Fas基因mRNA反义靶点筛选和体外验证

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-317nt、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。

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

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.