| A new DNA enzyme, the “Bipartite DNAzyme”, suitable for the sequence-specific cleavage of RNA, was obtained from a random DNA library by in vitro selection. A single family of catalytic molecules emerged from the selection, and a 22 nucleotide consensus sequence common to all clones defined a putative catalytic core. The most abundant clone self-cleaved at a single internal ribonucleotide phosphodiester with a relatively fast kobs value of 1.7 min−1, in 10 mM MgCl 2 at 23°C, pH 7.4. This DNAzyme (Clone 12–17) required divalent cations, with manganese most optimally supporting cleavage. The consensus sequence selected was responsible for catalysis, since a 37 nucleotide shorter version of the selected Clone 12–17, missing the entire 3′ fixed sequence and part of the 5′ fixed sequence was fully active. A re-selection from a mutagenized DNAzyme pool for the ability to cleave at extended RNA substrates yielded an unchanged catalytic core sequence. From this re-selection a DNAzyme (called “Bipartite”) capable of sequence-specifically cleaving extended stretches of RNA was derived. The Bipartite DNAzyme is able to cleave different ribonucleotide combinations, but the best cleavage site is A↓ANNN, (where N can be A, U, C, or G). For the best reaction rates, there is a requirement for these five ribonucleotides to be unpaired. Multiple-turnover kinetics, measured in 30 mM MgCl2, at 37°, pH 7.4, with an HIV derived RNA substrate (HIV-env), yielded a kcat value of ∼1.4 min−1 and a KM value of ∼230 nM, which were of the same order as kcat and KM values measured for other ribozymes in general use for RNA cleavage. The Bipartite was able to cleave in vitro a messenger RNA from C. elegans , the dpy-5 mRNA, and therefore could be used for mRNA ablation procedures. A rate versus pH analysis of the Bipartite DNAzyme in the presence of magnesium, revealed a two-phase profile, with an observed pKa of 5.95 ± 0.06. The pH versus rate profile in the presence of manganese and calcium also resulted in a two-phase profile, with measured pKa of 5.39 ± 0.17 and 6.24 ± 0.09, respectively. These results indicate that the divalent cation might have a direct role in catalysis. Kinetic solvent isotope effect experiments reveal that the pKa represents the ionization of a functional group, rather than a change in the rate-determining step of the reaction. Proton inventory experiments indicate that at least one proton is transferred in the transition state of the reaction. |
