In vitro selection of self-cleaving DNAs

Ribozymes catalyze an important set of chemical transformations in metabolism, and 'engineered' ribozymes have been made that catalyze a variety of additional reactions. The possibility that catalytic DNAs or 'deoxyribozymes' can be made has only recently been addressed. Specifically, it is unclear whether the absence of the 2' hydroxyl renders DNA incapable of exhibiting efficient enzyme-like activity, making it impossible to discover natural or create artificial DNA biocatalysts.

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 Cu2+ and ascorbate to mediate oxidative self-cleavage. Individual catalysts from class II use Cu2+ as the sole cofactor. Further optimization of a class II individual by in vitro selection yielded new catalytic DNAs that facilitate Cu2+-dependent self-cleavage with rate enhancements exceeding 1 000 000-fold relative to the uncatalyzed rate of DNA cleavage.

Despite the absence of 2' hydroxyls, single-stranded DNA can adopt structures that promote divalent-metal-dependent self-cleavage via an oxidative mechanism. These results suggest that an efficient DNA enzyme might be made to cleave DNA in a biological context.