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.
OBJECTIVE
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.


METHODS
Initially, transcripts representing 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.


RESULTS
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.


CONCLUSIONS
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.
Sun
Yuning
Y
Beijing Institute of Radiation Medicine, 27 Taiping Road, Beijing 100850, P.R. China. sunyuning70@yahoo.com.cn <sunyuning70@yahoo.com.cn>
Duan
Ming
M
Lin
Ruxian
R
Wang
Dan
D
Li
Chunrong
C
Bo
Xiaochen
X
Wang
Shengqi
S
D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D015536:Down-Regulation; D018390:Gene Targeting / Q000379:methods*; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins; D008113:Liver Neoplasms / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D008869:Microtubule-Associated Proteins / Q000187:drug effects / Q000235:genetics*; D009363:Neoplasm Proteins / Q000187:drug effects / Q000235:genetics*; D009368:Neoplasm Transplantation; D020411:Oligonucleotide Array Sequence Analysis; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000235:genetics*; D016914:Ribonuclease H / Q000494:pharmacology*; D016324:Sequence Tagged Sites; D000077022:Survivin; D014407:Tumor Cells, Cultured
C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D000077022:Survivin; D016914:Ribonuclease H