In vitro non-natural amino acid mutagenesis using a suppressor tRNA generated by the cis-acting hepatitis delta virus ribozyme.
In vitro non-natural amino acid mutagenesis requires aminoacyl-charged suppressor transfer RNAs which read an internal stop codon. For the synthesis of aminoacyl-tRNAs loaded with non-natural amino acids, T4 RNA ligase is used to ligate a chemically synthesised aminoacyl-dinucleotide to a truncated 74mer tRNA(-CA) lacking the two 3' end nucleotides. The 74mer tRNA(-CA) in turn is generated by run-off transcription from a linearised plasmid encoding the tRNA sequence under control of the T7 promoter. Transcripts with heterogeneous ends are commonly obtained, which interfere with subsequent reactions such as ligation or translation. Here we report an improved procedure for the generation and chromatographic purification of large amounts of homogeneous 3' end tRNA(-CA) by hepatitis delta virus ribozyme cis-cleavage and the first application of this tRNA to in vitro non-natural amino acid mutagenesis. Stop codon suppression is increased compared to conventionally synthesised suppressor tRNA; 2.5 microg of mutated protein was synthesised in a 50 microl batch reaction.
Röhrig
Christoph H
CH
Department of Chemistry, University of Konstanz, Fach M 725, D-78457 Konstanz, Germany.
Retz
Oliver A
OA
Meergans
Thomas
T
Schmidt
Richard R
RR
D019943:Amino Acid Substitution / Q000235:genetics*; D018014:Gene Transfer Techniques; D016153:Genes, Suppressor; D003698:Hepatitis Delta Virus / Q000235:genetics*; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D016337:RNA, Catalytic / Q000235:genetics*; D012343:RNA, Transfer / Q000235:genetics*; D014162:Transfection / Q000379:methods*
D016337:RNA, Catalytic; D012343:RNA, Transfer