tRNA 3′-amino-tailing for stable amino acid attachment

Stably charged amino acids attached to 3′-amino-tailed tRNA generated by protocol 2. (A) Levels of Arg-tRNA and Pro-tRNA, with or without the tail, are measured pre- and post-alkali hydrolysis. Charging levels of untailed tRNA^Arg and tRNA^Pro are 28% and 11%, respectively, whereas those of tailed tRNA^Arg and tRNA^Pro are 22% and 8%. After alkali hydrolysis, levels of stably charged tRNA are measured. (B) Percent stably charged and alkali-resistant aa-tRNAs generated by class I ArgRS, MetRS, and ValRS vs. class II LysRS and ProRS aminoacylation of the cognate amino-tailed tRNAs. The lower levels of Val and Lys retention (70%) relative to others (90%–100%) were likely attributable to the quality of the starting tRNA samples rather than the identity of the amino acid. The methodology is described in Figure 3; errors are standard deviation and n = 3.