Assay for Evaluating Ribonuclease H-Mediated Degradation of RNA-Antisense Oligonucleotide Duplexes

Ribonucleases H are complex enzymes whose functions are not clearly understood, further compounded by the fact that multiple forms of the enzyme are present in various organisms. They are known to recognize and degrade the ribonucleic acid (RNA) strand of numerous deoxyribonucleic acid (DNA)-RNA duplex substrates, and so may provide a unique mode of therapeutic intervention at the genetic level of virtually any disease. We have therefore set out detailed procedures for conducting routine assays with almost any one of this family of enzymes by a straightforward assay aimed at identifying novel enzyme-activating antisense oligonucleotides (AONs). The procedures described herein should enable easy identification of potent AON molecules, provided that the RNA is appropriately labeled for subsequent visualization following the guidelines set forth in this protocol.