A small oligodeoxyribonucleotide derived from in vitro selection has been shown to be capable of efficient sequence-specific cleavage of RNA at purine-pyrimidine junctions. As the reaction readily takes place under simulated physiologic conditions, this molecule described as the 10-23 general purpose RNA-cleaving DNA enzyme, has potential as a therapeutic agent. To further explore the character of this prototype, we examined the influence of base substitution and binding arm length asymmetry on its RNA cleaving activity. Surprisingly, substitution of the proximal nucleotide on the 3′-arm, to allow nonstandard Watson-Crick interactions, was found in some instances to improve the cleavage reaction rate. Although the identity of the unpaired purine in the RNA substrate cleavage site was found to have only a subtle influence on the rate of catalysis, with a slight decrease observed when a G at this position was changed to an A, nucleotide substitution (G to C) in the core motif at position 14 was found to completely abolish catalysis. The effect of arm length reduction varied with RNA substrate sequence and extent of helix asymmetry. Where the cleavage rate of one substrate was impaired by truncation of the deoxyribozymes 5′-arm (6 bp), the same modification in reactions with a different sequence produced a rate enhancement. Truncation of the 3′-arm, however, had no effect on the reaction rate of the one substrate tested yet nearly halved the cleavage rate in another substrate.