Inoue H, Hayase Y, Iwai S, Ohtsuka E
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Nucleic Acids Symp Ser. 1988(19):135-8.
To cleave RNA molecules using E. coli RNase H in a site-specific manner, a short oligodeoxyribonucleotide (3-5 mer) linked with oligo(2'-O-methyl)ribonucleotide(s) was designed to be used as a DNA splint. Our model experiments with ribooligomer the splint duplexes (9 mers) and RNase H demonstrated that a tetradeoxynucleotide cluster seems to be sufficient for the enzyme recognition and the short DNA-containing splint directs a unique cleavage of RNA by RNase H. The method could be applied to longer ribooligonucleotide substrates. For example, when 3'm (GA)d(AGAA)m(GGU)5' was used as a hybridization strand, 32pUCUUUCUUCUUCCAGGAU was cleaved specifically between U11 and C12 to yield 32pUCUUUCUUCUU. This method will have a variety of applications for the study of RNA.
为了使用大肠杆菌核糖核酸酶H以位点特异性方式切割RNA分子，设计了一种与寡聚（2'-O-甲基）核糖核苷酸连接的短寡脱氧核糖核苷酸（3-5聚体）用作DNA夹板。我们用核糖寡聚体夹板双链体（9聚体）和核糖核酸酶H进行的模型实验表明，一个四脱氧核苷酸簇似乎足以供酶识别，并且含短DNA的夹板可指导核糖核酸酶H对RNA进行独特的切割。该方法可应用于更长的核糖寡核苷酸底物。例如，当使用3'm（GA）d（AGAA）m（GGU）5'作为杂交链时，32pUCUUUCUUCUUCCAGGAU在U11和C12之间被特异性切割，产生32pUCUUUCUUCUU。该方法在RNA研究中将有多种应用。
