RNA-directed peptide synthesis across a nicked loop

Abstract

Ribosomal translation at the origin of life requires controlled aminoacylation to produce mono-aminoacyl esters of tRNAs. Herein, we show that transient annealing of short RNA oligo:amino acid mixed anhydrides to an acceptor strand enables the sequential transfer of aminoacyl residues to the diol of an overhang, first forming aminoacyl esters then peptidyl esters. Using N-protected aminoacyl esters prevents unwanted peptidyl ester formation in this manner. However, N-acyl-aminoacyl transfer is not stereoselective.

Graphical Abstract

Figure 1.

Peptide synthesis via alanyl mixed anhydride transfer. (A) Schematic representation of aminoacyl transfer; (B) HPLC trace of alanyl mixed anhydride transfer over time. The broken box shows a new peak other than the 10-mer acceptor strand and its 2′/3′-Ala-esters, (indicated by the dashed arrow); (C) time courses of the 2′/3′-Ala-esters (thin line) and the 2′/3′-dipeptidyl-esters (putatively) corresponding to the new peaks over time (corrected for the mixed anhydride yield); (D) MALDI-TOF results for the aminoacyl-transfer reaction, 2′/3′-Ala-Ala-esters, calculated 3260.6, found 3260.7; 2′/3′-trialanyl-esters, calculated 3331.7, found 3331.8. A and G in bold and coloured circles indicate amino acids alanine and glycine. Conditions: acceptor strand 100 μM, Ala-mixed anhydride donor strand (3 equivalents relative to the acceptor strand), HEPES 50 mM, NaCl 100 mM, MgCl₂ 5 mM, pH 6.8, 16°C.

Figure 2.

Peptide synthesis via transfer of an alanyl residue followed by N-formyl-aminoacyl transfer. HPLC traces of (A) fAla and (B) fGly transfer to 2′/3′-Ala-ester acceptors. The dotted lines show two new peaks corresponding to 2′/3′-N-formyl-dipeptidyl-ester acceptors over time. fAla and fGly ctrl indicate the reaction of fAla-/fGly-mixed anhydride donor transferred directly to the 2′/3′-diol of a 10-mer acceptor to give 2′/3′-fAla/fGly-esters. * indicates 2′/3′-fAla/fGly-esters. (C) The respective MALDI spectra and data. A and G in bold and coloured circles indicate amino acids alanine and glycine. Conditions: acceptor strand 100 μM, Ala-mixed anhydride donor strand (1 eq.) and N-formyl-aminoacyl mixed anhydride donor strand (2 eq.) to the acceptor strand, HEPES 50 mM, NaCl 100 mM, MgCl₂ 5 mM, pH 6.8, 10°C. fAla/fGly ctrl was performed at pH 8.0. Stacked HPLC traces are staggered by 15 s each for clarity.

Figure 3.

Time courses and MALDI characterization of 2′/3′-N-formyl-dipeptidyl RNA. Timepoint 0 was set at the peak yield of 2′/3′-aminoacyl-esters at which point the formylaminoacyl-mixed anhydride donor was added. The solid line in the time course represents the percentage yield of 2′/3′-fGly-/fAla-dipeptidyl-esters relative to the 2′/3′-aminoacyl-esters. The broken line represents the consumption due to hydrolysis and formylaminoacylation of the 2′/3′-aminoacyl-esters. A, G, L, P in bold and coloured circles indicate amino acids alanine, glycine, leucine, and proline. Conditions: acceptor strand 100 μM, aminoacyl-mixed anhydride tetramer donor strand (1 eq.) and N-formyl-aminoacyl mixed anhydride pentamer donor strand (2 eq.) to the acceptor strand, HEPES 50 mM, NaCl 100 mM, MgCl₂ 5 mM, pH 6.8, 10°C.

Figure 4.

Peptidyl-RNA synthesis is achieved by donor strand replacement. Time courses for the formation of 2′/3′-fGly-Ala-esters using different length Ala-mixed anhydride donor strands. A and G in bold and coloured circles indicate amino acids alanine and glycine. Conditions: acceptor strand 100 μM, Ala-mixed anhydride initial donor strand (1 eq.), N-formyl-glycyl-mixed anhydride donor strand (2 eq.) added at the peak formation of 2′/3′-Ala-esters, HEPES 50 mM, NaCl 100 mM, MgCl₂ 5 mM, pH 6.8, 10°C.