Deoxyribozyme-based, semisynthetic access to stable peptidyl-tRNAs exemplified by tRNAVal carrying a macrolide antibiotic resistance peptide.
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.
Institute of Organic Chemistry, Center for Molecular Biosciences (CMBI), University of Innsbruck, Innsbruck, Austria.
Dagmar
D
Graber
Krista
K
Trappl
Jessica
J
Steger
Anna-Skrollan
AS
Geiermann
Lukas
L
Rigger
Holger
H
Moroder
Norbert
N
Polacek
Ronald
R
Micura
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001483:Base Sequence; D021881:DNA, Catalytic / Q000378:metabolism*; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli; D018942:Macrolides / Q000494:pharmacology*; D013058:Mass Spectrometry; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D010455:Peptides; D019800:Phenol / Q000737:chemistry; D010766:Phosphorylation; D020871:RNA Stability; D012329:RNA, Bacterial / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000138:chemical synthesis*; D012366:RNA, Transfer, Val / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D000900:Anti-Bacterial Agents; D021881:DNA, Catalytic; D018942:Macrolides; D010455:Peptides; D012329:RNA, Bacterial; D012346:RNA, Transfer, Amino Acyl; D012366:RNA, Transfer, Val; C019037:tRNA, peptidyl-; D019800:Phenol