IN-VITRO SELECTION OF SELF-CLEAVING DNAS

Background: Ribozymes catalyze an important set of chemical transforma tions in metabolism, and 'engineered' ribozymes have been made that ca talyze a variety of additional reactions. The possibility that catalyt ic DNAs or 'deoxyribozymes' can be made has only recently been address ed. Specifically, it is unclear whether the absence of the 2' hydroxyl renders DNA incapable bf exhibiting efficient enzyme-like activity, m aking it impossible to discover natural or create artificial DNA bioca talysts. Results: We report the isolation by in vitro selection of two distinct classes of self-cleaving DNAs from a pool of random-sequence oligonucleotides. Individual catalysts from 'class I' require both Cu 2+ and ascorbate to mediate oxidative self-cleavage. Individual cataly sts from class II use Cu2+ as the sole cofactor. Further optimization of a Glass II individual by in vitro selection yielded new catalytic D NAs that facilitate Cu2+-dependent self-cleavage with rate enhancement s exceeding 1 000 000-fold relative to the uncatalyzed rate of DNA cle avage. Conclusions: Despite the absence of 2' hydroxyls, single-strand ed DNA can adopt structures that promote divalent-metal-dependent self -cleavage via an oxidative mechanism. These results suggest that an ef ficient DNA enzyme might be made to cleave DNA in a biological context . (C) Current Biology Ltd.