Cryo-specimen preparation and imaging of highly-structured and dynamic large non-coding RNAs

Abstract Background Many RNAs rely on their 3D structures for enabling critical biological functions, including catalytic reactions, such as splicing or protein synthesis, or regulation of gene expression, subcellular structure formation, and signaling. Yet, RNA structure determination still lags significantly behind protein structure determination. The main challenges are connected to the characterization of flexible, dynamic RNA structures, which can so far only be captured by secondary structure probing studies or low-resolution 3D imaging approaches. Methods Here, we present a methodological approach to produce, purify, and biochemically and structurally characterize both highly-structured and dynamic large non-coding RNAs. We specifically focus on identifying suitable conditions that enable RNA electron cryo-microscopy imaging at near-atomic resolution. Results By using a self-splicing group II intron ribozyme as a model system, we describe a pipeline that leads to the production and cryo-EM structure determination of one highly-structured and one dynamic RNAs of 327–388 nucleotides (100–133 kDa) in size. Discussion We discuss challenges in the optimization of buffer composition of the samples, and in the selection of the optimal grid vitrification conditions, both with respect to sample concentration, and of blot times and forces, which crucially impact on the grid quality. Our work establishes a reference to guide future characterization of large non-coding RNAs from a structure-functional perspective, with important implications for improving our understanding of fundamental biological and medically-relevant mechanisms.