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Nearest neighbor thermodynamic parameters are widely used for RNA and DNA secondary structure prediction and to model thermodynamic ensembles of secondary structures. The Nearest Neighbor Database (NNDB) is a freely available web resource (https://rna.urmc.rochester.edu/NNDB) that provides the functional forms, parameter values, and example calculations. The NNDB provides the 1999 and 2004 set of RNA folding nearest neighbor parameters. We expanded the database to include a set of DNA parameters and a set of RNA parameters that includes m 6 A in addition to the canonical RNA nucleobases. The site was redesigned using the Quarto open-source publishing system. A downloadable PDF version of the complete resource and downloadable sets of nearest neighbor parameters are available., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Influenza A kills hundreds of thousands of people globally every year and has the potential to generate more severe pandemics. Influenza A's RNA genome and transcriptome provide many potential therapeutic targets. Here, nuclear magnetic resonance (NMR) experiments suggest that one such target could be a hairpin loop of 8 nucleotides in a pseudoknot that sequesters a 3' splice site in canonical pairs until a conformational change releases it into a dynamic 2 × 2-nt internal loop. NMR experiments reveal that the hairpin loop is dynamic and able to bind oligonucleotides as short as pentamers. A 3D NMR structure of the complex contains 4 and likely 5 bp between pentamer and loop. Moreover, a hairpin sequence was discovered that mimics the equilibrium of the influenza hairpin between its structure in the pseudoknot and upon release of the splice site. Oligonucleotide binding shifts the equilibrium completely to the hairpin secondary structure required for pseudoknot folding. The results suggest this hairpin can be used to screen for compounds that stabilize the pseudoknot and potentially reduce splicing., (© 2022 Kauffmann et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Single-stranded regions of RNA are important for folding of sequences into 3D structures and for design of therapeutics targeting RNA. Prediction of ensembles of 3D structures for single-stranded regions often involves classical mechanical approximations of interactions defined by quantum mechanical calculations on small model systems. Nuclear magnetic resonance (NMR) spectra and molecular dynamics (MD) simulations of short single strands provide tests for how well the approximations model many of the interactions. Here, the NMR spectra for UCUCGU at 2, 15, and 30 °C are compared to simulations with the AMBER force fields, OL3 and ROC-RNA. This is the first such comparison to an oligoribonucleotide containing an internal guanosine nucleotide (G). G is particularly interesting because of its many H-bonding groups, large dipole moment, and proclivity for both syn and anti conformations. Results reveal formation of a G amino to phosphate non-bridging oxygen H-bond. The results also demonstrate dramatic differences in details of the predicted structures. The variations emphasize the dependence of predictions on individual parameters and their balance with the rest of the force field. The NMR data can serve as a benchmark for future force fields.

RNA and DNA are rapidly emerging as targets for therapeutics and as potential frameworks for nanotechnology. Accurate methods for predicting and designing structures and dynamics of nucleic acids would accelerate progress in these and other applications. Suitable approximations for modeling nucleic acids are being developed but require validation against disparate experimental observations. Here, nuclear magnetic resonance spectra for RNA and DNA single strands, CAAU and UCAAUC, are used as benchmarks to test molecular dynamics simulations with AMBER force fields OL3 and ROC-RNA for RNA and BSC1 for DNA. A detailed scheme for making comparisons is also presented. The results reflect recent progress in approximations and reveal remaining challenges.

The influenza A virus (IAV) is a continuous health threat to humans as well as animals due to its recurring epidemics and pandemics. The IAV genome is segmented and the eight negative-sense viral RNAs (vRNAs) are transcribed into positive sense complementary RNAs (cRNAs) and viral messenger RNAs (mRNAs) inside infected host cells. A role for the secondary structure of IAV mRNAs has been hypothesized and debated for many years, but knowledge on the structure mRNAs adopt in vivo is currently missing. Here we solve, for the first time, the in vivo secondary structure of IAV mRNAs in living infected cells. We demonstrate that, compared to the in vitro refolded structure, in vivo IAV mRNAs are less structured but exhibit specific locally stable elements. Moreover, we show that the targeted disruption of these high-confidence structured domains results in an extraordinary attenuation of IAV replicative capacity. Collectively, our data provide the first comprehensive map of the in vivo structural landscape of IAV mRNAs, hence providing the means for the development of new RNA-targeted antivirals., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Geometrical restraints provide key structural information for the determination of biomolecular structures at lower resolution by experimental methods such as crystallography or cryo-electron microscopy. In this work, restraint targets for nucleic acids bases are derived from three different sources and compared: small-molecule crystal structures in the Cambridge Structural Database (CSD), ultrahigh-resolution structures in the Protein Data Bank (PDB) and quantum-mechanical (QM) calculations. The best parameters are those based on CSD structures. After over two decades, the standard library of Parkinson et al. [(1996), Acta Cryst. D52, 57-64] is still valid, but improvements are possible with the use of the current CSD database. The CSD-derived geometry is fully compatible with Watson-Crick base pairs, as comparisons with QM results for isolated and paired bases clearly show that the CSD targets closely correspond to proper base pairing. While the QM results are capable of distinguishing between single and paired bases, their level of accuracy is, on average, nearly two times lower than for the CSD-derived targets when gauged by root-mean-square deviations from ultrahigh-resolution structures in the PDB. Nevertheless, the accuracy of QM results appears sufficient to provide stereochemical targets for synthetic base pairs where no reliable experimental structural information is available. To enable future tests for this approach, QM calculations are provided for isocytosine, isoguanine and the iCiG base pair., (open access.)

