Synthetic tRNA Preparation and Labeling

The template plasmid for human tRNA^iMet-CAT-2 encoding T7 promoter, hammerhead ribozyme, and nucleotides 9–72 of the tRNA followed by CCA was generated by overlap extension PCR and cloned in pGEM-1 (Promega). The templates for human tRNA^Asp-GTC-2 and the variant tRNA^Asp-GTC-A9G were generated by gene synthesis (BIOMATIK), and encoded T7 promoter, hammerhead ribozyme, nucleotides 9–72 of the tRNA followed by CCA, and a 3′ HDV ribozyme. The tRNA^iMet-CAT-2 and the two tRNA^Asp-GTC templates were cleaved with BstNI and HindIII, respectively, and the tRNA fragments were produced as run-off transcripts guided by T7 RNA polymerase. The in vitro transcribed tRNA fragments were 5′-end labelled and ligated by splint-guided ligation to a synthetic RNA corresponding to the first eight nucleotides of the respective tRNA. In vitro transcription, ³²P labeling at position 9, purification, and splint-guided ligation were carried out as previously described (10 (link)).