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Cyclometalated iridium(III) complexes are increasingly being developed for application in G-quadruplex (GQ) nucleic acid biosensors. We monitored the interactions of a GQ structure with an iridium(III) complex by nuclear magnetic resonance (NMR) titrations and subsequently compared the binding site inferred from NMR with binding positions modeled by molecular docking and molecular dynamics simulations. When titrated into a solution of G-quadruplex Pu22T , compound 1(PF 6 ), [Ir(ppy) 2 (pizp)](PF 6 ), where ppy is 2-phenylpyridine and pizp is 2-phenylimidazole[4,5f][1,10]phenanthroline, had the greatest impact on the hydrogen chemical shifts of G5, G8, G9, G13, and G17 residues of Pu22T , indicating end-stacking at the 5' tetrad. In blind cross-docking studies with Autodock 4, end-stacking at the 5' tetrad was found as the lowest energy binding position. AMBER molecular dynamics simulations resulted in a refined binding position at the 5' tetrad with improved pi stacking. For this model system, Pu22T-1 , molecular docking and molecular dynamics simulations are tools that are able to predict the experimentally determined binding position.

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.)

There is increasing interest in the roles of covalently modified nucleotides in RNA. There has been, however, an inability to account for modifications in secondary structure prediction because of a lack of software and thermodynamic parameters. We report the solution for these issues for N 6 -methyladenosine (m 6 A), allowing secondary structure prediction for an alphabet of A, C, G, U, and m 6 A. The RNAstructure software now works with user-defined nucleotide alphabets of any size. We also report a set of nearest neighbor parameters for helices and loops containing m 6 A, using experiments. Interestingly, N 6 -methylation decreases folding stability for adenosines in the middle of a helix, has little effect on folding stability for adenosines at the ends of helices, and increases folding stability for unpaired adenosines stacked on a helix. We demonstrate predictions for an N 6 -methylation-activated protein recognition site from MALAT1 and human transcriptome-wide effects of N 6 -methylation on the probability of adenosine being buried in a helix., (© 2022. The Author(s).)

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.

Basic neurophysiological research with monkeys has shown how neurons in the motor cortex have firing rates tuned to movement direction. This original finding would have been difficult to uncover without the use of a behaving primate paradigm in which subjects grasped a handle and moved purposefully to targets in different directions. Subsequent research, again using behaving primate models, extended these findings to continuous drawing and to arm and hand movements encompassing action across multiple joints. This research also led to robust extraction algorithms in which information from neuronal populations is used to decode movement intent. The ability to decode intended movement provided the foundation for neural prosthetics in which brain-controlled interfaces are used by paralyzed human subjects to control computer cursors or high-performance motorized prosthetic arms and hands. This translation of neurophysiological laboratory findings to therapy is a clear example of why using nonhuman primates for basic research is valuable for advancing treatment of neurological disorders. Recent research emphasizes the distribution of intention signaling through neuronal populations and shows how many movement parameters are encoded simultaneously. In addition to direction and velocity, the arm's impedance has now been found to be encoded as well. The ability to decode motion and force from neural populations will make it possible to extend neural prosthetic paradigms to precise interaction with objects, enabling paralyzed individuals to perform many tasks of daily living.

Triple-helix formation, using Hoogsteen hydrogen bonding of triplex-forming oligonucleotides, represents an attractive method for sequence-specific recognition of double-stranded nucleic acids. However, practical applications using triple-helix-forming oligonucleotides and their analogues are limited to long homopurine sequences. The key problem for recognition of pyrimidines is that they present only one hydrogen-bond acceptor or donor group in the major groove. Herein, we report our first attempt to overcome this problem by using peptide nucleic acids (PNAs) modified with extended nucleobases that form three hydrogen bonds along the entire Hoogsteen edge of the Watson-Crick base pair. New nucleobase triples (five) were designed, and their hydrogen bonding feasibility was confirmed by ab initio calculations. PNA monomers carrying the modified nucleobases were synthesized and incorporated in short model PNA sequences. Isothermal titration calorimetry showed that these nucleobases had a modest binding affinity for their double-stranded RNA (dsRNA) targets. Finally, molecular modeling of the modified triples in PNA-dsRNA helix suggested that the modest binding affinity was caused by subtle structural deviations from ideal hydrogen-bonding arrangements or disrupted π-stacking of the extended nucleobase scaffolds.

