A biologically stable DNAzyme that efficiently silences gene expression in cells

Efforts to use RNA-cleaving DNA enzymes (DNAzymes) as gene-silencing agents in therapeutic applications have stalled due to their low efficacy in clinical trials. Here we report a xeno-nucleic-acid-modified version of the classic DNAzyme 10-23 that achieves multiple-turnover activity under cellular conditions and resists nuclease digestion. The new reagent, X10-23, overcomes the problem of product inhibition, which limited previous 10-23 designs, using molecular chemotypes with DNA, 2'-fluoroarabino nucleic acid and α-L-threofuranosyl nucleic acid backbone architectures that balance the effects of enhanced biological stability with RNA hybridization and divalent metal ion coordination. In cultured mammalian cells, X10-23 facilitates persistent gene silencing by efficiently degrading exogenous and endogenous messenger RNA transcripts. Together, these results demonstrate that new molecular chemotypes can improve the activity and stability of DNAzymes, and may provide a new route for nucleic acid enzymes to reach the clinic.