| Antisense technology including antisense oligonucleotides(AS-ONs), ribozymes, DNA enzymes, and RNAi are becoming the important and potential tools for gene function announcement. It has either been vastly and deeply used in nucleic acid agents development.In theory, the sequence of AS-ONs should firstly match the mRNA sequence by Watson-Crick base pairing principle. But only those who could access the available sites on the folded mRNA structure can effectively bind on the molecules. This accessible sites are the "target sites" of a folded mRNA on the secondary and tertiary structures. So the target sites are of most important for effective AS-ONs design. As the CEO of ISIS Pharmaceuticals Stanley Crooke said in Apr 2000, there are two challenges in antisense: Selecting an appropriate portion of the mRNA to target has been a major challenge; Drug stability has also posed a challenge. Now that we have almost 40 more modifications for stabilizing the AS-ONs in which potential ones have been applied successfully such as 2'-O-methoxy ethyl modification, PNA, and LNA. We need to figure out the sites of the target mRNA to direct the AS-ONs at.In the past, the sites around the ATG, promoter, 5 prime cap sequence, or rbs(ribosome binding sequence) were taken as targets. But these AS-ONs obviously have no or unclear effects since they are not targeting the mRNA accessible sites.This study met the needs in nucleotides agents research and in functional genomics. Comparing to the newly developed mRNA accessible sites analysis protocols, a novel molecular biological method was set up and was either tested the practicality. Some primary work has done by using this method.Firstly, the novel method, RASS( RNA Accessible Sites Screening )was established by introducing poly A tail in any transcriptional mRNA (cRNA) which then was used to anneal on Biotin-poly T, streptavidin coated magnetic beads were used to immobilized the poly A/T coupled and naturally folded mRNA in vitro. A single stranded deoxynucleotide library containing 18nt random sequence flanked by two fixed sequences was applied to the immobilized mRNA. After washing, the binding deoxynucleotide library tags were then achieved, amplified, subcloned and sequenced. The tags were analyzed and the matching sites on the mRNA were obtained. It could rapidly figure out all the sites by precisely giving the matched nucleotide bases.Secondly, the sites of a model gene-green fluorescence protein(GFP) was selected by using RASS and MAST(mRNA accessible sites tagging) simultaneously to evaluate the RASS protocol. Four accessible sites(1,2,3,4) were obtained by MAST in which three( 1,2,4) were the same with those from RASS. AS-ONs and 10-23 type DNA enzymes were designed and phosphorathioate oligos were synthesized for all. The effects of the AS-ONs of these four sites induced RNaseH splicing to cRNA were all examined in vitro, and in vivo by co-transfecting the pEGFPNl plasmid with the every oligos in Hela cells.AS-ONs of sites 1,2,4 induced RNaseH splicing on the GFP cRNA were markedly strong in vitro, but the other one(#3) yet had a lower splicing efficacy and the control AS-ON of site 2 with two bases mismatched without. Two of the four AS-ONs (A2, A4) have the best efficacy for site-binding so induced the highest RNaseH splicing. Showed the RASS selected AS-ONs have specific and effective binding capacities because of they are directed to the accessible sites.To test if the sites precisely gave the nucleotide sequences for the most efficiency AS-ONs, a best site (No.2) were choose for designed a serious AS-ONs shifted to the 5 prime and to the 3 prime by every 4 bases(A2-8, A2-4, A2+4, A2+8). Only the original AS-ONs A2 had the best efficacy on induced RNaseH splicing. Showed the RASS selected AS-ONs had exactly sequence information for design the best AS-ONs because of they were selected by the oligo library.In vivo, the AS-ONs except A3 could inhibit the GFP expression by co-transfected the pEGFPNl plasmid with every AS-ON. A2 and A4 have obvious effects to inhi...
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