Screening Of Antisense Oligodeoxynucleotides Targeted To Survivin

Screening of antisense oligodeoxynucleotides targeted to survivinSurvivin, a novel inhibitor of apoptosis, is undetectable in normal adult tissues but becomes notably expressed in the most common human cancers, and is recognized as a potential target in anticancer therapy. To find out effective antisense oligodeoxynucleotides (ASODNs) targeting survivin for cancer therapy, we performed a new approach called full length gene targeting (FLGT).First, prepare the full length sequence of target gene survivin, and transcript it. Target mRNA was hybridized to oligodeoxynucleotide libraries. Then the ASODNs which can bound with target mRNA were separated from those who can not when RNA was extracted from the solution. Those binding ASODNs was PCR amplified and sequenced. Fifteen mRNA accessible sites (MASs) was conformed. Then fifteen ASODN probes complementary to these MASs was synthesized and used to manufacture oligonucleotide micoarrays. a-32P-UTP labeled target mRNA was hybridized to the microarray, thirteen probes gave significant heteroduplex yield. Substrate heteroduplex was obtained by hybridization under the same condition, followed by RNase H catalytic reaction on microarrays in situ. Good correlation was found between the DNA/RNA binding strength on microarrays in situ and the antisense activities in vivo.Survivin ASODN was transfected into A549 cells mediated by lipofectin reagent. The expression of survivin protein and mRNA was detected by western-blot and RT-PCR method. Three of them significantly inhibit the expression of survivin mRNA, and one of them inhibit the expression of survivin protein as well.These findings indicated that the ASODNs screened through FLGT can effectively bind to targeted mRNA in situ and in vitro, and may have a potential role in cancer therapy.