Kinetic characterization of hairpin ribozyme variants.
Kinetic analysis of ribozyme reactions is a common method to evaluate and compare activities of catalytic RNAs. The hairpin ribozyme catalyzes the reversible cleavage of a suitable RNA substrate at a specific site. Hairpin ribozyme variants as an allosteric ribozyme responsive to flavine mononucleotide and a hairpin-derived twin ribozyme that catalyzes two cleavage reactions and two ligation events with the result of a fragment exchange have been developed by rational design and were kinetically characterized. Herein, protocols for preparation of ribozymes and dye-labeled substrates as well as for analysis of cleavage, ligation, and fragment exchange reactions are provided.
Institut für Biochemie, Universität Greifswald, Greifswald, Germany.
Bettina
B
Appel
Thomas
T
Marschall
Anne
A
Strahl
Sabine
S
Müller
D000096:Acetone / Q000737:chemistry; D000494:Allosteric Regulation; D001483:Base Sequence; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D056148:Chromatography, Reverse-Phase; D004396:Coloring Agents / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D000431:Ethanol / Q000737:chemistry; D005486:Flavin Mononucleotide / Q000378:metabolism; D055029:Inverted Repeat Sequences; D007700:Kinetics; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
D004396:Coloring Agents; D016337:RNA, Catalytic; D000096:Acetone; D000431:Ethanol; D005486:Flavin Mononucleotide