Nondenaturing Purification of Co-Transcriptionally Folded RNA Avoids Common Folding Heterogeneity

Abstract

Due to the energetic frustration of RNA folding, tertiary structured RNA is typically characterized by a rugged folding free energy landscape where deep kinetic barriers separate numerous misfolded states from one or more native states. While most in vitro studies of RNA rely on (re)folding chemically and/or enzymatically synthesized RNA in its entirety, which frequently leads into kinetic traps, nature reduces the complexity of the RNA folding problem by segmental, co-transcriptional folding starting from the 5′ end. We here have developed a simplified, general, nondenaturing purification protocol for RNA to ask whether avoiding denaturation of a co-transcriptionally folded RNA can reduce commonly observed in vitro folding heterogeneity. Our protocol bypasses the need for large-scale auxiliary protein purification and expensive chromatographic equipment and involves rapid affinity capture with magnetic beads and removal of chemical heterogeneity by cleavage of the target RNA from the beads using the ligand-induced glmS ribozyme. For two disparate model systems, the Varkud satellite (VS) and hepatitis delta virus (HDV) ribozymes, we achieve >95% conformational purity within one hour of enzymatic transcription, without the need for any folding chaperones. We further demonstrate that in vitro refolding introduces severe conformational heterogeneity into the natively-purified VS ribozyme but not into the compact, double-nested pseudoknot fold of the HDV ribozyme. We conclude that conformational heterogeneity in complex RNAs can be avoided by co-transcriptional folding followed by nondenaturing purification, providing rapid access to chemically and conformationally pure RNA for biologically relevant biochemical and biophysical studies.

HDV Family of Self-Cleaving Ribozymes

“…These results, however, are artifacts of sample preparation, whereas ribozymes studied under cotranscriptional conditions or purified without denaturation tend to exhibit fast, monophasic self-scission and uniform hydrodynamic radii, respectively. 62,63 Overall, these data suggest that the ribozymes are properly folded and highly active under in vivo-like conditions, but can sample many inhibitory conformations resulting from alternative base-pairing interactions.…”

Single-molecule studies of riboswitch folding

“…The folding competition between these structures shifts upon ligand binding, and this coupling forms the physical basis for gene regulation [40, 41]. If an associated gene’s regulatory fate is sensitive to the exact timing of folding events, for example, through co-transcriptional folding or via transcription-translation coupling [42, 43], then the riboswitch is kinetically controlled (section 4.1 ). If regulation depends upon the increased structural stability of the ligand-bound aptamer, and the aptamer is given sufficient time to out-compete the expression platform (that is, the system has time to reach thermal equilibrium), the riboswitch is thermodynamically controlled (section 4.2 ).…”

RNA synthesis and purification for structural studies

“…28,42,43 Target RNA is fused to a ribozyme and an affinity sequence. The affinity sequence is bound by a specific protein and allows the immobilization of the transcribed target RNA-ribozyme fusion to a resin.…”