Hayase Y, Inoue H, Ohtsuka E
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Biochemistry. 1990 Sep 18;29(37):8793-7. doi: 10.1021/bi00489a041.
In order to cleave RNA at specific positions in Escherichia coli formylmethionine tRNA, RNase H and complementary chimeric oligonucleotides consisting of DNA and 2'-O-methyl-RNA (Inoue et al. (1987) FEBS Lett. 215, 327] were used. Specific cleavages in the D loop, anticodon loop, T psi C loop, anticodon stem, and acceptor stem were investigated. Virtually unique hydrolyses with RNase H were observed at the T psi C loop, anticodon stem, and acceptor stem when relatively longer chimeric oligonucleotides (20-mer) were used. An efficient cleavage at the anticodon was obtained with a chimeric 13-mer when the higher structure of the tRNA was broken by hybridization with a 20-mer at the acceptor as well as the T psi C stem region. It was found that stabilities of hybrids with chimeric oligonucleotides and the presence of minor nucleosides affect the cleavage of tRNA by this approach.
为了在大肠杆菌甲酰甲硫氨酸tRNA的特定位置切割RNA，使用了核糖核酸酶H和由DNA与2'-O-甲基-RNA组成的互补嵌合寡核苷酸（井上等人，（1987年），《欧洲生物化学学会联合会快报》215, 327]）。研究了D环、反密码子环、TψC环、反密码子茎和受体茎中的特异性切割。当使用相对较长的嵌合寡核苷酸（20聚体）时，在TψC环、反密码子茎和受体茎处观察到核糖核酸酶H几乎独特的水解作用。当tRNA的高级结构通过在受体以及TψC茎区域与20聚体杂交而被破坏时，使用13聚体的嵌合寡核苷酸可在反密码子处获得有效切割。发现与嵌合寡核苷酸杂交体的稳定性以及稀有核苷的存在会影响通过这种方法对tRNA的切割。
