Assay of ribozyme-substrate cleavage by anion-exchange high-performance liquid chromatography.
An HPLC procedure has been developed that can be used to monitor the rate of phosphodiester cleavage of an oligoribonucleotide substrate by an RNA ribozyme with catalytic activity. The method operates at high substrate concentrations (far beyond K(m)), thus allowing the reactions to approach Vmax of the reaction. This method is an efficient alternative to radioisotope labeling methods.
Vinayak
R
R
Department of Biological Sciences, Northern Illinois University, DeKalb 60115, USA.
Andrus
A
A
Sinha
N D
ND
Hampel
A
A
D001483:Base Sequence; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D005456:Fluorescent Dyes; D015497:HIV-1 / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D016337:RNA, Catalytic / Q000138:chemical synthesis / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D009843:Oligoribonucleotides; D016337:RNA, Catalytic; D012367:RNA, Viral