During manipulation, force is exerted with the expectation that an object will move in an intended manner. This prediction is a learned coordination between force and movement. Mechanically, impedance is a way to describe this coordination, and object interaction could be anticipated by setting impedance before the hand moves the object. This strategy would be especially important at the end of a reach, because feedback is ineffective for rapid force changes. Since mechanical impedance is not subject to the time delays of feedback, it can, if set properly, produce the desired motion on impact. We examined this possibility by instructing subjects to move a handle to a specific target position along a track. The handle was locked in place until the subject exerted enough force to cross a threshold; the handle was then released abruptly to move along the track. We hypothesized that this ballistic release task would encourage subjects to modify impedance in anticipation of the upcoming movement and found that one component of impedance, stiffness, varied in a way that matched the behavioral demands of the task. Analysis suggests that this stiffness was set before the handle moved and governed the subsequent motion. We also found separate components of muscle activity that corresponded to stiffness and to changes in force. Our results show that subjects used a robust and efficient strategy to coordinate force and displacement by modulating muscle activity in a way that was behaviorally relevant in the task. NEW & NOTEWORTHY The arm can behave like a spring, and this mechanical behavior can be advantageous in situations requiring rapid changes in force and/or displacement. Selection of a proper "virtual" spring before the occurrence of a rapid transient could facilitate a desired responsive movement. We show that these spring-like arm mechanics, set in anticipation of an instantaneous force change, function as an efficient strategy to control movement when feedback is ineffective.

The pre-mRNA branch point sequence (BPS) anneals with a pseudouridine-modified region of the U2 small nuclear (sn)RNA, and offers a 2' hydroxyl group of a bulged adenosine as the nucleophile for the first catalytic step of pre-mRNA splicing. To increase our structural understanding of branch site selection, we characterized a duplex containing a BPS sequence that is common among multicellular eukaryotes (5'-UACUGAC-3') and the complementary U2 snRNA site using NMR. A major conformation of the expected branch site adenosine stacked within the duplex and paired with the conserved pseudouridine of the U2 snRNA strand. In contrast, the guanosine preceding the branch site appeared flexible and had weak contacts with the surrounding nucleotides. Pseudouridine-modified and unmodified U2 snRNA-BPS-containing duplexes remained structurally similar. These results highlight the importance of auxiliary factors to achieve the active bulged conformation of the branch site nucleophile for the first step of pre-mRNA splicing., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The development of new RNA-binding ligands is attracting increasing interest in fundamental science and the pharmaceutical industry. The goal of this study was to improve the RNA binding properties of triplex-forming peptide nucleic acids (PNAs) by further increasing the pK a of 2-aminopyridine (M). Protonation of M was the key for enabling triplex formation at physiological pH in earlier studies. Substitution on M by an electron-donating 4-methoxy substituent resulted in slight destabilization of the PNA-dsRNA triplex, contrary to the expected stabilization due to more favorable protonation. To explain this unexpected result, the first NMR structural studies were performed on an M-modified PNA-dsRNA triplex which, combined with computational modeling identified unfavorable steric and electrostatic repulsion between the 4-methoxy group of M and the oxygen of the carbonyl group connecting the adjacent nucleobase to PNA backbone. The structural studies also provided insights into hydrogen-bonding interactions that might be responsible for the high affinity and unusual RNA-binding preference of PNAs., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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 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.