RNA thermodynamics play an important role in determining the two- and three-dimensional structures of RNA. Internal loops of the sequence 5'-GMNU/3'-UNMG are relatively unstable thermodynamically. Here, five duplexes with GU-flanked 2 × 2 nucleotide internal loops were structurally investigated to reveal determinants of their instability. The following internal loops were investigated: 5'-GCAU/3'-UACG, 5'-UUCG/3'-GCUU, 5'-GCUU/3'-UUCG, 5'-GUCU/3'-UCUG, and 5'-GCCU/3'-UCCG. Two-dimensional nuclear magnetic resonance spectra indicate the absence of GU wobble base pairing in 5'-GCUU/3'-UUCG, 5'-GUCU/3'-UCUG, and 5'-GCCU/3'-UCCG. The 5'-GCUU/3'-UUCG loop has an unusual conformation of the GU base pairs, in which U's O2 carbonyl forms a bifurcated hydrogen bond with G's amino and imino protons. The internal loop of 5'-GUCU/3'-UCUG displays a shifted configuration in which GC pairs flank a U-U pair and several U's are in fast exchange between positions inside and outside the helix. In contrast, 5'-GCAU/3'-UACG and 5'-UUCG/3'-GCUU both have the expected GU wobble base pairs flanking the internal loop. Evidently, GU base pairs flanking internal loops are more likely to display atypical structures relative to Watson-Crick base pairs flanking internal loops. This appears to be more likely when the G of the GU pair is 5' to the loop. Such unusual structures could serve as recognition elements for biological function and as benchmarks for structure prediction methods.

RNA folding free energy change nearest neighbor parameters are widely used to predict folding stabilities of secondary structures. They were determined by linear regression to datasets of optical melting experiments on small model systems. Traditionally, the optical melting experiments are analyzed assuming a two-state model, i.e. a structure is either complete or denatured. Experimental evidence, however, shows that structures exist in an ensemble of conformations. Partition functions calculated with existing nearest neighbor parameters predict that secondary structures can be partially denatured, which also directly conflicts with the two-state model. Here, a new approach for determining RNA nearest neighbor parameters is presented. Available optical melting data for 34 Watson-Crick helices were fit directly to a partition function model that allows an ensemble of conformations. Fitting parameters were the enthalpy and entropy changes for helix initiation, terminal AU pairs, stacks of Watson-Crick pairs and disordered internal loops. The resulting set of nearest neighbor parameters shows a 38.5% improvement in the sum of residuals in fitting the experimental melting curves compared to the current literature set.

RNA molecules are key players in numerous cellular processes and are characterized by a complex relationship between structure, dynamics, and function. Despite their apparent simplicity, RNA oligonucleotides are very flexible molecules, and understanding their internal dynamics is particularly challenging using experimental data alone. We show how to reconstruct the conformational ensemble of four RNA tetranucleotides by combining atomistic molecular dynamics simulations with nuclear magnetic resonance spectroscopy data. The goal is achieved by reweighting simulations using a maximum entropy/Bayesian approach. In this way, we overcome problems of current simulation methods, as well as in interpreting ensemble- and time-averaged experimental data. We determine the populations of different conformational states by considering several nuclear magnetic resonance parameters and point toward properties that are not captured by state-of-the-art molecular force fields. Although our approach is applied on a set of model systems, it is fully general and may be used to study the conformational dynamics of flexible biomolecules and to detect inaccuracies in molecular dynamics force fields.

The RNA "GAGU" duplex, (5'GAC GAGU GUCA) 2 , contains the internal loop (5'-GAGU-3') 2 , which has two conformations in solution as determined by NMR spectroscopy. The major conformation has a loop structure consisting of trans -Watson-Crick/Hoogsteen GG pairs, A residues stacked on each other, U residues bulged outside the helix, and all sugars with a C2'- endo conformation. This differs markedly from the internal loops, (5'-G AG C-3') 2 , (5'-A AG U-3') 2 , and (5'-UAGG-3') 2 , which all have cis -Watson-Crick/Watson-Crick AG "imino" pairs flanked by cis -Watson-Crick/Watson-Crick canonical pairs resulting in maximal hydrogen bonding. Here, molecular dynamics was used to test whether the Amber force field (ff99 + bsc0 + OL3) approximates molecular interactions well enough to keep stable the unexpected conformation of the GAGU major duplex structure and the NMR structures of the duplexes containing (5'-G AG C-3') 2 , (5'-A AG U-3') 2 , and (5'-U AG G-3') 2 internal loops. One-microsecond simulations were repeated four times for each of the duplexes starting in their NMR conformations. With the exception of (5'-UAGG-3') 2 , equivalent simulations were also run starting with alternative conformations. Results indicate that the Amber force field keeps the NMR conformations of the duplexes stable for at least 1 µsec. They also demonstrate an unexpected minor conformation for the (5'-GAGU-3') 2 loop that is consistent with newly measured NMR spectra of duplexes with natural and modified nucleotides. Thus, unrestrained simulations led to the determination of the previously unknown minor conformation. The stability of the native (5'-GAGU-3') 2 internal loop as compared to other loops can be explained by changes in hydrogen bonding and stacking as the flanking bases are changed., (© 2018 Spasic et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

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.

