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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. Only 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 MgCl2 at 23°C. This DNAzyme (“Bipartite I”) required divalent cations, with magnesium and manganese most optimally supporting cleavage. A reselection 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 (“Bipartite II”) capable of sequence-specifically cleaving extended stretches of RNA was derived. A rate versus pH analysis of the Bipartite II DNAzyme revealed a two-phase profile, similar to that reported for the hepatitis delta virus (HDV) ribozyme, suggesting that the Bipartite II DNAzyme and the HDV ribozyme may share similar catalytic strategies. Multiple-turnover kinetics, measured in 30 mM MgCl2, at 37°C, with an HIV-1-derived RNA substrate, 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 and DNAzymes in general use for RNA cleavage. The Bipartite DNAzyme therefore represents a new and potentially useful reagent, both for the processing of RNA transcripts in vitro and for mRNA ablation procedures in vivo.