A novel integrated strategy (full length gene targeting) for mRNAaccessible site tagging combined with microarray hybridization/RNase Hcleavage to screen effective antisense oligonucleotides

摘要

Purpose: Down regulation of targeted gene by antisenseoligonucleotides (ASOs) has been an effective approach for moleculartherapy and the study of gene function. However, it is difficult to findoptimal and effective ASOs. We describe a novel integrated strategycalled full length gene targeting (FLGT), involving mRNA accessible sitetagging combined with microarray hybridization/RNase H cleavage forscreening effective ASOs in full length of target gene.Methods: Initially, transcripts representing mRNA (cRNA) werehybridized with randomized oligonucleotides library, thenoligonucleotides tags were sequenced, aligned to target mRNA, and foundto be able to precisely define the accessible sites of the mRNA byTargetFinder softerware. Further, selected ASO probes were synthesizedand used to construct microarrays. Target mRNA labeledα-32P-UTP was hybridized to the microarrays, and thesubstrate heteroduplexes were followed by RNase H catalytic reaction onmicroarrays. Those ASOs with strong signal and shorter T1/2 (timeof 50% heteroduplex cleavage by RNase H) were selected in thecombinatorial assays. Survivin, an inhibitor of apoptosis, was chosen asa target to screen ASOs by the FLGT process.Results: Using the integrated strategy, five ASOs against survivinwere selected and showed significant down regulation of survivinexpression and inhibition of tumor cells growth in vitro. Furthermore,one ASO was used to further investigate its antitumor activity on Humanhepatocellular carcinoma (HCC) orthotopic transplant model in mice.Conclusions: This study demonstrated that FLGT is useful forscreening effective ASOs. FLGT may become a useful tool for screeningmore effective ASOs in full length of target gene.