The database of RNA sequences is exploding, but knowledge of energetics, structures, and dynamics lags behind. All-atom computational methods, such as molecular dynamics, hold promise for closing this gap. New algorithms and faster computers have accelerated progress in improving the reliability and accuracy of predictions. Currently, the methods can facilitate refinement of experimentally determined nuclear magnetic resonance and x-ray structures, but are 'unreliable' for predictions based only on sequence. Much remains to be discovered, however, about the many molecular interactions driving RNA folding and the best way to approximate them quantitatively. The large number of parameters required means that a wide variety of experimental results will be required to benchmark force fields and different approaches. As computational methods become more reliable and accessible, they will be used by an increasing number of biologists, much as x-ray crystallography has expanded. Thus, many fundamental physical principles underlying the computational methods are described. This review presents a summary of the current state of molecular dynamics as applied to RNA. It is designed to be helpful to students, postdoctoral fellows, and faculty who are considering or starting computational studies of RNA. WIREs RNA 2017, 8:e1422. doi: 10.1002/wrna.1422., (© 2017 Wiley Periodicals, Inc.)

The prediction of RNA three-dimensional structure from sequence alone has been a long-standing goal. High-resolution, experimentally determined structures of simple noncanonical pairings and motifs are critical to the development of prediction programs. Here, we present the nuclear magnetic resonance structure of the (5'CCAGAAACGGAUGGA) 2 duplex, which contains an 8 × 8 nucleotide internal loop flanked by three Watson-Crick pairs on each side. The loop is comprised of a central 5'AC/3'CA nearest neighbor flanked by two 3RRs motifs, a known stable motif consisting of three consecutive sheared GA pairs. Hydrogen bonding patterns between base pairs in the loop, the all-atom root-mean-square deviation for the loop, and the deformation index were used to compare the structure to automated predictions by MC-sym, RNA FARFAR, and RNAComposer.

Nowadays different experimental techniques, such as single molecule or relaxation experiments, can provide dynamic properties of biomolecular systems, but the amount of detail obtainable with these methods is often limited in terms of time or spatial resolution. Here we use state-of-the-art computational techniques, namely, atomistic molecular dynamics and Markov state models, to provide insight into the rapid dynamics of short RNA oligonucleotides, to elucidate the kinetics of stacking interactions. Analysis of multiple microsecond-long simulations indicates that the main relaxation modes of such molecules can consist of transitions between alternative folded states, rather than between random coils and native structures. After properly removing structures that are artificially stabilized by known inaccuracies of the current RNA AMBER force field, the kinetic properties predicted are consistent with the time scales of previously reported relaxation experiments.

In this unit, protocols are provided for predicting RNA secondary structure with the user-friendly RNAstructure desktop computer program and the RNAstructure Web server. The minimum free energy structure and a set of suboptimal structures with similar free energies are predicted. Prediction of high-affinity oligonucleotide binding sites to a structured RNA target is also presented. © 2016 by John Wiley & Sons, Inc., (Copyright © 2016 John Wiley & Sons, Inc.)

Influenza A virus (IAV) affects 5%-10% of the world's population every year. Through genome changes, many IAV strains develop resistance to currently available anti-influenza therapeutics. Therefore, there is an urgent need to find new targets for therapeutics against this important human respiratory pathogen. In this study, 2'-O-methyl and locked nucleic acid antisense oligonucleotides (ASOs) were designed to target internal regions of influenza A/California/04/2009 (H1N1) genomic viral RNA segment 8 (vRNA8) based on a base-pairing model of vRNA8. Ten of 14 tested ASOs showed inhibition of viral replication in Madin-Darby canine kidney cells. The best five ASOs were 11-15 nucleotides long and showed inhibition ranging from 5- to 25-fold. In a cell viability assay they showed no cytotoxicity. The same five ASOs also showed no inhibition of influenza B/Brisbane/60/2008 (Victoria lineage), indicating that they are sequence specific for IAV. Moreover, combinations of ASOs slightly improved anti-influenza activity. These studies establish the accessibility of IAV vRNA for ASOs in regions other than the panhandle formed between the 5' and 3' ends. Thus, these regions can provide targets for the development of novel IAV antiviral approaches., Competing Interests: Author Disclosure Statement No competing financial interests exist.

