Establishing broad generality of DNA catalysts for site-specific hydrolysis of single-stranded DNA

We recently reported that a DNA catalyst (deoxy-ribozyme) can site-specifically hydrolyze DNA on the minutes time scale. Sequence specificity is pro-vided by Watson-Crick base pairing between the DNA substrate and two oligonucleotide binding arms that flank the 40-nt catalytic region of the deoxyribozyme. The DNA catalyst from our recent in vitro selection effort, 10MD5, can cleave a single-stranded DNA substrate sequence with the aid of Zn2+ and Mn2+ cofactors, as long as the sub-strate cleavage site encompasses the four particu-lar nucleotides ATG^T. Thus, 10MD5 can cleave only 1 out of every 256 (44) arbitrarily chosen DNA sites, which is rather poor substrate sequence tolerance. In this study, we demonstrated substantially broader generality of deoxyribozymes for site-specific DNA hydrolysis. New selection experiments were per-formed, revealing the optimality of presenting only one or two unpaired DNA substrate nucleotides to the N40 DNA catalytic region. Comprehensive selec-tions were then performed, including in some cases a key selection pressure to cleave the substrate at a predetermined site. These efforts led to identifica-tion of numerous new DNA-hydrolyzing deoxyribo-zymes, many of which require merely two particular nucleotide identities at the cleavage site (e.g. T^G), while retaining Watson-Crick sequence generality beyond those nucleotides alongwith useful cleavage rates. These findings establish experimentally that broadly sequence-tolerant and site-specific deoxy-ribozymes are readily identified for hydrolysis of single-stranded DNA