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

Yuning Sun, Ming Duan, Ruxian Lin, Dan Wang, Chunrong Li, Xiaochen Bo, Shengqi Wang
2006 Molecular Vision  
Down regulation of targeted gene by antisense oligonucleotides (ASOs) has been an effective approach for molecular therapy and the study of gene function. However, it is difficult to find optimal and effective ASOs. We describe a novel integrated strategy called full length gene targeting (FLGT), involving mRNA accessible site tagging combined with microarray hybridization/RNase H cleavage for screening effective ASOs in full length of target gene. Initially, transcripts representing mRNA
more » ... esenting mRNA (cRNA) were hybridized with randomized oligonucleotides library, then oligonucleotides tags were sequenced, aligned to target mRNA, and found to be able to precisely define the accessible sites of the mRNA by TargetFinder softeware. Further, selected ASO probes were synthesized and used to construct microarrays. Target mRNA labeled alpha-(32)P-UTP was hybridized to the microarrays, and the substrate heteroduplexes were followed by RNase H catalytic reaction on microarrays. Those ASOs with strong signal and shorter T(1/2) (time of 50% heteroduplex cleavage by RNase H) were selected in the combinatorial assays. Survivin, an inhibitor of apoptosis, was chosen as a target to screen ASOs by the FLGT process. Using the integrated strategy, five ASOs against survivin were selected and showed significant down regulation of survivin expression and inhibition of tumor cells growth in vitro. Furthermore, one ASO was used to further investigate its antitumor activity on Human hepatocellular carcinoma (HCC) orthotopic transplant model in mice. This study demonstrated that FLGT is useful for screening effective ASOs. FLGT may become a useful tool for screening more effective ASOs in full length of target gene.
pmid:17149362 fatcat:lctlvrrhvbdorc5s2jrplooyni