We have solved at 1. 07: Å resolution the X-ray crystal structure of a polyriboadenylic acid (poly(rA)) parallel and continuous double helix. Fifty-nine years ago, double helices of poly(rA) were first proposed to form at acidic pH. Here, we show that 7-mer oligo(rA), i.e. rA7, hybridizes and overlaps in all registers at pH 3.5 to form stacked double helices that span the crystal. Under these conditions, rA7 forms well-ordered crystals, whereas rA6 forms fragile crystalline-like structures, and rA5, rA8 and rA11 fail to crystallize. Our findings support studies from ∼50 years ago: one showed using spectroscopic methods that duplex formation at pH 4.5 largely starts with rA7 and begins to plateau with rA8; another proposed a so-called 'staggered zipper' model in which oligo(rA) strands overlap in multiple registers to extend the helical duplex. While never shown, protonation of adenines at position N1 has been hypothesized to be critical for helix formation. Bond angles in our structure suggest that N1 is protonated on the adenines of every other rAMP-rAMP helix base pair. Our data offer new insights into poly(rA) duplex formation that may be useful in developing a pH sensor., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Influenza A virus is a significant public health threat, but little is understood about the viral RNA structure and function. Current vaccines and therapeutic options to control influenza A virus infections are mostly protein-centric and of limited effectiveness. Here, we report using an ensemble defect approach to design mutations to misfold regions of conserved mRNA structures in influenza A virus segments 7 and 8. Influenza A mutant viruses inhibit pre-mRNA splicing and attenuate viral replication in cell culture, thus providing evidence for functions of the targeted regions. Targeting these influenza A viral RNA regions provides new possibilities for designing vaccines and therapeutics against this important human respiratory pathogen. The results also demonstrate that the ensemble defect approach is an efficient way to test for function of RNA sequences.

Influenza A is a negative sense RNA virus that kills hundreds of thousands of humans each year. Base pairing in RNA is very favorable, but possibilities for RNA secondary structure of the influenza genomic RNA have not been investigated. This work presents the first experimentally-derived exploration of potential secondary structure in an influenza A naked (protein-free) genomic segment. Favorable folding regions are revealed by in vitro chemical structure mapping, thermodynamics, bioinformatics, and binding to isoenergetic microarrays of an entire natural sequence of the 875 nt segment 8 vRNA and of a smaller fragment. Segment 8 has thermodynamically stable and evolutionarily conserved RNA structure and encodes essential viral proteins NEP and NS1. This suggests that vRNA self-folding may generate helixes and loops that are important at one or more stages of the influenza life cycle.

Dynamic programming methods for predicting RNA secondary structure often use thermodynamics and experimental restraints and/or constraints to limit folding space. Chemical mapping results typically restrain certain nucleotides not to be in AU or GC pairs. Two-dimensional nuclear magnetic resonance (NMR) spectra can reveal the order of AU, GC, and GU pairs in double helixes. This chapter describes a program, NMR-assisted prediction of secondary structure and chemical shifts (NAPSS-CS), that constrains possible secondary structures on the basis of the NMR determined order and 5'-3' direction of AU, GC, and GU pairs in helixes. NAPSS-CS minimally requires input of the order of base pairs as determined from nuclear Overhauser effect spectroscopy (NOESY) of imino protons. The program deduces the 5'-3' direction of the base pairs if certain chemical shifts are also input. Secondary structures predicted by the program provide assignments of input chemical shifts to particular nucleotides in the sequence, thus facilitating an important step for determination of the three dimensional structure by NMR. The method is particularly useful for revealing pseudoknots and an example is provided. The method may also allow determination of secondary structures when a sequence folds into two structures that exchange slowly.

Knowledge of RNA structure is necessary to determine structure-function relationships and to facilitate design of potential therapeutics. RNA secondary structure prediction can be improved by applying constraints from nuclear magnetic resonance (NMR) experiments to a dynamic programming algorithm. Imino proton walks from NOESY spectra reveal double-stranded regions. Chemical shifts of protons in GH1, UH3, and UH5 of GU pairs, UH3, UH5, and AH2 of AU pairs, and GH1 of GC pairs were analyzed to identify constraints for the 5' to 3' directionality of base pairs in helices. The 5' to 3' directionality constraints were incorporated into an NMR-assisted prediction of secondary structure (NAPSS-CS) program. When it was tested on 18 structures, including nine pseudoknots, the sensitivity and positive predictive value were improved relative to those of three unrestrained programs. The prediction accuracy for the pseudoknots improved the most. The program also facilitates assignment of chemical shifts to individual nucleotides, a necessary step for determining three-dimensional structure.

Influenza A is a negative-sense RNA virus with an eight-segment genome. Some segments encode more than one polypeptide product, but how the virus accesses alternate internal open reading frames (ORFs) is not completely understood. In segment 2, ribosomal scanning produces two internal ORFs, PB1-F2 and N40. Here, chemical mapping reveals a Mg(2+)-dependent pseudoknot structure that includes the PB1-F2 and N40 start codons. The results suggest that interactions of the ribosome with the pseudoknot may affect the level of translation for PB1-F2 and N40.

Molecular dynamics (MD) simulations for RNA tetramers r(AAAA), r(CAAU), r(GACC), and r(UUUU) are benchmarked against 1 H- 1 H NOESY distances and 3 J scalar couplings to test effects of RNA torsion parametrizations. Four different starting structures were used for r(AAAA), r(CAAU), and r(GACC), while five starting structures were used for r(UUUU). On the basis of X-ray structures, criteria are reported for quantifying stacking. The force fields, AMBER ff99, parmbsc0, parm99χ_Yil, ff10, and parmTor, all predict experimentally unobserved stacks and intercalations, e.g., base 1 stacked between bases 3 and 4, and incorrect χ, ϵ, and sugar pucker populations. The intercalated structures are particularly stable, often lasting several microseconds. Parmbsc0, parm99χ_Yil, and ff10 give similar agreement with NMR, but the best agreement is only 46%. Experimentally unobserved intercalations typically are associated with reduced solvent accessible surface area along with amino and hydroxyl hydrogen bonds to phosphate nonbridging oxygens. Results from an extensive set of MD simulations suggest that recent force field parametrizations improve predictions, but further improvements are necessary to provide reasonable agreement with NMR. In particular, intramolecular stacking and hydrogen bonding interactions may not be well balanced with the TIP3P water model. NMR data and the scoring method presented here provide rigorous benchmarks for future changes in force fields and MD methods.

