Giles R V, Tidd D M
Department of Biochemistry, University of Liverpool, Merseyside, UK.
Anticancer Drug Des. 1992 Feb;7(1):37-48.
Chimeric oligodeoxynucleotides, comprised of internal phosphodiester and terminal methylphosphonodiester sections, possess many beneficial characteristics as antisense effectors. We have investigated the effects of progressive replacement of phosphodiester by methylphosphonodiester linkages on hybrid stability with complementary RNA and DNA. The melting temperatures (Tms) of oligodeoxynucleotide/RNA heteroduplexes were found to decrease dramatically with increasing methylphosphonate substitution. In contrast, a smaller reduction in Tm was observed for comparable DNA heteroduplexes. This disparate reduction in hybrid stability was found with both the G + C-rich human c-myc and A + T-rich human c-Ha-ras sequences used, suggesting that methylphosphonate oligodeoxynucleotide analogues generally hybridize with less affinity to RNA than DNA. RNase H assays were employed to determine if the noted decreases in Tm impaired the ability of chimeric oligodeoxynucleotides to direct the degradation of RNA. Contrary to expectation, increasing methylphosphonate substitution gave rise to increasing rates of RNA degradation for both the c-myc and c-Ha-ras series. The present results suggest that chimeric oligodeoxynucleotide analogues may be of considerable utility as antisense agents in systems where RNase H is thought to make a major contribution to inhibition of gene expression.
由内部磷酸二酯和末端甲基磷酸二酯部分组成的嵌合寡脱氧核苷酸，作为反义效应物具有许多有益特性。我们研究了用甲基磷酸二酯键逐步取代磷酸二酯对与互补RNA和DNA杂交稳定性的影响。发现随着甲基膦酸酯取代的增加，寡脱氧核苷酸/RNA异源双链体的解链温度（Tm）急剧下降。相比之下，对于类似的DNA异源双链体，观察到Tm的降低较小。在使用的富含G + C的人c-myc和富含A + T的人c-Ha-ras序列中都发现了这种杂交稳定性的不同降低，这表明甲基膦酸酯寡脱氧核苷酸类似物与RNA杂交的亲和力通常低于与DNA杂交的亲和力。采用RNase H测定法来确定所观察到的Tm降低是否会损害嵌合寡脱氧核苷酸指导RNA降解的能力。与预期相反，对于c-myc和c-Ha-ras系列，甲基膦酸酯取代的增加导致RNA降解速率增加。目前的结果表明，在认为RNase H对基因表达抑制起主要作用的系统中，嵌合寡脱氧核苷酸类似物作为反义剂可能具有相当大的实用性。
