716. Differential Effects of Secondary Structure on Antisense Oligonucleotide (AON) and Short Interfering RNA (siRNA) Mediated mRNA Cleavage
Authors: Stephen I. Rudnick, Alan M. Gewirtz
Journal: Molecular Therapy (2006)

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′-O-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. In order to better represent mRNA secondary structure, the 21nt target site was fully blocked by a single annealed 2′OMe ON. Bulges from 2 to 16nt in the induced double strand were introduced by incorporating non-complementary bases between two fully complementary 15nt regions. In all bulge model experiments, the siRNA pathway retained at least 48% of its activity. However, even with the largest bulge of 16nt, siRNA activity was limited to 75% of the control with no 2′OMe ON. In the presence of a 10nt or larger bulge, the AON pathway retained 74% of its activity, but was reduced to as little as 28% with smaller bulges. These data reveal a difference in the effect of mRNA secondary structure on siRNA and AON activity in vitro. The AON consistently degraded more mRNA than the siRNA, which may reflect a difference in the concentration of RNaseH and the siRNA incorporated in RISC. Any structure at the target site reduced siRNA activity, but it was never fully inhibited. AON could cleave the mRNA target if a minimal binding site of eight to ten adjacent nucleotides were accessible. If the target site was fully double stranded, the AON activity was affected to a greater extent than the siRNA. This suggests that there is an advantage to siRNA based target hybridization in comparison to AON when the target site is involved in complex secondary structure. In addition, the data supports the hypothesis that no processive scanning of the mRNA is involved in RNAi, but argues that hybridization occurs in more than a simple diffusion based model.