Influenza A is an RNA virus with a genome of eight negative sense segments. Segment 7 mRNA contains a 3' splice site for alternative splicing to encode the essential M2 protein. On the basis of sequence alignment and chemical mapping experiments, the secondary structure surrounding the 3' splice site has an internal loop, adenine bulge, and hairpin loop when it is in the hairpin conformation that exposes the 3' splice site. We report structural features of a three-dimensional model of the hairpin derived from nuclear magnetic resonance spectra and simulated annealing with restrained molecular dynamics. Additional insight was provided by modeling based on (1)H chemical shifts. The internal loop containing the 3' splice site has a dynamic guanosine and a stable imino (cis Watson-Crick/Watson-Crick) GA pair. The adenine bulge also appears to be dynamic with the A either stacked in the stem or forming a base triple with a Watson-Crick GC pair. The hairpin loop is a GAAA tetraloop closed by an AC pair.

Oligonucleotide microarrays are widely used in various biological studies. In this review, application of oligonucleotide microarrays for identifying binding sites and probing structure of RNAs is described. Deep sequencing allows fast determination of DNA and RNA sequence. High-throughput methods for determination of secondary structures of RNAs have also been developed. Those methods, however, do not reveal binding sites for oligonucleotides. In contrast, microarrays directly determine binding sites while also providing structural insights. Microarray mapping can be used over a wide range of experimental conditions, including temperature, pH, various cations at different concentrations and the presence of other molecules. Moreover, it is possible to make universal microarrays suitable for investigations of many different RNAs, and readout of results is rapid. Thus, microarrays are used to provide insight into oligonucleotide sequences potentially able to interfere with biological function. Better understanding of structure-function relationships of RNA can be facilitated by using microarrays to find RNA regions capable to bind oligonucleotides. That information is extremely important to design optimal sequences for antisense oligonucleotides and siRNA because both bind to single-stranded regions of target RNAs., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Influenza A virus utilizes RNA throughout infection. Little is known, however, about the roles of RNA structures. A previous bioinformatics survey predicted multiple regions of influenza A virus that are likely to generate evolutionarily conserved and stable RNA structures. One predicted conserved structure is in the pre-mRNA coding for essential proteins, M1 and M2. This structure starts 79 nucleotides downstream of the M2 mRNA 5' splice site. Here, a combination of biochemical structural mapping, mutagenesis, and NMR confirms the predicted three-way multibranch structure of this RNA. Imino proton NMR spectra reveal no change in secondary structure when 80 mM KCl is supplemented with 4 mM MgCl2. Optical melting curves in 1 M NaCl and in 100 mM KCl with 10 mM MgCl2 are very similar, with melting temperatures ∼14 °C higher than that for 100 mM KCl alone. These results provide a firm basis for designing experiments and potential therapeutics to test for function in cell culture.

Structured noncoding RNAs underlie fundamental cellular processes, but determining their three-dimensional structures remains challenging. We demonstrate that integrating ¹H NMR chemical shift data with Rosetta de novo modeling can be used to consistently determine high-resolution RNA structures. On a benchmark set of 23 noncanonical RNA motifs, including 11 'blind' targets, chemical-shift Rosetta for RNA (CS-Rosetta-RNA) recovered experimental structures with high accuracy (0.6-2.0 Å all-heavy-atom r.m.s. deviation) in 18 cases.

Thermodynamic data are reported revealing that pseudouridine (Ψ) can stabilize RNA duplexes when replacing U and forming Ψ-A, Ψ-G, Ψ-U and Ψ-C pairs. Stabilization is dependent on type of base pair, position of Ψ within the RNA duplex, and type and orientation of adjacent Watson-Crick pairs. NMR spectra demonstrate that for internal Ψ-A, Ψ-G and Ψ-U pairs, the N3 imino proton is hydrogen bonded to the opposite strand nucleotide and the N1 imino proton may also be hydrogen bonded. CD spectra show that general A-helix structure is preserved, but there is some shifting of peaks and changing of intensities. Ψ has two hydrogen donors (N1 and N3 imino protons) and two hydrogen bond acceptors because the glycosidic bond is C-C rather than C-N as in uridine. This greater structural potential may allow Ψ to behave as a kind of structurally driven universal base because it can enhance stability relative to U when paired with A, G, U or C inside a double helix. These structural and thermodynamic properties may contribute to the biological functions of Ψ.

Locked Nucleic Acids (LNAs) are RNA analogues with an O2'-C4' methylene bridge which locks the sugar into a C3'-endo conformation. This enhances hybridization to DNA and RNA, making LNAs useful in microarrays and potential therapeutics. Here, the LNA, L(CAAU), provides a simplified benchmark for testing the ability of molecular dynamics (MD) to approximate nucleic acid properties. LNA χ torsions and partial charges were parametrized to create AMBER parm99_LNA. The revisions were tested by comparing MD predictions with AMBER parm99 and parm99_LNA against a 200 ms NOESY NMR spectrum of L(CAAU). NMR indicates an A-Form equilibrium ensemble. In 3000 ns simulations starting with an A-form structure, parm99_LNA and parm99 provide 66% and 35% agreement, respectively, with NMR NOE volumes and (3)J-couplings. In simulations of L(CAAU) starting with all χ torsions in a syn conformation, only parm99_LNA is able to repair the structure. This implies methods for parametrizing force fields for nucleic acid mimics can reasonably approximate key interactions and that parm99_LNA will improve reliability of MD studies for systems with LNA. A method for approximating χ population distribution on the basis of base to sugar NOEs is also introduced.

