The influence of arm length asymmetry and base substitution on the activity of the 10-23 DNA enzyme

A small oligodeoxyribonucleotide derived from in vitro selection has been s hown 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 purpos e 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 a llow nonstandard Watson-Crick interactions, was found in some instances to improve the cleavage reaction rate. Although the identity of the unpaired p urine in the RNA substrate cleavage site was found to have only a subtle in fluence 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 t he core motif at position 14 was found to completely abolish catalysis. The effect of arm length reduction varied with RNA substrate sequence and exte nt of helix asymmetry. Where the cleavage rate of one substrate was impaire d by truncation of the deoxyribozymes 5'-arm (6 bp), the same modification in reactions with a different sequence produced a rate enhancement. Truncat ion of the 3'-arm, however, had no effect on the reaction rate of the one s ubstrate tested yet nearly halved the cleavage rate in another substrate.