Understanding the function of complex RNA molecules depends critically on understanding their structure. However, creating three-dimensional (3D) structural models of RNA remains a significant challenge. We present a protocol (the nucleic acid simulation tool [NAST]) for RNA modeling that uses an RNA-specific knowledge-based potential in a coarse-grained molecular dynamics engine to generate plausible 3D structures. We demonstrate NAST's capabilities by using only secondary structure and tertiary contact predictions to generate, cluster, and rank structures. Representative structures in the best ranking clusters averaged 8.0 6 0.3 Å and 16.3 6 1.0 Å RMSD for the yeast phenylalanine tRNA and the P4-P6 domain of the Tetrahymena thermophila group I intron, respectively. The coarse-grained resolution allows us to model large molecules such as the 158-residue P4-P6 or the 388-residue T. thermophila group I intron. One advantage of NAST is the ability to rank clusters of structurally similar decoys based on their compatibility with experimental data. We successfully used ideal small-angle X-ray scattering data and both ideal and experimental solvent accessibility data to select the best cluster of structures for both tRNA and P4-P6. Finally, we used NAST to build in missing loops in the crystal structures of the Azoarcus and Twort ribozymes, and to incorporate crystallographic data into the Michel-Westhof model of the T. thermophila group I intron, creating an integrated model of the entire molecule. Our software package is freely available at https://simtk.org/home/nast.

The biological behaviors of ribozymes, riboswitches, and numerous other functional RNA molecules are critically dependent on their tertiary folding and their ability to sample multiple functional states. The conformational heterogeneity and partially folded nature of most of these states has rendered their characterization by high-resolution structural approaches difficult or even intractable. Here we introduce a method to rapidly infer the tertiary helical arrangements of large RNA molecules in their native and non-native solution states. Multiplexed hydroxyl radical (⅐OH) cleavage analysis (MOHCA) enables the high-throughput detection of numerous pairs of contacting residues via random incorporation of radical cleavage agents followed by two-dimensional gel electrophoresis. We validated this technology by recapitulating the unfolded and native states of a well studied model RNA, the P4 -P6 domain of the Tetrahymena ribozyme, at subhelical resolution. We then applied MOHCA to a recently discovered third state of the P4 -P6 RNA that is stabilized by high concentrations of monovalent salt and whose partial order precludes conventional techniques for structure determination. The three-dimensional portrait of a compact, non-native RNA state reveals a well ordered subset of native tertiary contacts, in contrast to the dynamic but otherwise similar molten globule states of proteins. With its applicability to nearly any solution state, we expect MOHCA to be a powerful tool for illuminating the many functional structures of large RNA molecules and RNA/protein complexes.hydroxyl radical ͉ molten globule ͉ Tetrahymena ribozyme ͉ two-dimensional gel T he discoveries of catalytic RNAs, silencing RNAs, riboswitches, and a panoply of functional RNA molecules have sweeping implications for our views of evolution from an early ''RNA World'' and for the potential of structured RNAs to act in roles beyond the simple transmission of information laid out in the Central Dogma of Molecular Biology (1). The functions of these RNAs in primitive and modern life are being elucidated at an explosive pace. Nevertheless, a deep understanding of these fundamental biopolymers and their biological roles requires structural portraits of their functional states, and, in this respect, progress has been slow.Our understanding of RNA structure has greatly lagged behind that of protein structure: compared with nearly 40,000 protein structures in the Protein Data Bank, there are currently Ͻ1,000 experimentally determined RNA structures, most of which are small fragments (2). High-resolution approaches using NMR spectroscopy (NMR) and x-ray crystallography have the potential to describe RNA structure at the atomic level, but have been considerably hampered by numerous factors, including limited chemical shift dispersion, the large sizes of structured RNAs, and the poor behavior of RNA at high concentrations.Further enriching and complicating the modeling of RNA behavior is the seemingly pervasive tendency of RNA to form alternative secondary and tert...

We distinguish the contribution of the electrostatic environment, initial conformational ensemble, and macromolecular stability on the folding mechanism of a large RNA using a combination of time-resolved ''Fast Fenton'' hydroxyl radical footprinting and exhaustive kinetic modeling. This integrated approach allows us to define the folding landscape of the L-21 Tetrahymena thermophila group I intron structurally and kinetically from its earliest steps with unprecedented accuracy. Distinct parallel pathways leading the RNA to its native form upon its Mg 2؉ -induced folding are observed. The structures of the intermediates populating the pathways are not affected by variation of the concentration and type of background monovalent ions (electrostatic environment) but are altered by a mutation that destabilizes one domain of the ribozyme. Experiments starting from different conformational ensembles but folding under identical conditions show that whereas the electrostatic environment modulates molecular flux through different pathways, the initial conformational ensemble determines the partitioning of the flux. This study showcases a robust approach for the development of kinetic models from collections of local structural probes.pathway ͉ ribozyme ͉ salt ͉ assembly ͉ topology M ost of the studied RNA folding pathways are populated with multiple kinetic intermediates (1-9) whose prevalence suggests their important role in the regulation of RNA function (10-14). The structural properties of folding intermediates and the factors that influence their population and lifetime are incompletely understood. Among the known factors are intermediate stability, electrostatic environment, nativestate topology, and the initial conformational ensemble (15). RNA's negative charge results in counterion concentration strongly influencing the folding landscape and the relative stability of kinetic intermediates (16)(17)(18)(19)(20). The native-state topology of structured RNA consisting of Watson-Crick duplexes connected by semiflexible junctions and specific long-range tertiary contacts constrains the biopolymer's conformational degrees of freedom (21-23). The ensemble of macromolecular conformations present at the initial state directs the RNA commitment to a particular folding pathway (16): a compact ensemble with significant tertiary structure may be more favorable to conformational search but can also yield barriers that impede resolution of misfolded molecules. The distinct roles of each of these influences in the determination of the structure, population, and lifetime of RNA folding intermediates have yet to be established.In this study we investigate the interplay among electrostatics, initial conformation, and macromolecular stability in RNA folding. We analyze the solution condition dependence of the intermediate structures and molecular flux through them on the Mg 2ϩ -mediated folding of the L-21 Tetrahymena thermophila group I intron and a mutant of reduced stability by varying the background monovalent cation type and co...

Motivation: The recent development of methods for modeling RNA 3D structures using coarse-grain approaches creates a need to bridge low- and high-resolution modeling methods. Although they contain topological information, coarse-grain models lack atomic detail, which limits their utility for some applications.Results: We have developed a method for adding full atomic detail to coarse-grain models of RNA 3D structures. Our method [Coarse to Atomic (C2A)] uses geometries observed in known RNA crystal structures. Our method rebuilds full atomic detail from ideal coarse-grain backbones taken from crystal structures to within 1.87–3.31 Å RMSD of the full atomic crystal structure. When starting from coarse-grain models generated by the modeling tool NAST, our method builds full atomic structures that are within 1.00 Å RMSD of the starting structure. The resulting full atomic structures can be used as starting points for higher resolution modeling, thus bridging high- and low-resolution approaches to modeling RNA 3D structure.Availability: Code for the C2A method, as well as the examples discussed in this article, are freely available at www.simtk.org/home/c2a.Contact: russ.altman@stanford.edu

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