Background: Influenza B and C are single-stranded RNA viruses that cause yearly epidemics and infections. Knowledge of RNA secondary structure generated by influenza B and C will be helpful in further understanding the role of RNA structure in the progression of influenza infection., Findings: All available protein-coding sequences for influenza B and C were analyzed for regions with high potential for functional RNA secondary structure. On the basis of conserved RNA secondary structure with predicted high thermodynamic stability, putative structures were identified that contain splice sites in segment 8 of influenza B and segments 6 and 7 of influenza C. The sequence in segment 6 also contains three unused AUG start codon sites that are sequestered within a hairpin structure., Conclusions: When added to previous studies on influenza A, the results suggest that influenza splicing may share common structural strategies for regulation of splicing. In particular, influenza 3' splice sites are predicted to form secondary structures that can switch conformation to regulate splicing. Thus, these RNA structures present attractive targets for therapeutics aimed at targeting one or the other conformation.

The stability of RNA secondary structure can be predicted using a set of nearest neighbor parameters. These parameters are widely used by algorithms that predict secondary structure. This contribution introduces the UV optical melting experiments that are used to determine the folding stability of short RNA strands. It explains how the nearest neighbor parameters are chosen and how the values are fit to the data. A sample nearest neighbor calculation is provided. The contribution concludes with new methods that use the database of sequences with known structures to determine parameter values.

Interpreting the tsunami of sequence information for RNA would be facilitated by an understanding of all the physical principles determining RNA structure. In principle, a complete understanding would make it computationally possible to find RNA sequences that fold for function and to predict their three-dimensional structure. It would, thus, also facilitate discovery of new principles relating structure to function. This review covers some of the progress in understanding RNA over roughly the preceding 40 years and suggests progress still to be made., (Copyright © 2013 Wiley Periodicals, Inc.)

Influenza A virus is a segmented single-stranded (-)RNA virus that causes substantial annual morbidity and mortality. The transcriptome of influenza A is predicted to have extensive RNA secondary structure. The smallest genome segment, segment 8, encodes two proteins, NS1 and NEP, via alternative splicing. A conserved RNA domain in the intron of segment 8 may be important for regulating production of NS1. Two different multi-branch loop structures have been proposed for this region. A combination of in vitro chemical mapping and isoenergetic microarray techniques demonstrate that the consensus sequence for this region folds into a hairpin conformation. These results provide an alternative folding for this region and a foundation for designing experiments to probe its functional role in the influenza life cycle.

Background: Influenza A virus contributes to seasonal epidemics and pandemics and contains Global Ordered RNA structure (GORS) in the nucleoprotein (NP), non-structural (NS), PB2, and M segments. A related virus, influenza B, is also a major annual public health threat, but unlike influenza A is very selective to human hosts. This study extends the search for GORS to influenza B., Findings: A survey of all available influenza B sequences reveals GORS in the (+) and (-)RNAs of the NP, NS, PB2, and PB1 gene segments. The results are similar to influenza A, except GORS is observed for the M1 segment of influenza A but not for PB1. In general, the folding free energies of human-specific influenza B RNA segments are less stable than allowable by the encoded amino acid sequence. This is consistent with findings in influenza A, where human-specific influenza RNA folds are less stable than avian and swine strains., Conclusions: These results reveal fundamental molecular similarities and differences between Influenza A and B and suggest a rational basis for choosing segments to target with therapeutics and for viral attenuation for live vaccines by altering RNA folding stability.

The sequence dependence of RNA energetics is important for predicting RNA structure. Hairpins with C(n) loops are consistently less stable than hairpins with other loops, which suggests the structure of C(n) regions could be unusual in the "unfolded" state. For example, previous nuclear magnetic resonance (NMR) evidence suggested that polycytidylic acid forms a left-handed helix. In this study, UV melting experiments show that the hairpin formed by r(5'GGACCCCCGUCC) is less stable than r(5'GGACUUUUGUCC). NMR spectra for single-stranded C(4) oligonucleotide, mimicking the unfolded hairpin loop, are consistent with a right-handed A-form-like helix. Comparisons between NMR spectra and molecular dynamics (MD) simulations suggest that recent reparametrizations, parm99χ_YIL and parm99TOR, of the AMBER parm99 force field improve the agreement between structural features for C(4) determined by NMR and predicted by MD. Evidently, the force field revisions to parm99 improve the modeling of RNA energetics and therefore structure.

The RNA duplex, (5'GACGAGUGUCA)(2), has two conformations in equilibrium. The nuclear magnetic resonance solution structure reveals that the major conformation of the loop, 5'GAGU/3'UGAG, is novel and contains two unusual Watson-Crick/Hoogsteen GG pairs with G residues in the syn conformation, two A residues stacked on each other in the center of the helix with inverted sugars, and two bulged-out U residues. The structure provides a benchmark for testing approaches for predicting local RNA structure and a sequence that allows the design of a unique arrangement of functional groups and/or a conformational switch into nucleic acids.

