Unnatural amino acid mutagenesis requires the in vitro production of aminoacyl tRNAs. Bacteriophage T4 RNA ligase is used to ligate α-amino-protected dCA amino acids to 74mer tRNA. Previously, there has been no facile method for evaluating the efficiency of this reaction prior to using the tRNA in translation. We report a novel use of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry in monitoring the formation of aminoacyl 76mer tRNA. This method is more efficient and precise than the traditional technique of gel electrophoresis. These MALDI conditions should also prove useful for analyzing aminoacyl tRNAs produced through aminoacyl tRNA synthetases and other methods.

The CCA end is common to all tRNAs as the universal site for amino acid attachment. It is also conserved in the 3′-terminal tRNA-like structure of viral genomes that can be aminoacylated by an aminoacyl-tRNA synthetase (Florentz & Giegé, 1995). During aminoacylation, the CCA end enters the catalytic center of an aminoacyl-tRNA synthetase and provides the site for chemistry to take place. The CCA end is also widely used in replication of retroviruses, the bacterial single-stranded RNA viruses, and duplex DNA plasmids of fungal mitochondria. During replication, the CCA end interacts with the template-specificity domain of reverse transcriptase or replicase and provides the initiation site for primer binding and extension (Maizels & Weiner, 1994). The importance of the CCA end in translation and in replication suggests that its conformation will play a role in these two fundamental processes.