A universal approach for determining three-dimensional RNA structures

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Summary RNA molecules participate in the most fundamental cellular processes implicated in human disease. Many RNAs contain structured modules that make critical contributions to RNA functions and represent attractive drug targets, especially for diseases without a cure and associated with “undruggable” proteins. Knowledge of the three-dimensional structures of these RNAs would help to understand the mechanism of the RNA function and could greatly accelerate drug discovery efforts. However, traditional methods for RNA structure determination, X-ray crystallography and NMR spectroscopy, are laborious and have serious technical limitations. Single-particle cryogenic electron microscopy (cryo-EM) has many advantages over crystallography and NMR but is applicable only for large molecules or macromolecular assemblies and cannot be used for the majority of natural RNAs because of their insufficient size. This proposal is focused on developing a novel approach for preparing cryo-EM samples of RNA that circumvents the size restrictions, omits the RNA purification and RNA refolding steps, and allows facile cryo-EM data processing and structure solution. The proposed proof-of-concept study combines three specific aims. Specific Aim 1 is devoted to the development of the novel biochemical approach for preparing uniform RNA species by in vitro transcription. Specific Aim 2 will use computational modelling, biochemical assays, and single-particle cryo-EM experimentation to develop a methodology for preparing cryo-EM samples compatible with the structure solution of small- and medium- sized RNAs. Aim 3 will validate the methodology using model RNA molecules and conventional single particle cryo-EM structure solution pipeline. The proposal integrates computational methods, biochemical assays, and cryo-EM-based structure determination to develop a universal and simple approach for solving structures of the majority of RNA and RNA-drug complexes. The proposed technology is anticipated to be superior to the existing methods in labor, cost, and applicability.