一种通过RNA连接酶磷酸化对多聚核糖核苷酸进行3'标记的新方法及其在连接反应的3'修饰中的应用。
A new method for 3'-labelling of polyribonucleotides by phosphorylation with RNA ligase and its application to the 3'-modification for joining reactions.
作者信息
Ohtsuka E, Uemura H, Doi T, Miyake T, Nishikawa S, Ikehara M
出版信息
Nucleic Acids Res. 1979 Feb;6(2):443-54. doi: 10.1093/nar/6.2.443.
P1-Adenosine 5'-P2-o-nitrobenzyl pyrophosphate (nbzlppA) has been synthesized as a substrate for T4 RNA ligase catalyzed 3'-phosphorylation. Incubation of oligoribonucleotides and nbzlppA with RNA ligase yielded oligoribonucleotides having a 3'-o-(o-nitrobenzyl) phosphate. Photochemical removal of the o-nitrobenzyl group provided the free 3'-phosphate. Using [P2-32P] nbzlppA, 3'-termini of oligoribonucleotides could be labelled with 32P. This reaction was applied to modify the 3'-end of donor molecules in joining reaction with RNA ligase. A trinucleotide U-A-G was converted to U-A-Gpnbzl and phosphorylated with polynucleotide kinase. pU-A-Gpnbzl was then joined to an acceptor trinucleotide A-U-G to yield A-U-G-U-A-Gp.
P1-腺苷5'-P2-邻硝基苄基焦磷酸（nbzlppA）已被合成为T4 RNA连接酶催化3'-磷酸化的底物。将寡核糖核苷酸和nbzlppA与RNA连接酶一起温育，得到具有3'-O-（邻硝基苄基）磷酸的寡核糖核苷酸。邻硝基苄基的光化学去除提供了游离的3'-磷酸。使用[P2-32P] nbzlppA，寡核糖核苷酸的3'-末端可用32P进行标记。该反应应用于在与RNA连接酶的连接反应中修饰供体分子的3'-末端。三核苷酸U-A-G被转化为U-A-Gpnbzl并用多核苷酸激酶进行磷酸化。然后将pU-A-Gpnbzl与受体三核苷酸A-U-G连接，得到A-U-G-U-A-Gp。
Zotero 插件