimization approaches to predicting RNA structures with more than one functional fold, as well as two methods for analysis of a thermodynamics-based Boltzmann ensemble of structures. The review then highlights two examples of viral RNA 3'-UTR regions that fold into more than one conformation and have been characterized by single molecule fluorescence energy resonance transfer or NMR spectroscopy. These examples highlight the different approaches and challenges in predicting structure and function from sequence for RNA with multiple biological roles and folds. More well-defined examples and new metrics for measuring differences in RNA structures will guide future improvements in prediction of RNA structure and function from sequence.

Surprising Sequence Effects on GU Closure of Symmetric 2 × 2 Nucleotide RNA Internal Loops.

GU base pairs are important RNA structural motifs and often close loops. Accurate prediction of RNA structures relies upon understanding the interactions determining structure. The thermodynamics of some 2 × 2 nucleotide internal loops closed by GU pairs are not well understood. Here, several self-complementary oligonucleotide sequences expected to form duplexes with 2 × 2 nucleotide internal loops closed by GU pairs were investigated. Surprisingly, nuclear magnetic resonance revealed that many of the sequences exist in equilibrium between hairpin and duplex conformations. This equilibrium is not observed with loops closed by Watson-Crick pairs. To measure the thermodynamics of some 2 × 2 nucleotide internal loops closed by GU pairs, non-self-complementary sequences that preclude formation of hairpins were designed. The measured thermodynamics indicate that some internal loops closed by GU pairs are unusually unstable. This instability accounts for the observed equilibria between duplex and hairpin conformations. Moreover, it suggests that future three-dimensional structures of loops closed by GU pairs may reveal interactions that unexpectedly destabilize folding.

Swellix: a computational tool to explore RNA conformational space.

BACKGROUND: The sequence of nucleotides in an RNA determines the possible base pairs for an RNA fold and thus also determines the overall shape and function of an RNA. The Swellix program presented here combines a helix abstraction with a combinatorial approach to the RNA folding problem in order to compute all possible non-pseudoknotted RNA structures for RNA sequences. The Swellix program builds on the Crumple program and can include experimental constraints on global RNA structures such as the minimum number and lengths of helices from crystallography, cryoelectron microscopy, or in vivo crosslinking and chemical probing methods. RESULTS: The conceptual advance in Swellix is to count helices and generate all possible combinations of helices rather than counting and combining base pairs. Swellix bundles similar helices and includes improvements in memory use and efficient parallelization. Biological applications of Swellix are demonstrated by computing the reduction in conformational space and entropy due to naturally modified nucleotides in tRNA sequences and by motif searches in Human Endogenous Retroviral (HERV) RNA sequences. The Swellix motif search reveals occurrences of protein and drug binding motifs in the HERV RNA ensemble that do not occur in minimum free energy or centroid predicted structures. CONCLUSIONS: Swellix presents significant improvements over Crumple in terms of efficiency and memory use. The efficient parallelization of Swellix enables the computation of sequences as long as 418 nucleotides with sufficient experimental constraints. Thus, Swellix provides a practical alternative to free energy minimization tools when multiple structures, kinetically determined structures, or complex RNA-RNA and RNA-protein interactions are present in an RNA folding problem.

Advancing viral RNA structure prediction: measuring the thermodynamics of pyrimidine-rich internal loops.

Accurate thermodynamic parameters improve RNA structure predictions and thus accelerate understanding of RNA function and the identification of RNA drug binding sites. Many viral RNA structures, such as internal ribosome entry sites, have internal loops and bulges that are potential drug target sites. Current models used to predict internal loops are biased toward small, symmetric purine loops, and thus poorly predict asymmetric, pyrimidine-rich loops with >6 nucleotides (nt) that occur frequently in viral RNA. This article presents new thermodynamic data for 40 pyrimidine loops, many of which can form UU or protonated CC base pairs. Uracil and protonated cytosine base pairs stabilize asymmetric internal loops. Accurate prediction rules are presented that account for all thermodynamic measurements of RNA asymmetric internal loops. New loop initiation terms for loops with >6 nt are presented that do not follow previous assumptions that increasing asymmetry destabilizes loops. Since the last 2004 update, 126 new loops with asymmetry or sizes greater than 2 × 2 have been measured. These new measurements significantly deepen and diversify the thermodynamic database for RNA. These results will help better predict internal loops that are larger, pyrimidine-rich, and occur within viral structures such as internal ribosome entry sites.

Thermodynamic stabilities of three-way junction nanomotifs in prohead RNA.

The thermodynamic stabilities of four natural prohead or packaging RNA (pRNA) three-way junction (3WJ) nanomotifs and seven phi29 pRNA 3WJ deletion mutant nanomotifs were investigated using UV optical melting on a three-component RNA system. Our data reveal that some pRNA 3WJs are more stable than the phi29 pRNA 3WJ. The stability of the 3WJ contributes to the unique self-assembly properties of pRNA. Thus, ultrastable pRNA 3WJ motifs suggest new scaffolds for pRNA-based nanotechnology. We present data demonstrating that pRNA 3WJs differentially respond to the presence of metal ions. A comparison of our data with free energies predicted by currently available RNA secondary structure prediction programs shows that these programs do not accurately predict multibranch loop stabilities. These results will expand the existing parameters used for RNA secondary structure prediction from sequence in order to better inform RNA structure-function hypotheses and guide the rational design of functional RNA supramolecular assemblies.

