Flanagan W M, Kothavale A, Wagner R W
Gilead Sciences, Foster City, CA 94404, USA.
Nucleic Acids Res. 1996 Aug 1;24(15):2936-41. doi: 10.1093/nar/24.15.2936.
To understand the parameters required for designing potent and specific antisense C-5 propynyl-pyrimidine-2'-deoxyphosphorothioate-modified oligonucleotides (C-5 propyne ONs), we have utilized a HeLa line that stably expresses luciferase under tight control of a tetracycline-responsive promoter. Using this sensitive and regulatable cell-based system we have identified five distinct antisense ONs targeting luciferase and have investigated the role that ON length, target mismatches, compound stability and intracellular RNA levels play in affecting antisense potency. We demonstrate that C-5 propyne ONs as short as 11 bases retained 66% of the potency demonstrated by the parent 15 base compound, that a one base internal mismatch between the antisense ON and the luciferase target reduced the potency of the antisense ON by 43% and two or more mismatches completely inactivated the antisense ON and that C-5 propyne ONs have a biologically active half-life in tissue culture of 35 h. In addition, by regulating the intracellular levels of the luciferase mRNA over 20-fold, we show that the potency of C-5 propyne ONs is unaffected by changes in the expression level of the target RNA. These data suggest that low and high copy messages can be targeted with equivalent potency using C-5 propyne ONs.
为了了解设计高效且特异的反义C-5炔基嘧啶-2'-脱氧硫代磷酸酯修饰寡核苷酸（C-5炔基寡核苷酸）所需的参数，我们利用了一种在四环素反应性启动子的严格控制下稳定表达荧光素酶的HeLa细胞系。使用这种灵敏且可调节的基于细胞的系统，我们鉴定出了5种针对荧光素酶的不同反义寡核苷酸，并研究了寡核苷酸长度、靶标错配、化合物稳定性和细胞内RNA水平在影响反义活性方面所起的作用。我们证明，短至11个碱基的C-5炔基寡核苷酸保留了亲本15个碱基化合物所显示活性的66%，反义寡核苷酸与荧光素酶靶标之间一个碱基的内部错配使反义寡核苷酸的活性降低了43%，两个或更多错配则完全使反义寡核苷酸失活，并且C-5炔基寡核苷酸在组织培养中的生物活性半衰期为35小时。此外，通过将荧光素酶mRNA的细胞内水平调节20倍以上，我们表明C-5炔基寡核苷酸的活性不受靶标RNA表达水平变化的影响。这些数据表明，使用C-5炔基寡核苷酸可以以同等效力靶向低拷贝和高拷贝的信息。
