RNA Structure Analysis

Many RNA molecules can fold into three-dimensional complex structures and possess basic biological functions, including regulating gene expression, sensing small molecules, and catalyzing reactions. The heterogeneity of RNA molecule conformations, especially in the absence of protein factors, is too complicated to be analyzed by traditional techniques such as X-ray crystallography and NMR.

Cryo-electron microscopy (Cryo-EM), which resolves protein structures at atomic resolution, is a powerful tool. Structural insights into biological samples that are not available from conventional X-ray crystallography and nuclear magnetic resonance (NMR) can be explored by Cryo-EM. Despite the important role of RNA in various biological processes, RNA remains under-explored in Cryo-EM.

Figure 1. Cryo-EM-guided RNA structure determination. (Ma, H. et al., 2022)

• Ribosolve

RNA structural analysis is mainly for RNA-protein complexes, and pure RNA structural analysis has not received much attention. The main reason may be that RNA sample preparation is somewhat different from protein, and RNA itself is more flexible, so it is not easy to judge the homogeneity of the formed structure, which hinders the resolution of Cryo-EM structures.

Ribosolve is a hybrid method that integrates Cryo-EM, biochemical method M2-seq, and Rosetta computational modeling to rapidly resolve the structures of pure RNAs. The entire process is significant labor and material savings, allowing researchers to resolve a larger number of RNA structures of varying lengths (60 to 400 nt) in a few months. This includes several riboswitches, ribozymes, and synthetic RNA structures.

Although not at the atomic-level resolution, Ribosolve has been able to reveal the overall three-dimensional structures of RNA molecules at resolutions of 4.7 to 10 Å. The Ribosolve method allows analysis of conformational changes caused by the binding of RNA to other factors and validates the accuracy of RNA structure prediction.

• ROCK

To understand the role of individual RNA molecules, their 3D structures need to be deciphered at the atomic and molecular levels. Techniques used to study the structure of DNA and proteins cannot be applied to RNA molecules with nearly the same effect. This is because their molecular composition and structural flexibility prevent them from easily forming crystals. In addition to Ribosolve, a completely new method for studying the structure of RNA molecules has now been reported as ROCK.

ROCK combines RNA nanotechnology and Cryo-EM methods to assemble multiple identical RNA molecules into highly ordered structures, thus significantly reducing the flexibility of individual RNA molecules and increasing their molecular weight exponentially. ROCK is breaking the current limitations of RNA structure studies and can unlock, with the near-atomic resolution, 3D RNA molecules that are difficult or impossible to obtain with existing methods structures that are difficult or impossible to obtain with existing methods.

Creative Biostructure always focuses on the latest Cryo-EM methods, and we aim to help you obtain structural information on your target RNA samples. We are more than happy to help you achieve breakthroughs in many areas of basic research and drug development, including the emerging field of RNA therapeutics. Please contact us for more information about our services.

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Reference

1. Ma H, et al. Cryo-EM advances in RNA structure determination. Signal Transduction and Targeted Therapy. 2022. 7(1): 1-6.