The 3' splice site of the influenza A segment 7 transcript is utilized to produce mRNA for the critical M2 ion-channel protein. In solution a 63 nt fragment that includes this region can adopt two conformations: a pseudoknot and a hairpin. In each conformation, the splice site, a binding site for the SF2/ASF exonic splicing enhancer and a polypyrimidine tract, each exists in a different structural context. The most dramatic difference occurs for the splice site. In the hairpin the splice site is between two residues that are involved in a 2 by 2 nucleotide internal loop. In the pseudoknot, however, these bases are canonically paired within one of the pseudoknotted helices. The conformational switching observed in this region has implications for the regulation of splicing of the segment 7 mRNA. A measure of stability of the structures also shows interesting trends with respect to host specificity: avian strains tend to be the most stable, followed by swine and then human.

Preceding NMR experiments show that the conformation of tandem GA base pairs, an important recurrent non-canonical building block in RNA duplexes, is context dependent. The GA base pairs adopt "sheared" N3(G)-N6(A), N2(G)-N7(A) geometry in the r(CGAG)(2) and r(iGGAiC)(2) contexts while switching to "imino" N1(G)-N1(A), O6(G)-N6(A) geometry in the r(GGAC)(2) and r(iCGAiG)(2) contexts (iC and iG stand for isocytosine and isoguanine, respectively). As base stacking is likely to be one of the key sources of the context dependence of the conformation of GA base pairs, we calculated base stacking energies in duplexes containing such base pairs, to see if this dependence can be predicted by stacking energy calculations. When investigating the context dependence of the GA geometry two different conformations of the same duplex were compared (imino vs. sheared). The geometries were generated via explicit solvent MD simulations of the respective RNA duplexes, while the subsequent QM energy calculations focused on base stacking interactions of the four internal base pairs. Geometrical relaxation of nucleobase atoms prior to the stacking energy computations has a non-negligible effect on the results. The stacking energies were derived at the DFT-D/6-311++G(3df,3pd) level. We show a rather good correspondence between the intrinsic gas-phase stacking energies and the NMR-determined GA geometries. The conformation with more favorable gas-phase stacking is in most cases the one observed in experiments. This correlation is not improved when including solvent effects via the COSMO method. On the other side, the stacking calculations do not predict the relative thermodynamic stability of duplex formation for different sequences.

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.

All-atom force fields are important for predicting thermodynamic, structural, and dynamic properties of RNA. In this paper, results are reported for thermodynamic integration calculations of free energy differences of duplex formation when CG pairs in the RNA duplexes r(CCGG)(2), r(GGCC)(2), r(GCGC)(2), and r(CGCG)(2) are replaced by isocytidine-isoguanosine (iCiG) pairs. Agreement with experiment was improved when ε/ζ, α/γ, β, and χ torsional parameters in the AMBER99 force field were revised on the basis of quantum mechanical calculations. The revised force field, AMBER99TOR, brings free energy difference predictions to within 1.3, 1.4, 2.3, and 2.6 kcal/mol at 300 K, respectively, compared to experimental results for the thermodynamic cycles of CCGG → iCiCiGiG, GGCC → iGiGiCiC, GCGC → iGiCiGiC, and CGCG → iCiGiCiG. In contrast, unmodified AMBER99 predictions for GGCC → iGiGiCiC and GCGC → iGiCiGiC differ from experiment by 11.7 and 12.6 kcal/mol, respectively. In order to test the dynamic stability of the above duplexes with AMBER99TOR, four individual 50 ns molecular dynamics (MD) simulations in explicit solvent were run. All except r(CCGG)(2) retained A-form conformation for ≥82% of the time. This is consistent with NMR spectra of r(iGiGiCiC)(2), which reveal an A-form conformation. In MD simulations, r(CCGG)(2) retained A-form conformation 52% of the time, suggesting that its terminal base pairs may fray. The results indicate that revised backbone parameters improve predictions of RNA properties and that comparisons to measured sequence dependent thermodynamics provide useful benchmarks for testing force fields and computational methods.

The 3' splice site of influenza A segment 7 is used to produce mRNA for the M2 ion-channel protein, which is critical to the formation of viable influenza virions. Native gel analysis, enzymatic/chemical structure probing, and oligonucleotide binding studies of a 63 nt fragment, containing the 3' splice site, key residues of an SF2/ASF splicing factor binding site, and a polypyrimidine tract, provide evidence for an equilibrium between pseudoknot and hairpin structures. This equilibrium is sensitive to multivalent cations, and can be forced towards the pseudoknot by addition of 5 mM cobalt hexammine. In the two conformations, the splice site and other functional elements exist in very different structural environments. In particular, the splice site is sequestered in the middle of a double helix in the pseudoknot conformation, while in the hairpin it resides in a two-by-two nucleotide internal loop. The results suggest that segment 7 mRNA splicing can be controlled by a conformational switch that exposes or hides the splice site.

