Institute of Organic Chemistry, Center for Molecular Biosciences, University of Innsbruck, 6020 Innsbruck, Austria.
Nucleic Acids Res. 2010 Oct;38(19):6796-802. doi: 10.1093/nar/gkq508. Epub 2010 Jun 4.
The 3'-peptidyl-tRNA conjugates that possess a hydrolysis-resistant ribose-3'-amide linkage instead of the natural ester linkage would represent valuable substrates for ribosomal studies. Up to date, access to these derivatives is severely limited. Here, we present a novel approach for the reliable synthesis of non-hydrolyzable 3'-peptidyl-tRNAs that contain all the respective genuine nucleoside modifications. In short, the approach is based on tRNAs from natural sources that are site-specifically cleaved within the TΨC loop by using DNA enzymes to obtain defined tRNA 5'-fragments carrying the modifications. After dephosphorylation of the 2',3'-cyclophosphate moieties from these fragments, they are ligated to the respective 3'-peptidylamino-tRNA termini that were prepared following the lines of a recently reported solid-phase synthesis. By this novel concept, non-hydrolyzable 3'-peptidyl-tRNA conjugates possessing all natural nucleoside modifications are accessible in highly efficient manner.
具有水解抗性的核糖 3'-酰胺键而不是天然酯键的 3'-肽基-tRNA 缀合物将成为核糖体研究的有价值的底物。迄今为止，这些衍生物的获取受到严重限制。在这里，我们提出了一种可靠合成含有所有天然核苷修饰的非水解 3'-肽基-tRNA 的新方法。简而言之，该方法基于天然来源的 tRNA，这些 tRNA 在 TΨC 环内通过 DNA 酶进行特异性切割，以获得带有修饰的定义明确的 tRNA 5'-片段。从这些片段中去除 2'、3'-环磷酸酯部分后，将它们连接到相应的 3'-肽基氨基-tRNA 末端，这些末端是按照最近报道的固相合成方法制备的。通过这个新的概念，可以高效地获得具有所有天然核苷修饰的非水解 3'-肽基-tRNA 缀合物。
