Fluorescence resonance energy transfer analysis of ribozyme kinetics reveals the mode of action of a facilitator oligonucleotide.
A defining characteristic of catalysts is the rate at which they can process multiple copies of substrate. In the case of synthetic hammerhead ribozymes that cleave an RNA sequence, binding of the ribozyme to the substrate and products is through base-paired duplexes. The kinetics of formation and dissociation of these duplexes can determine the turnover of the ribozyme. We have followed these processes in real time by using fluorescent labels that can interact through fluorescence resonance energy transfer (FRET). This approach has been used to identify the rate-limiting steps for a particular ribozyme and to reveal how turnover was improved by a facilitator oligonucleotide. It was found that dissociation of the ribozyme-substrate complex is faster than cleavage to products. Hence, to undergo cleavage, most substrate molecules must interact with a ribozyme more than once. In the presence of a facilitator oligonucleotide, the complex is stabilized so that cleavage is faster than dissociation. Under these circumstances, cleavage of the substrate becomes the most likely outcome following binding to the ribozyme.
Perkins
T A
TA
Hybridon, Inc., Worcester, Massachusetts 01605, USA.
Wolf
D E
DE
Goodchild
J
J
D001483:Base Sequence; D002151:Calorimetry; D004735:Energy Transfer; D019793:Fluorescein; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007700:Kinetics; D008956:Models, Chemical; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism*; D012235:Rhodamines; D013050:Spectrometry, Fluorescence
D005452:Fluoresceins; D005456:Fluorescent Dyes; D009838:Oligodeoxyribonucleotides; D016337:RNA, Catalytic; D012235:Rhodamines; C005358:tetramethylrhodamine; D019793:Fluorescein