Deoxyribozyme-based, semisynthetic access to stable peptidyl-tRNAs exemplified by tRNAVal carrying a macrolide antibiotic resistance peptide

Methods Mol Biol. 2012:848:201-13. doi: 10.1007/978-1-61779-545-9_13.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Bacterial Agents / pharmacology
  • Base Sequence
  • DNA, Catalytic / metabolism*
  • Drug Resistance, Bacterial*
  • Escherichia coli
  • Macrolides / pharmacology*
  • Mass Spectrometry
  • Models, Molecular
  • Nucleic Acid Conformation
  • Peptides*
  • Phenol / chemistry
  • Phosphorylation
  • RNA Stability*
  • RNA, Bacterial / metabolism
  • RNA, Transfer, Amino Acyl / chemical synthesis*
  • RNA, Transfer, Val / chemical synthesis
  • RNA, Transfer, Val / chemistry*
  • RNA, Transfer, Val / isolation & purification
  • RNA, Transfer, Val / metabolism

Substances

  • Anti-Bacterial Agents
  • DNA, Catalytic
  • Macrolides
  • Peptides
  • RNA, Bacterial
  • RNA, Transfer, Amino Acyl
  • RNA, Transfer, Val
  • tRNA, peptidyl-
  • Phenol