High-resolution structures and conformational dynamics of RNA origami during folding

Abstract Cotranscriptional folding of RNA is a fundamental self-assembly process of nature, important for the biological assembly of complex molecular machines like the ribosome. Inspired by this folding process, we developed the cotranscriptional RNA origami design method to efficiently produce RNA nanostructures by enzymatic synthesis, advantageous for large-scale production in vitro or expression in vivo. However, advancing this technology further will require high-resolution characterization and a better understanding of the cotranscriptional folding process. Here, we use cryogenic electron microscopy to study a panel of RNA origami structures at local resolutions up to 3.4 Å, revealing details of kissing loop and crossover structural modules used to compose RNA origami. The derived structural parameters are used to reduce internal strain and global twist to obtain more ideal shapes. In three-dimensional bundle designs, we discover a novel kinetic folding trap that forms during cotranscriptional folding, and is only released 6-8 hours after transcription. Finally, pushing both scale and complexity, we design the first multi-domain RNA origami and characterize the conformational variability of its domains by individual particle electron tomography. Our results improve understanding of RNA structure, dynamics, and folding, providing a basis for future applications of cotranscriptional folding in RNA medicine and synthetic biology.