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Deoxyribozyme-Based, Semisynthetic Access to Stable Peptidyl-tRNAs Exemplified by tRNAVal Carrying a Macrolide Antibiotic Resistance Peptide

2012; Springer Science+Business Media; Linguagem: Inglês

10.1007/978-1-61779-545-9_13

1940-6029

Dagmar Graber, Krista Trappl, Jessica Steger, Anna‐Skrollan Geiermann, Lukas Rigger, Holger Moroder, Norbert Polacek, Ronald Micura,

Genomics and Phylogenetic Studies

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 tRNAVal-3′-NH-VFLVM-NH2 and relies on commercially available Escherichia coli tRNAVal. 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, tRNAVal-3′-NH-VFLVM-NH2 is accessible in efficient manner. Furthermore, we point out that the approach is applicable to other types of tRNA.