RNA engineering approaches to aid high-resolution structure determination of medium- to large-sized RNAs.
Abstract
High-resolution structure determination of RNA remains a major challenge due to its conformational flexibility, intrinsic heterogeneity, and susceptibility to degradation. Although RNA plays central roles in gene regulation, viral replication, and cellular homeostasis, RNA-only structures represent a small fraction of entries in the Protein Data Bank. Overcoming the limitations associated with RNA has therefore required the development of innovative engineering strategies to stabilize defined conformations and facilitate structural analysis. In this review, we describe RNA engineering approaches that enable structure determination of medium to large RNAs by X-ray crystallography and cryo-electron microscopy. These advances include the use of stable tetraloops to promote proper folding and crystal packing, scaffold-based strategies employing tRNA and 5S rRNA for recombinant RNA production and stabilization, and bacteriophage Φ29 prohead RNA (pRNA) as well as Group I and Group II intron scaffolds to facilitate cryo-EM structure determination. Together, these approaches repurpose natural RNA structural features and host processing mechanisms into practical tools for RNA stabilization, large-scale production, and structural characterization.