Crumple: An Efficient Tool to Explore Thoroughly the RNA Folding Landscape.

The folding landscape for an RNA sequence contains many diverse structures and motifs, which are often sampled rather than completely explored. Today's supercomputers make the complete enumeration of all possible folds for an RNA and a detailed description of the RNA folding landscape a more feasible task. This chapter provides protocols for using the Crumple folding algorithm, an efficient tool to generate all possible non-pseudoknotted folds for an RNA sequence. Crumple in conjunction with Sliding Windows and Assembly can incorporate experimental constraints on the global features of an RNA, such as the minimum number and lengths of helices, which may be determined by crystallography or cryo-electron microscopy. This complete enumeration method is independent of free-energy minimization and allows the user to incorporate experimental data such as chemical probing, SELEX data on RNA-protein binding motifs, and phylogenetic covariation.

NMR Structures and Dynamics in a Prohead RNA Loop that Binds Metal Ions.

Metal ions are critical for RNA structure and enzymatic activity. We present the structure of an asymmetric RNA loop that binds metal ions and has an essential function in a bacteriophage packaging motor. Prohead RNA is a noncoding RNA that is required for genome packaging activity in phi29-like bacteriophage. The loops in GA1 and phi29 bacteriophage share a conserved adenine that forms a base triple, although the structural context for the base triple differs. NMR relaxation studies and femtosecond time-resolved fluorescence spectroscopy reveal the dynamic behavior of the loop in the metal ion bound and unbound forms. The mechanism of metal ion binding appears to be an induced conformational change between two dynamic ensembles rather than a conformational capture mechanism. These results provide experimental benchmarks for computational models of RNA-metal ion interactions.

Prohead RNA: a noncoding viral RNA of novel structure and function.

Prohead RNA (pRNA) is an essential component of the powerful Φ29-like bacteriophage DNA packaging motor. However, the specific role of this unique RNA in the Φ29 packaging motor remains unknown. This review examines pRNA as a noncoding RNA of novel structure and function. In order to highlight the reasons for exploring the structure and function of pRNA, we (1) provide an overview of Φ29-like bacteriophage and the Φ29 DNA packaging motor, including putative motor mechanisms and structures of its component parts; (2) discuss pRNA structure and possible roles for pRNA in the Φ29 packaging motor; (3) summarize pRNA self-assembly; and (4) describe the prospective therapeutic applications of pRNA. Many questions remain to be answered in order to connect what is currently known about pRNA structure to its novel function in the Φ29 packaging motor. The knowledge gained from studying the structure, function, and sequence variation in pRNA will help develop tools to better navigate the conformational landscapes of RNA. WIREs RNA 2016, 7:428-437. doi: 10.1002/wrna.1330 For further resources related to this article, please visit the WIREs website.

Structures and Energetics of Four Adjacent G·U Pairs That Stabilize an RNA Helix.

Consecutive G·U base pairs inside RNA helices can be destabilizing, while those at the ends of helices are thermodynamically stabilizing. To determine if this paradox could be explained by differences in base stacking, we determined the high-resolution (1.32 Å) crystal structure of (5'-GGUGGCUGUU-3')2 and studied three sequences with four consecutive terminal G·U pairs by NMR spectroscopy. In the crystal structure of (5'-GGUGGCUGUU-3')2, the helix is overwound but retains the overall features of A-form RNA. The penultimate base steps at each end of the helix have high base overlap and contribute to the unexpectedly favorable energetic contribution for the 5'-GU-3'/3'-UG-5' motif in this helix position. The balance of base stacking and helical twist contributes to the positional dependence of G·U pair stabilities. The energetic stabilities and similarity to A-form RNA helices suggest that consecutive G·U pairs would be recognized by RNA helix binding proteins, such as Dicer and Ago. Thus, these results will aid future searches for target sites of small RNAs in gene regulation.

A parallel implementation of the Wuchty algorithm with additional experimental filters to more thoroughly explore RNA conformational space.

We present new modifications to the Wuchty algorithm in order to better define and explore possible conformations for an RNA sequence. The new features, including parallelization, energy-independent lonely pair constraints, context-dependent chemical probing constraints, helix filters, and optional multibranch loops, provide useful tools for exploring the landscape of RNA folding. Chemical probing alone may not necessarily define a single unique structure. The helix filters and optional multibranch loops are global constraints on RNA structure that are an especially useful tool for generating models of encapsidated viral RNA for which cryoelectron microscopy or crystallography data may be available. The computations generate a combinatorially complete set of structures near a free energy minimum and thus provide data on the density and diversity of structures near the bottom of a folding funnel for an RNA sequence. The conformational landscapes for some RNA sequences may resemble a low, wide basin rather than a steep funnel that converges to a single structure.

Probing viral genomic structure: alternative viewpoints and alternative structures for satellite tobacco mosaic virus RNA.

