Application of Antisense Oligonucleotides Designed to Screened Green Fluorescence Protein mRNA Accessible Sites
MAO Jian-Ping 1)*, WANG Quan-Hui 1), SHI Shui-Lan 2), YUAN Guo-Gang 1), ZUO Gang 1), CUI Yu-Fang 1), MAO Bing-Zhi 1)( 1)Department of Immunology, Institute of Radiation Medicine, Beijing 100850, China; 2) Department of Immunology, PLA 476 Hospital, FuZhou 350002, China)
The efficacies of antisense oligonucleotides(ODNs) is greatly dependent on the accessibility of their mRNA targets. To find the target sequences is important for antisense oligonucleotides design. Poly-A anchored RNA accessible sites screening (PARASS) was set up for identifying the accessible target sites of green fluorescence protein (GFP) mRNA. Poly-A was introduced to transcripted mRNA molecules and annealed to biotin-poly-T by which immobilizing mRNA molecules on streptoavidin coated magnetic beads; so that the terminal fixed mRNA molecules could be stretched out and folded naturally. After they were hybridized with oligonucleotide library, the eluted binding library tags could present the accessible sites. By PARASS, three accessible sites of GFP mRNA were derived. Comparing with the control groups, phosphorothioate antisense oligonucleotides targeted to these sites induced RNase H dependent cleaving on the mRNA effectively. In HeLa cells,they showed knock down effects on GFP expression which supported by Northern blotting. These results showed that PARASS is useful tool for antisense oliogonucleotides design.