Probing fast ribozyme reactions under biological conditions with rapid quench-flow kinetics.
Reaction kinetics on the millisecond timescale pervade the protein and RNA fields. To study such reactions, investigators often perturb the system with abiological solution conditions or substrates in order to slow the rate to timescales accessible by hand mixing; however, such perturbations can change the rate-limiting step and obscure key folding and chemical steps that are found under biological conditions. Mechanical methods for collecting data on the millisecond timescale, which allow these perturbations to be avoided, have been developed over the last few decades. These methods are relatively simple and can be conducted on affordable and commercially available instruments. Here, we focus on using the rapid quench-flow technique to study the fast reaction kinetics of RNA enzymes, or ribozymes, which often react on the millisecond timescale under biological conditions. Rapid quench of ribozymes is completely parallel to the familiar hand-mixing approach, including the use of radiolabeled RNAs and fractionation of reactions on polyacrylamide gels. We provide tips on addressing and preventing common problems that can arise with the rapid-quench technique. Guidance is also offered on ensuring the ribozyme is properly folded and fast-reacting. We hope that this article will facilitate the broader use of rapid-quench instrumentation to study fast-reacting ribozymes under biological reaction conditions.
Department of Chemistry and Center for RNA Molecular Biology, The Pennsylvania State University, University Park, PA 16802, United States. Electronic address: jamieleebingaman@gmail.com.
Jamie L
JL
Bingaman
Department of Chemistry and Center for RNA Molecular Biology, The Pennsylvania State University, University Park, PA 16802, United States.
Kyle J
KJ
Messina
Department of Chemistry and Center for RNA Molecular Biology, The Pennsylvania State University, University Park, PA 16802, United States; Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, United States. Electronic address: pcb5@psu.edu.
Philip C
PC
Bevilacqua
D002384:Catalysis; D002623:Chemistry Techniques, Analytical / Q000295:instrumentation / Q000379:methods*; D007700:Kinetics; D008954:Models, Biological; D008956:Models, Chemical; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry*; D016337:RNA, Catalytic / Q000737:chemistry*; D011849:Radioactive Tracers; D013997:Time Factors
D016337:RNA, Catalytic; D011849:Radioactive Tracers; D012313:RNA
RNA enzymology; Rapid-quench; Ribozyme; Ribozyme kinetics