21] Kinetic oligonucleotide hybridization for monitoring kinetic folding of large RNAs

Abstract

Kinetic oligonucleotide hybridization is a relatively simple in vitro method for monitoring RNA folding transitions that occur with a half-time of 10 seconds or greater. Kinetic oligonucleotide hybridization have been used to characterize the Mg 2+ dependent kinetic folding pathways of the Tetrahymena group I ribozyme and the RNA component of RNase P. Kinetic oligonucleotide hybridization exploits the observation that many large RNAs, such as group I and II ribozymes and ribosomal RNA, require divalent metal ions, proteins, or both to acquire the native tertiary fold. In the absence of these folding effectors, the RNA adopts an extended, partially denatured structure consisting primarily of short-range secondary interactions. The tertiary folding transitions that occur once the effector is added include compaction of the structure and formation and/or stabilization of long- and short-range interactions. Structural changes in RNA, including those that accompany folding, can be visualized experimentally as a change in reactivity toward small chemical probes and larger probes, such as complementary oligodeoxynucleotides. Kinetic oligonucleotide hybridization takes snapshots of the kinetic folding process by monitoring time-dependent changes in accessibility to binding by short, sequence-specific oligodeoxynucleotide probes.