Viral RNA structure prediction is a valuable tool for development of drugs against viral disease. This work discusses different approaches to predicting encapsidated viral RNA and highlights satellite tobacco mosaic virus (STMV) RNA as a model system with excellent crystallography data. Fundamentally important issues for debate include thermodynamic versus kinetic control of virus assembly and the possible consequences of quasi-species in the primary structure on RNA secondary structure prediction of a single structure or an ensemble of structures. Multiple computational tools and chemical reagents are now available for improved viral RNA structure prediction. Two different predicted structures for encapsidated STMV RNA result from differences in three main areas: a different approach and philosophy to studying encapsidated viral RNA, an emphasis on different RNA motifs, and technical differences in computational methods and chemical reagents. The experiments with traditional chemical probing and SHAPE reagents are compared in terms of chemistry, results, and interpretation for STMV RNA as well as other RNA protein assemblies, such as the 5'UTR of HIV and the ribosome. This discussion of the challenges of viral RNA structure prediction will lead to new experiments and improved future predictions for viral RNA.

Effects of salt, polyethylene glycol, and locked nucleic acids on the thermodynamic stabilities of consecutive terminal adenosine mismatches in RNA duplexes.

Consecutive terminal mismatches add thermodynamic stability to RNA duplexes and occur frequently in microRNA-mRNA interactions. Accurate thermodynamic stabilities of consecutive terminal mismatches contribute to the development of specific, high-affinity siRNA therapeutics. Consecutive terminal adenosine mismatches (TAMS) are studied at different salt concentrations, with polyethylene glycol cosolutes, and with locked nucleic acid (LNA) substitutions. These measurements provide benchmarks for the application of thermodynamic predictions to different physiological or therapeutic conditions. The salt dependence for RNA duplex stability is similar for TAMS, internal loops, and Watson-Crick duplexes. A unified model for predicting the free energy of an RNA duplex with or without loops and mismatches at lower sodium concentrations is presented. The destabilizing effects of PEG 200 are larger for TAMS than internal loops or Watson-Crick duplexes, which may result from different base stacking conformations, dynamics, and water hydration. In contrast, LNA substitutions stabilize internal loops much more than TAMS. Surprisingly, the average per adenosine increase in stability for LNA substitutions in internal loops is -1.82 kcal/mol and only -0.20 kcal/mol for TAMS. The stabilities of TAMS and internal loops with LNA substitutions have similar favorable free energies. Thus, the unfavorable free energy of adenosine internal loops is largely an entropic effect. The favorable stabilities of TAMS result mainly from base stacking. The ability of RNA duplexes to form extended terminal mismatches in the absence of proteins such as argonaute and identifying the enthalpic contributions to terminal mismatch stabilities provide insight into the physical basis of microRNA-mRNA molecular recognition and specificity.

Incorporating global features of RNA motifs in predictions for an ensemble of secondary structures for encapsidated MS2 bacteriophage RNA.

The secondary structure of encapsidated MS2 genomic RNA poses an interesting RNA folding challenge. Cryoelectron microscopy has demonstrated that encapsidated MS2 RNA is well-ordered. Models of MS2 assembly suggest that the RNA hairpin-protein interactions and the appropriate placement of hairpins in the MS2 RNA secondary structure can guide the formation of the correct icosahedral particle. The RNA hairpin motif that is recognized by the MS2 capsid protein dimers, however, is energetically unfavorable, and thus free energy predictions are biased against this motif. Computer programs called Crumple, Sliding Windows, and Assembly provide useful tools for prediction of viral RNA secondary structures when the traditional assumptions of RNA structure prediction by free energy minimization may not apply. These methods allow incorporation of global features of the RNA fold and motifs that are difficult to include directly in minimum free energy predictions. For example, with MS2 RNA the experimental data from SELEX experiments, crystallography, and theoretical calculations of the path for the series of hairpins can be incorporated in the RNA structure prediction, and thus the influence of free energy considerations can be modulated. This approach thoroughly explores conformational space and generates an ensemble of secondary structures. The predictions from this new approach can test hypotheses and models of viral assembly and guide construction of complete three-dimensional models of virus particles.

Nucleotide Dynamics at the A-Site Cleft in the Peptidyltransferase Center of H. marismortui 50S Ribosomal Subunits.

Resistance mutations to antibiotics targeting rRNA can be far from the drug-binding site. Crystallography studies revealed that the antibiotic resistance mutation G2482A (G2447A in E. coli ) in Haloarcula marismortui 50S ribosomes does not directly contact the drug or introduce changes to the ribosomal structure except for losing a potassium ion coordinated to a base triple at the drug-binding site. Using molecular dynamics simulations, we tested hypotheses regarding the effects of the G2482A mutation and ion coordination on the conformational dynamics of the 50S ribosome. Simulations show that the mutation enhances conformational fluctuation at the antibiotic binding site, weakens the hydrogen-bonding network, and increases flexibility at the 50S peptidyl transferase center (PTC). Our data supports the view that distant mutations can perturb the dynamic network in the ribosomal PTC, thereby raising the entropic cost of antibiotic binding. These results underscore the importance of considering conformational dynamics in rational drug design.