A reparameterization of the torsional parameters for the glycosidic dihedral angle, χ, for the AMBER99 force field in RNA nucleosides is used to provide a modified force field, AMBER99χ. Molecular dynamics simulations of cytidine, uridine, adenosine, and guanosine in aqueous solution using the AMBER99 and AMBER99χ force fields are compared with NMR results. For each nucleoside and force field, 10 individual molecular dynamics simulations of 30 ns each were run. For cytidine with AMBER99χ force field, each molecular dynamics simulation time was extended to 120 ns for convergence purposes. Nuclear magnetic resonance (NMR) spectroscopy, including one-dimensional (1D) 1H, steady-state 1D 1H nuclear Overhauser effect (NOE), and transient 1D 1H NOE, was used to determine the sugar puckering and preferred base orientation with respect to the ribose of cytidine and uridine. The AMBER99 force field overestimates the population of syn conformations of the base orientation and of C2′-endo sugar puckering of the pyrimidines, while the AMBER99χ force field’s predictions are more consistent with NMR results. Moreover, the AMBER99 force field prefers high anti conformations with glycosidic dihedral angles around 310° for the base orientation of purines. The AMBER99χ force field prefers anti conformations around 185°, which is more consistent with the quantum mechanical calculations and known 3D structures of folded ribonucleic acids (RNAs). Evidently, the AMBER99χ force field predicts the structural characteristics of ribonucleosides better than the AMBER99 force field and should improve structural and thermodynamic predictions of RNA structures.

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, 1H–31P 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.

The sequence dependence of RNA energetics is important for predicting RNA structure. Hairpins with Cn loops are consistently less stable than hairpins with other loops, which suggests the structure of Cn 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 C4 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 C4 determined by NMR and predicted by MD. Evidently, the force field revisions to parm99 improve the modeling of RNA energetics and therefore structure.

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.

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