Potential in vivo applications of RNA interference (RNAi) require suppression of various off-target activities. Herein, we report that replacement of a single phosphate linkage between the first and second nucleosides of the passenger strand with an amide linkage almost completely abolished its undesired activity and restored the desired activity of guide strands that had been compromised by unfavorable amide modifications. Molecular modeling suggested that the observed effect was most likely due to suppressed loading of the amide-modified strand into Ago2 caused by inability of amide to adopt the conformation required for the backbone twist that docks the first nucleotide of the guide strand in the MID domain of Ago2. Eliminating off-target activity of the passenger strand will be important for improving therapeutic potential of RNAi.

The development of nucleic acid base-pair analogues that use new modes of molecular recognition is important both for fundamental research and practical applications. The goal of this study was to evaluate 2-methoxypyridine as a cationic thymidine mimic in the A-T base pair. The hypothesis was that including protonation in the Watson-Crick base pairing scheme would enhance the thermal stability of the DNA double helix without compromising the sequence selectivity. DNA and peptide nucleic acid (PNA) sequences containing the new 2-methoxypyridine nucleobase (P) were synthesized and studied by using UV thermal melting and NMR spectroscopy. Introduction of P nucleobase caused a loss of thermal stability of ≈10 °C in DNA-DNA duplexes and ≈20 °C in PNA-DNA duplexes over a range of mildly acidic to neutral pH. Despite the decrease in thermal stability, the NMR structural studies showed that P-A formed the expected protonated base pair at pH 4.3. Our study demonstrates the feasibility of cationic unnatural base pairs; however, future optimization of such analogues will be required., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

While the use of RNA interference (RNAi) in molecular biology and functional genomics is a well-established technology, in vivo applications of synthetic short interfering RNAs (siRNAs) require chemical modifications. We recently found that amides as non-ionic replacements for phosphodiesters may be useful modifications for optimization of siRNAs. Herein, we report a comprehensive study of systematic replacement of a single phosphate with an amide linkage throughout the guide strand of siRNAs. The results show that amides are surprisingly well tolerated in the seed and central regions of the guide strand and increase the silencing activity when placed between nucleosides 10 and 12, at the catalytic site of Argonaute. A potential explanation is provided by the first crystal structure of an amide-modified RNA-DNA with Bacillus halodurans RNase H1. The structure reveals how small changes in both RNA and protein conformation allow the amide to establish hydrogen bonding interactions with the protein. Molecular dynamics simulations suggest that these alternative binding modes may compensate for interactions lost due to the absence of a phosphodiester moiety. Our results suggest that an amide can mimic important hydrogen bonding interactions with proteins required for RNAi activity and may be a promising modification for optimization of biological properties of siRNAs., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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.

We report a disagreement between the predicted structures of the dTpT thymine pair (thymidylyl(3' → 5')thymidine) using nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations using the AMBER ff14SB and ff14 + ε/ζOL1 + χOL4 force fields for DNA. The NMR structure was determined using NOE couplings to thymine's H6 and J(HH) couplings between sugar protons. The MD simulation used replica exchange methods to produce converged statistics in a 500 ns trajectory. NMR data indicate that both thymine nucleotides in the pair display an anti conformation of B-DNA, while the MD simulations predict a structure in which the 5'-thymine is flipped into a syn conformation and the 3'-thymine is in an anti conformation. The syn conformation of the 5'-thymine predicted by MD appears by a ∼ 180-deg flip of the glycosidic angle in comparison to the B-form anti structure. Differences in the distortion of the sugar pucker between 5'-thymine and 3'-thymine further highlighted the surprisingly different conformation of the 5'- and 3'-ends. While both MD and NMR indicate the deoxyribose sugars to be primarily in the 2'-endo conformation typical of B-form DNA, the MD simulations predict a more twisted conformation (2'-endo/1'-exo) for the 5'-sugar and significant flexibility of C3' of the 3'-sugar. We conclude that the current AMBER force field does not accurately predict the conformation of single-stranded thymine, in agreement with previous work investigating single-stranded DNA.