Nondenaturing purification of co-transcriptionally folded RNA avoids common folding heterogeneity.
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.
Pereira
Miguel J B
MJ
Single Molecule Analysis Group, Department of Chemistry, University of Michigan, Ann Arbor, Michigan, United States of America.
Behera
Vivek
V
Walter
Nils G
NG
D003698:Hepatitis Delta Virus / Q000737:chemistry* / Q000235:genetics; D008280:Magnetics; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D052616:Solid Phase Extraction / Q000379:methods*; D014158:Transcription, Genetic
D016337:RNA, Catalytic; D012367:RNA, Viral