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 availableEscherichia colitRNAVal . 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.
