We began by exploring the extent to which tRNAs could be acylated with monomers carrying a reactive dicarbonyl motif, and the extent to which these acylated tRNAs could initiate the in vitro translation of short peptides (Fig. ). N-formyl methionine (fM) and monomers 1-4 (Fig. ) were converted into activated 3,5-dinitrobenzyl (DNB) esters (Supplementary Fig. ) and their ability to acylate a model tRNA microhelix (MH, Supplementary Fig. and) in the presence of flexizyme dFx was evaluated as a function of time and pH. The extent of MH acylation was monitored using both acidic polyacrylamide gel electrophoresis (aPAGE) (Supplementary Fig. ) and intact tRNA LC-MS (Supplementary Fig. ). Although gel electrophoresis failed to resolve any acylated MH product from the non-acylated MH substrate, presumably due to similarity in mass and charge, intact tRNA LC-MS revealed that both 2-DNB and 3-DNB were substrates for dFx in MH-acylation reactions, especially at higher pH and longer incubation times (Supplementary Table ). It is likely that the activity of 3-DNB as a substrate for dFx was previously missed because of the low resolving power of gel electrophoresis . Although we were unable to identify conditions that led to detectable MH acylation by the 3,5-dinitrobenzyl ester of monomer 1 (Fig. ), in the case of monomer 4, intact tRNA LC-MS detected formation of acylated MH in 0.6% yield (Supplementary Fig. and DYKDDDDK is a FLAG-tag that enables peptide isolation and enrichment prior to LC-HRMS analysis. b, Yields for flexizyme-promoted acylation of MH and tRNA fMet by the DNB esters of fM and monomers 1-4. Yields were determined using intact tRNA LC-MS . ND indicates that an acylated product was not detected. A 260 chromatograms and deconvoluted mass spectra (inset box) of tRNA fMet acylated with the DNB esters of c, fM (fM-DNB), d, monomer 2 (2-DNB), and e, monomer 3 (3-DNB). Extracted ion chromatograms of peptide P1 containing the N-terminal residue f, fM, g, 2, or h, 3 in the presence of acylated or non-acylated tRNA fMet . The inset shows the mass spectrum of each peptide. All acylations were performed in triplicate. Data shown is representative.