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Updated: May 25, 2026

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses
Published on: February 25, 2011
Deoxyribozyme-based, semisynthetic access to stable peptidyl-tRNAs exemplified by tRNAVal carrying a macrolide
Dagmar Graber1, Krista Trappl, Jessica Steger
1Institute of Organic Chemistry, Center for Molecular Biosciences (CMBI), University of Innsbruck, Innsbruck, Austria.
Abstract:
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.
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