Lingner J, Keller W
Department of Cell Biology, Biozentrum of the University of Basel, Switzerland.
Nucleic Acids Res. 1993 Jun 25;21(12):2917-20. doi: 10.1093/nar/21.12.2917.
Two commonly used methods to end-label RNA-molecules are 5'-end labeling by polynucleotide kinase and 3'-end labeling with pCp and T4 RNA ligase. We show here that RNA 3'-ends can also be labeled with the chain-terminating analogue cordycepin 5'-triphosphate (3'-deoxy-ATP) which is added by poly(A) polymerase. For a synthetic RNA it is shown that 40% of cordycepin becomes incorporated when the nucleotide is used at limiting concentrations and that with an excess of cordycepin 5'-triphosphate essentially all the RNA becomes modified at its 3'-end. The reaction is complete within minutes and the RNA product is uniform and suitable for sequence analysis. The efficiency of labeling varies with different RNA-molecules and is different from RNA ligase. Poly(A) polymerase preferentially labels longer RNA-molecules whereas short RNA-molecules are labeled more efficiently by T4 RNA ligase.
两种常用的RNA分子末端标记方法是用多核苷酸激酶进行5'末端标记以及用对氯苯磷酸（pCp）和T4 RNA连接酶进行3'末端标记。我们在此表明，RNA的3'末端也可用链终止类似物虫草素5'-三磷酸（3'-脱氧-ATP）进行标记，该类似物由聚腺苷酸聚合酶添加。对于合成RNA，结果表明，当核苷酸以限量浓度使用时，40%的虫草素会被掺入，而在过量的虫草素5'-三磷酸存在下，基本上所有RNA的3'末端都会被修饰。反应在几分钟内完成，RNA产物均匀且适合进行序列分析。标记效率因不同的RNA分子而异，且与RNA连接酶不同。聚腺苷酸聚合酶优先标记较长的RNA分子，而短RNA分子则被T4 RNA连接酶更有效地标记。
