摘要
基因mRNA的结构靶点筛选是反义核酸药物研发的一个难题 .兔 (Oryctolaguscuniculus) β珠蛋白基因mRNA的结构靶位点通过运用MAST技术筛选获得 ,和计算机软件RNAstructure3 71模拟分析的位点进行了比较 ,也和寡核苷酸微阵列杂交技术筛选获得的靶点结果 (M .Natalie ,1 997)进行了比较 ,显示 :据MAST技术获得的兔 β珠蛋白基因 2个反义核酸结合靶位点 ,和用RNAstructure3 71软件给出的模拟分析的 2个靶位点相同 ,且它们与寡核苷酸微阵列杂交技术的结果完全一致 .运用MAST技术筛选获得绿色荧光蛋白 (GFP)mRNA有 4个结构靶位点 ,体外分析表明这 4个靶位点均有效 ,其中有 3个与RNAstructure3 71软件分析的靶点相同 ,但计算机模拟推荐的结构靶位点较多 ,而且随着基因长度增加确认靶位点的难度增大 ,获得的靶位点还需要实验验证 ,计算机软件模拟分析对实验筛选靶点、设计反义核酸有辅助价值 .MAST方法能筛选各种长度基因mRNA的全部可及位点和准确给定核苷酸的起止位置以供设计反义核酸 ,具有简单快捷的优点 ,将能为反义核酸设计起重要作用 .
Selecting the target sites of the mRNA has been a major challenge in antisense drug research. Two accessible target sites of rabbit ( Oryctolagus cuniculus )β globin mRNA fragment were obtained by using MAST. From the lowest free energy (Δ G 37 ) structure folded by RNAstructure3 71, two sites were achieved by oligo walking and were identical to those obtained from MAST. Moreover, they were the same with those ones reported by combinatorial oligonucleotide arrays hybridization. For green fluorescence protein(GFP), four accessible target sites were obtained by MAST. Antisense oligonucleotides designed to these sites were tested to be effective in vitro. By using oligo walking function of RNAstructure3 71, 3 among the 8 recommended sites were similar with 3 sites of MAST selected. The validating of sites computer aid predicted among many recommended ones need to be tested in vitro and it would work more difficultly if for long sequence length gene. But it is yet helpful to target sites screening and antisense designing. Effective antisense oligonucleotides could be designed based on sequence data from MAST. MAST is a simplified protocol and could screen mRNAs of any size, and be a good tool in antisense drugs designing.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2004年第3期399-407,共9页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金 (No.3 0 2 715 46)
北京市自然科学基金 (No.5 0 3 3 0 2 0 )
军事医学科学院回国启动基金资助~~
