716. Differential Effects of Secondary Structure on Antisense Oligonucleotide (AON) and Short Interfering RNA (siRNA) Mediated mRNA Cleavage

Abstract

Post-transcriptional gene silencing (PTGS) impairs protein's synthesis by targeting its messenger RNA (mRNA). The antisense oligonucleotide (AON) and RNA interference (RNAi) pathways can both accomplish PTGS by hybridization of a reverse complementary oligonucleotide and subsequent degradation of the mRNA by an RNaseH mechanism. Here we investigate the influence of specific mRNA secondary structure on targeting efficiency bymonitoring AON and siRNA mediated mRNA cleavage in Drosophila embryo whole cell lysates. Structure was induced within a short mRNA target by annealing 2'|[ndash]|O|[ndash]|methyl oligonucleotides (2'OMe ON) followed by in vitro cleavage assays. We demonstrate that annealing a single 2'OMe ON upstream or downstream of a 21 nucleotide (nt) target site enhances mRNA cleavage within both PTGS pathways by 7% to 30%. Inducing structure on both sides of the target site with two or more 2'OMe ON resulted in 23% to 76%, or 18% to 92% more product formation for the siRNA and AON pathways respectively when compared to controls without induced structure. When two 2'OMe ON were annealed incrementally closer to the center of the target site from either side, it was found that AON retained 80% or more of their activity as long as eight or more nucleotides were unblocked. When less than 8nt remained available, AON activity was reduced to half of its potential. In the same fashion, it was found that the siRNA retained 90% of its activity when 16nt or more were available. Otherwise, siRNA catalyzed cleavage was reduced to 52% to 61% as more of the target site was encroached upon.