Murakami A, Blake K R, Miller P S
Biochemistry. 1985 Jul 16;24(15):4041-6. doi: 10.1021/bi00336a036.
Oligodeoxynucleoside methylphosphonates, nucleic acid analogues that contain nonionic, 3'-5'-linked methylphosphonate internucleotide bonds, can be used to control mRNA function in living cells. In order to use analogues of defined sequence in biochemical and biological experiments, methods have been developed to characterize the chain length and sequence of oligodeoxyribonucleoside methylphosphonates and to study their interaction with mRNA. Methylphosphonate oligomers that terminate at the 5' end with a 3'-5' internucleotide phosphodiester bond are readily phosphorylated by polynucleotide kinase. Treatment of these 32P end labeled oligomers with aqueous piperidine randomly hydrolyzes the methylphosphonate linkage and upon gel electrophoresis produces a ladder of oligomers, which allows the chain length of the oligomer to be determined. The sequence of 32P end labeled oligonucleoside methylphosphonates can be determined by a modified chemical sequencing procedure. The interaction of the oligomers with rabbit globin mRNA was studied. The oligomers hybridize with mRNA in agarose gels. The stability of the hybrids increases with increasing chain length of the oligomer. The binding site of the oligomers on mRNA can be determined by using the oligomer as a primer for reverse transcriptase. The length of the resulting transcript is determined by polyacrylamide gel electrophoresis after removal of the methylphosphonate primer by treatment with piperidine. The results indicate that binding and priming ability of the oligonucleoside methylphosphonates are affected by the secondary structure of the mRNA.
寡脱氧核苷甲基膦酸酯是一类核酸类似物，其含有非离子型的、3'-5'-连接的甲基膦酸酯核苷酸间键，可用于控制活细胞中的mRNA功能。为了在生化和生物学实验中使用特定序列的类似物，已开发出方法来表征寡脱氧核糖核苷甲基膦酸酯的链长和序列，并研究它们与mRNA的相互作用。在5'端以3'-5'核苷酸间磷酸二酯键终止的甲基膦酸酯寡聚物很容易被多核苷酸激酶磷酸化。用哌啶水溶液处理这些32P末端标记的寡聚物会随机水解甲基膦酸酯键，在凝胶电泳时会产生一系列寡聚物条带，从而可以确定寡聚物的链长。32P末端标记的寡核苷甲基膦酸酯的序列可以通过改良的化学测序程序来确定。研究了这些寡聚物与兔珠蛋白mRNA的相互作用。这些寡聚物在琼脂糖凝胶中与mRNA杂交。杂交体的稳定性随着寡聚物链长的增加而增加。寡聚物在mRNA上的结合位点可以通过使用寡聚物作为逆转录酶的引物来确定。在用哌啶处理去除甲基膦酸酯引物后，通过聚丙烯酰胺凝胶电泳确定所得转录本的长度。结果表明，寡核苷甲基膦酸酯的结合和引发能力受mRNA二级结构的影响。
