Graber Dagmar, Trappl Krista, Steger Jessica, Geiermann Anna-Skrollan, Rigger Lukas, Moroder Holger, Polacek Norbert, Micura Ronald
Institute of Organic Chemistry, Center for Molecular Biosciences (CMBI), University of Innsbruck, Innsbruck, Austria.
Methods Mol Biol. 2012;848:201-13. doi: 10.1007/978-1-61779-545-9_13.
We present a protocol for the reliable synthesis of non-hydrolyzable 3'-peptidyl-tRNAs that contain all the respective genuine nucleoside modifications. The approach is exemplified by tRNA(Val)-3'-NH-VFLVM-NH(2) and relies on commercially available Escherichia coli tRNA(Val). This tRNA was cleaved site-specifically within the TΨC loop using a 10-23 type DNA enzyme to obtain a 58 nt tRNA 5'-fragment which contained the modifications. After cleavage of the 2',3'-cyclophosphate moiety from the 5'-fragment, it was ligated to the 18 nt RNA-pentapeptide conjugate which had been chemically synthesized. By this methodology, tRNA(Val)-3'-NH-VFLVM-NH(2) is accessible in efficient manner. Furthermore, we point out that the approach is applicable to other types of tRNA.
我们提出了一种可靠合成不可水解的3'-肽基-tRNA的方案，该tRNA包含所有各自的天然核苷修饰。以tRNA(Val)-3'-NH-VFLVM-NH(2)为例说明了该方法，该方法依赖于市售的大肠杆菌tRNA(Val)。使用10-23型DNA酶在TΨC环内进行位点特异性切割，得到一个58 nt的tRNA 5'-片段，该片段含有修饰。从5'-片段上切割掉2',3'-环磷酸部分后，将其连接到化学合成的18 nt RNA-五肽缀合物上。通过这种方法，可以高效地获得tRNA(Val)-3'-NH-VFLVM-NH(2)。此外，我们指出该方法适用于其他类型的tRNA。
