Nuclear magnetic resonance reveals a two hairpin equilibrium near the 3'-splice site of influenza A segment 7 mRNA that can be shifted by oligonucleotides.

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.

Distributed processing of movement signaling.

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.

Molecular dynamics correctly models the unusual major conformation of the GAGU RNA internal loop and with NMR reveals an unusual minor conformation.

The RNA "GAGU" duplex, (5'GACGAGUGUCA)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'-GAGC-3')2, (5'-AAGU-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'-GAGC-3')2, (5'-AAGU-3')2, and (5'-UAGG-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.

Nuclear Magnetic Resonance Reveals That GU Base Pairs Flanking Internal Loops Can Adopt Diverse Structures.

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.

Stiffness as a control factor for object manipulation.

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.

Dynamic stacking of an expected branch point adenosine in duplexes containing pseudouridine-modified or unmodified U2 snRNA sites.

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.

Synthetic, Structural, and RNA Binding Studies on 2-Aminopyridine-Modified Triplex-Forming Peptide Nucleic Acids.

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 pKa 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.

Synthesis and RNA-Binding Properties of Extended Nucleobases for Triplex-Forming Peptide Nucleic Acids.

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.

Amide linkages mimic phosphates in RNA interactions with proteins and are well tolerated in the guide strand of short interfering RNAs.

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.

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.

Nuclear Magnetic Resonance Structure of an 8 × 8 Nucleotide RNA Internal Loop Flanked on Each Side by Three Watson-Crick Pairs and Comparison to Three-Dimensional Predictions.

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.

2-Methoxypyridine as a Thymidine Mimic in Watson-Crick Base Pairs of DNA and PNA: Synthesis, Thermal Stability, and NMR Structural Studies.

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.

Structure determination of noncanonical RNA motifs guided by ¹H NMR chemical shifts.

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.

Motor cortical correlates of arm resting in the context of a reaching task and implications for prosthetic control.

Prosthetic devices are being developed to restore movement for motor-impaired individuals. A robotic arm can be controlled based on models that relate motor-cortical ensemble activity to kinematic parameters. The models are typically built and validated on data from structured trial periods during which a subject actively performs specific movements, but real-world prosthetic devices will need to operate correctly during rest periods as well. To develop a model of motor cortical modulation during rest, we trained monkeys (Macaca mulatta) to perform a reaching task with their own arm while recording motor-cortical single-unit activity. When a monkey spontaneously put its arm down to rest between trials, our traditional movement decoder produced a nonzero velocity prediction, which would cause undesired motion when applied to a prosthetic arm. During these rest periods, a marked shift was found in individual units' tuning functions. The activity pattern of the whole population during rest (Idle state) was highly distinct from that during reaching movements (Active state), allowing us to predict arm resting from instantaneous firing rates with 98% accuracy using a simple classifier. By cascading this state classifier and the movement decoder, we were able to predict zero velocity correctly, which would avoid undesired motion in a prosthetic application. Interestingly, firing rates during hold periods followed the Active pattern even though hold kinematics were similar to those during rest with near-zero velocity. These findings expand our concept of motor-cortical function by showing that population activity reflects behavioral context in addition to the direct parameters of the movement itself.

Structural features of a 3' splice site in influenza a.

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.

Nuclear Magnetic Resonance-Assisted Prediction of Secondary Structure for RNA: Incorporation of Direction-Dependent Chemical Shift Constraints.

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.

Secondary structure of a conserved domain in an intron of influenza A M1 mRNA.

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.

Synthesis, biophysical studies and RNA interference activity of RNA having three consecutive amide linkages.

RNA sequences having up to three consecutive internal amide linkages were synthesized and studied using UV and NMR spectroscopy. The amide modifications did not interfere with normal base-pairing and A-type RNA conformation. Three consecutive amides were well tolerated in the passenger strand of siRNA and caused little change in RNAi activity.

The nuclear magnetic resonance of CCCC RNA reveals a right-handed helix, and revised parameters for AMBER force field torsions improve structural predictions from molecular dynamics.

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.

NMR structure of a 4 x 4 nucleotide RNA internal loop from an R2 retrotransposon: identification of a three purine-purine sheared pair motif and comparison to MC-SYM predictions.

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.