绿色荧光蛋白基因mRNA反义寡核苷酸的筛选和应用  

Application of Antisense Oligonucleotides Designed to Screened Green Fluorescence Protein mRNA Accessible Sites

作　　者：毛建平[1] 王全会[1] 施水兰[2] 袁国刚[1] 左刚[1] 崔玉芳[1] 毛秉智[1] 

机构地区：[1]军事医学科学院放射医学研究所,北京100850 [2]解放军476医院免疫学研究室,福州350002

出　　处：《中国生物化学与分子生物学报》2005年第4期529-536,共8页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金(No.30271546);北京市自然科学基金(No.5033020)资助项目.~~

摘　　要：基因mRNA的靶点筛选是设计反义寡核苷酸的关键.建立了PARASS(polyAanchoredRNAaccessiblesitesscreening)方法,即通过在mRNA末端引入polyA,与生物素标记的polyT退火结合,将其同链亲和素磁珠混合,使mRNA通过3’末端得到固定,保持mRNA的自然伸展和折叠,与寡核苷酸文库杂交筛选mRNA的结合靶点.PARASS筛选获得了绿色荧光蛋白(GFP)mRNA的3个反义寡核苷酸结合靶点,据其设计了多条反义寡核苷酸,与对照组相比,体外RNaseH分析显示3个靶点均为有效,在HeLa细胞内针对靶点的反义寡核苷酸能抑制GFP的表达,得到了Northern印迹结果支持.PARASS对反义寡核苷酸药物设计具有应用价值.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.

关 键 词：MRNA 反义寡核苷酸 靶点 PARASS GFP 

分 类 号：Q523[生物学—生物化学] Q751