Fas基因mRNA反义靶点筛选和体外验证  

Antisense Sites Screening of Fas Gene mRNA and Its Validation in vitro

作　　者：左刚[1] 韩慧明[1] 田晓丽[2] 王全会[1] 毛建平[1] 

机构地区：[1]军事医学科学院放射与辐射医学研究所,北京100850 [2]军事医学科学院附属307医院,北京100850

出　　处：《中国生物工程杂志》2006年第4期20-26,共7页China Biotechnology

基　　金：国家自然科学基金资助项目(30271546)

摘　　要：应用PARASS(poly-A anchored RNA accessible sites screening)技术筛选Fas基因mRNA获得3个潜在反义作用靶点,靶点1、2、3分别位于Fas基因297nt-317m、619nt～639nt和662nt- 682nt。设计了对应靶点的反义寡核苷酸A1、A2、A3和10-23型DNAzyme D1、D2和D3。将反义寡核苷酸和Fas基因RNA结合再加入RNase H进行反应,10-23型DNAzyme则直接与Fas基因 RNA作用,结果表明:3个靶点的反义寡核苷酸组及DNAzyme均能降解Fas基因RNA,为有效靶点,其靶点反应优势次序为靶点3>靶点1>靶点2。而非靶点对照组和有效靶点突变了2个碱基的对照组均没有反应。靶点2和靶点3与ISIS公司经过多次实验筛选到的Fas反义作用靶点位置基本相同,表明PARASS技术的有效性和可靠性。获得的有效反义寡核苷酸和DNAzyme为后续研究打下基础。Three candidate antisense target sites of mouse Fas gene were screened by PARASS （poly-A anchored RNA accessible sites screening） technology. They were target at Fas gene 297nt～317nt, 618nt～ 638nt and 662nt～682nt. Antisense oligos （ A1, A2 and A3 ） and DNAzymes （ D1, D2, and D3 ） for every target site were designed and synthesized. In vitro, the validation of the sites were judged by antisense oligos included RNase H splicing and the DNAzyme degradation. The results indicated that A1, A2 and A3 introduced RNase H degradation. DNAzymes D1, D2 and D3 cleaved Fas mRNA effectively. Neither degradation observed in antisense oligo RNase H group in non-target site （ 1211～1231nt） and 2 bases mismatched of A3, nor splicing occurred in DNzyme group in non-target site （1211～1231nt） and 2 bases mismatched of D3. Site 2 and 3 were at the same positions with those of ISIS Pharmaceuticals. The effective antisense oligos and DNAzymes for Fas gene could be used for the research subsequently.

关 键 词：靶点反义寡核苷酸DNAzyme FAS PARASS 

分 类 号：Q756[生物学—分子生物学]