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

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 tRNA^Asp, 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.