Fluorescence polarization for monitoring ribozyme reactions in real time.
Fluorescence polarization has been used recently to monitor diverse macromolecular interactions. In this report, the application of fluorescence polarization has been extended to monitor ribozyme reactions in real time. With fluorescently labeled substrate RNAs, group I ribozyme ligation and hammerhead ribozyme cleavage reactions were studied by fluorescence polarization in substrate excess (multiple turnover) conditions. These results also show that fluorescently labeled RNAs remain active substrates for ribozymes. Furthermore, a direct comparison of fluorescence polarization with fluorescence resonance energy transfer showed that both techniques were comparable for monitoring ribozyme reactions.
Singh
K K
KK
Institute for Hematopathology, University of Kiel, Germany. kumudks@scripps.edu
Rücker
T
T
Hanne
A
A
Parwaresch
R
R
Krupp
G
G
D001331:Automation / Q000295:instrumentation; D002384:Catalysis; D003199:Computer Systems; D005452:Fluoresceins / Q000032:analysis; D005454:Fluorescence Polarization / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis; D006151:Guanosine / Q000032:analysis; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000378:metabolism*; D012235:Rhodamines / Q000032:analysis; D012264:Ribonucleotide Reductases / Q000378:metabolism; D012680:Sensitivity and Specificity; D013379:Substrate Specificity
C114557:5-carboxytetramethylrhodamine succinimidyl ester; D005452:Fluoresceins; D005456:Fluorescent Dyes; D016337:RNA, Catalytic; D012235:Rhodamines; C418822:sunY ribozyme; D006151:Guanosine; C024098:6-carboxyfluorescein; D012264:Ribonucleotide Reductases; C101141:anaerobic ribonucleotide reductase