Influenza A is a significant public health threat, partially because of its capacity to readily exchange gene segments between different host species to form novel pandemic strains. An understanding of the fundamental factors providing species barriers between different influenza hosts would facilitate identification of strains capable of leading to pandemic outbreaks and could also inform vaccine development. Here, we describe the difference in predicted RNA secondary structure stability that exists between avian, swine and human coding regions. The results predict that global ordered RNA structure exists in influenza A segments 1, 5, 7 and 8, and that ranges of free energies for secondary structure formation differ between host strains. The predicted free energy distributions for strains from avian, swine, and human species suggest criteria for segment reassortment and strains that might be ideal candidates for viral attenuation and vaccine development.

Conformational changes are important in RNA for binding and catalysis and understanding these changes is important for understanding how RNA functions. Computational techniques using all-atom molecular models can be used to characterize conformational changes in RNA. These techniques are applied to an RNA conformational change involving a single base pair within a nine base pair RNA duplex. The Adenine-Adenine (AA) non-canonical pair in the sequence 5'GGUGAAGGCU3' paired with 3'PCCGAAGCCG5', where P is Purine, undergoes conformational exchange between two conformations on the timescale of tens of microseconds, as demonstrated in a previous NMR solution structure [Chen, G., et al., Biochemistry, 2006. 45: 6889-903]. The more populated, major, conformation was estimated to be 0.5 to 1.3 kcal/mol more stable at 30 °C than the less populated, minor, conformation. Both conformations are trans-Hoogsteen/sugar edge pairs, where the interacting edges on the adenines change with the conformational change. Targeted Molecular Dynamics (TMD) and Nudged Elastic Band (NEB) were used to model the pathway between the major and minor conformations using the AMBER software package. The adenines were predicted to change conformation via intermediates in which they are stacked as opposed to hydrogen-bonded. The predicted pathways can be described by an improper dihedral angle reaction coordinate. Umbrella sampling along the reaction coordinate was performed to model the free energy profile for the conformational change using a total of 1800 ns of sampling. Although the barrier height between the major and minor conformations was reasonable, the free energy difference between the major and minor conformations was the opposite of that expected based on the NMR experiments. Variations in the force field applied did not improve the misrepresentation of the free energies of the major and minor conformations. As an alternative, the Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) approximation was applied to predict free energy differences between the two conformations using a total of 800 ns of sampling. MM-PBSA also incorrectly predicted the major conformation to be higher in free energy than the minor conformation.

Analysis of the R2 retrotransposons from multiple silkmoth and fruitfly species have revealed three segments that contain conserved RNA secondary structures. These conserved structures play important roles in the propagation of the R2 element, including R2 RNA processing and transposon integration into the host genome as well as a likely role in translation. Two of the structured regions comprise protein binding sites: one is located in the 3' UTR and the other is in the 5' UTR close to the putative start of the R2 open reading frame (ORF). The 3' structure was deduced from chemical mapping and sequence comparison. The 5' structure was determined using a combination of chemical mapping, oligonucleotide binding, NMR and sequence analysis and contains an unusual pseudoknot structure. The third structure occurs at the 5' end of the R2 RNA and is responsible for self-cleavage of the 5' end of the element from a 28S ribosomal RNA co-transcript. A structure for this fragment was proposed based on motif searching and sequence comparison. There is remarkable similarity in sequence and structure to the hepatitis delta virus (HDV) ribozyme. Seed alignments for the 5' structure and the R2 ribozyme, containing representative sequences and consensus structures, have been submitted to the Rfam database.

The NMR solution structure is reported of a duplex, 5'GUGAAGCCCGU/3'UCACAGGAGGC, containing a 4 × 4 nucleotide internal loop from an R2 retrotransposon RNA. The loop contains three sheared purine-purine pairs and reveals a structural element found in other RNAs, which we refer to as the 3RRs motif. Optical melting measurements of the thermodynamics of the duplex indicate that the internal loop is 1.6 kcal/mol more stable at 37°C than predicted. The results identify the 3RRs motif as a common structural element that can facilitate prediction of 3D structure. Known examples include internal loops having the pairings: 5'GAA/3'AGG, 5'GAG/3'AGG, 5'GAA/3'AAG, and 5'AAG/3'AGG. The structural information is compared with predictions made with the MC-Sym program.

Accurately modeling unpaired regions of RNA is important for predicting structure, dynamics, and thermodynamics of folded RNA. Comparisons between NMR data and molecular dynamics simulations provide a test of force fields used for modeling. Here, NMR spectroscopy, including NOESY, (1)H-(31)P HETCOR, DQF-COSY, and TOCSY, was used to determine conformational preferences for single-stranded GACC RNA. The spectra are consistent with a conformational ensemble containing major and minor A-form-like structures. In a series of 50 ns molecular dynamics (MD) simulations with the AMBER99 force field in explicit solvent, initial A-form-like structures rapidly evolve to disordered conformations. A set of 50 ns simulations with revised χ torsions (AMBER99χ force field) gives two primary conformations, consistent with the NMR spectra. A single 1.9 μs MD simulation with the AMBER99χ force field showed that the major and minor conformations are retained for almost 68% of the time in the first 700 ns, with multiple transformations from A-form to non-A-form conformations. For the rest of the simulation, random-coil structures and a stable non-A-form conformation inconsistent with NMR spectra were seen. Evidently, the AMBER99χ force field improves structural predictions for single-stranded GACC RNA compared to the AMBER99 force field, but further force field improvements are needed., (© 2011 American Chemical Society)