Work

RNA engineering approaches to aid high-resolution structure determination of medium- to large-sized RNAs

Year: 2026

Type: review

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-sized RNAs by X-ray crystallography and cryo-EM. 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 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.

Source: Journal of Biological Chemistry

DOI: 10.1016/j.jbc.2026.113616

Authors: Jerricho Tipo, Keerthi Gottipati, Marc C. Morais, Kyung H. Choi

Institutions: Indiana University Bloomington, The University of Texas Medical Branch at Galveston, Indiana University

Language: English

Topic: RNA and protein synthesis mechanisms

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $3,430

Funders: National Institutes of Health, National Institute of General Medical Sciences, National Institute of Allergy and Infectious Diseases