A continuous kinetic assay for RNA-cleaving deoxyribozymes, exploiting ethidium bromide as an extrinsic fluorescent probe.
We describe a rapid and inexpensive method to monitor the kinetics of small RNA-cleaving deoxyribozymes, based on the exogenous fluorophore ethidium bromide. Ethidium binds preferentially to double-stranded nucleic acids, and its fluorescence emission increases dramatically upon intercalation. Thus, ethidium can be used in single-turnover experiments to measure both annealing of the deoxyribozyme to its substrate and release of the products. Under conditions in which dissociation of the product is fast compared with cleavage, the apparent rate of product release reflects the cleavage step. The method was developed for characterizing the so-called 8-17 catalytic DNA, but its general applicability in the deoxyribozyme field was verified using the 10-23 RNA-cleaving construct. Catalysis by both deoxyribozymes was not inhibited in the presence of substoichiometric amounts of ethidium, and the rates obtained through the ethidium assay were virtually identical to the rates determined using radiolabeled substrates. In contrast, the assay cannot be applied to the large, structured ribozymes, and its use to study the kinetics of the small hammerhead ribozyme was hampered by the presence on the catalyst of at least one high-affinity ethidium binding site.
Ferrari
Davide
D
Department of Biochemistry and Molecular Biology, University of Parma, 43100 Parma, Italy.
Peracchi
Alessio
A
D001483:Base Sequence; D001665:Binding Sites; D021881:DNA, Catalytic / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism; D004996:Ethidium / Q000737:chemistry* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D005470:Fluorometry / Q000379:methods*; D007700:Kinetics; D046911:Macromolecular Substances; D012313:RNA / Q000378:metabolism*; D016337:RNA, Catalytic / Q000378:metabolism
D021881:DNA, Catalytic; D004277:DNA, Single-Stranded; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D016337:RNA, Catalytic; C402582:RNA-cleaving DNA 10-23; C407482:RNA-cleaving DNA 8-17; C427204:hammerhead ribozyme; D012313:RNA; D004996:Ethidium