Angelin Prasad

Synthesizing peptide sequences from non-natural amino acids is a difficult task for a number of reasons, including but not limited to issues maintaining a correct reading frame, size of the amino acid, and charging tRNAs with their non-cognate amino acids. Flexizyme is an enzyme that helps charge tRNAs with non natural amino acids and is typically used in reactions with in-vitro transcribed tRNAs. In-vivo transcribed tRNA’s may be a better option for flexizyme reactions due to hypothesized better stability from their post-transcriptional modifications, which are not present in in-vitro transcribed tRNA’s. A more stable tRNA should theoretically be able to participate in translation reactions at a higher rate and produce more peptides. The relative stabilities of in-vitro vs in-vivo tRNA’s were assessed for tRNAfMET by assessing their Tm values through UV-Vis Spectroscopy. Then, both in-vitro and in-vivo transcribed tRNAfMET were used in Hibit luminescence assays to determine their yields and efficiency. Results indicated that in-vivo transcribed tRNAs had more consistent and higher Tm values. However, the Hibit luminescence assays failed to indicate high translation yields and significant differences in translation rates between in-vitro and in-vivo transcribed tRNAs. Therefore, we are unable to conclude that the presence of post-transcriptional modifications makes a significant difference in the improvement of the flexizyme reactions. Future steps include assessing for limiting factors and issues with the template being used to determine whether the issue lies with the system or the tRNAs itself.