# Scaffolds with optimized quaternary symmetry for de novo cryoEM structure determination of small RNAs

## TL;DR

Researchers developed symmetric RNA scaffolds to determine high-resolution structures of small RNAs using cryoEM, enabling detailed analysis of their architecture.

## Contribution

The novel use of symmetric RNA scaffolds allows cryoEM structure determination of small RNAs previously too tiny for this method.

## Key findings

- C2 and D2-symmetric scaffolds enabled cryoEM structures of small RNAs at resolutions beyond 3 Å.
- The method was applied to tRNAAsp, Mango-III, and new aptamers, revealing molecular details of RNA-ligand interactions.
- The scaffolds provide a general tool for studying compact RNA folds at the atomic level.

## Abstract

Structured RNAs play many roles in cells and emerging biotechnology. While large RNAs and ribonucleoprotein complexes often benefit from high-resolution structural analysis through cryogenic-sample electron microscopy (cryoEM), single-domain RNAs, particularly those smaller than ~100 nt (33 kDa), have proven challenging. Here we address this methodological gap by engineering two- and fourfold symmetric scaffolds that enable de novo structure solution of covalently attached RNA guests to beyond 3 Å overall resolution for the best resolved guests. We apply C2 and D2-symmetric scaffolds to post-transcriptionally unmodified tRNAAsp, the fluorogenic aptamer Mango-III, and previously uncharacterized quinine- and 8-oxoguanine-binding aptamers. Experimental Coulomb potential maps with quality sufficient for small-molecule ligand, cation and water molecule placement reveal the molecular basis for specificity and suggest routes for structure-guided RNA engineering. Optimized scaffolds with intrinsic quaternary structure are a new general tool to interrogate the atomistic architecture of natural and designed compact RNA folds by single-particle cryoEM.

Engineered RNA domains that can adopt two- and fourfold symmetry are shown to be effective scaffolds for structure determination for RNAs otherwise too small for cryoEM.