Triplet-Encoded Prebiotic RNA Aminoacylation

Abstract

The encoding step of translation involves attachment of amino acids to cognate tRNAs by aminoacyl-tRNA synthetases, themselves the product of coded peptide synthesis. So, the question arises─before these enzymes evolved, how were primordial tRNAs selectively aminoacylated? Here, we demonstrate enzyme-free, sequence-dependent, chemoselective aminoacylation of RNA. We investigated two potentially prebiotic routes to aminoacyl-tRNA acceptor stem-overhang mimics and analyzed those oligonucleotides undergoing the most efficient aminoacylation. Overhang sequences do not significantly influence the chemoselectivity of aminoacylation by either route. For aminoacyl-transfer from a mixed anhydride donor strand, the chemoselectivity and stereoselectivity of aminoacylation depend on the terminal three base pairs of the stem. The results support early suggestions of a second genetic code in the acceptor stem.

Figure 1

RNA aminoacylation via activated phosphates. (A) Activated phosphates required for RNA ligation chemistry also react with amino acids to give phosphoramidates and/or mixed anhydrides depending on pH; (B) production of 3′-aminoacyl-RNA by indirect and direct transfer of aminoacyl groups from phosphoramidates and mixed anhydrides, respectively.

Figure 2

Divergent patterns in the first three nucleotides in the stem. (A–F) Time courses showing the aminoacyl-transfer kinetics with identical overhang “UUCCA”, but base pairs changed in the stem (indicated by the red font); solid lines represent the best fits from nonlinear regression analyses. (G) Plot of sequencing results after mixed anhydride aminoacylation selection using a partially randomized stem (5′-GAUUCNNNUUCCA) showing correlation between amino acids. Sequence logo represents the top 10% of trinucleotides from the raw reading numbers. Axes of the scatter plots are read numbers and are omitted for simplicity. Pearson correlation coefficients are shown in the upper right corner. Aminoacyl-transfer conditions: both oligos (100 μM), NaCl (100 mM), MgCl₂ (5 mM), HEPES (50 mM, pH 6.8).

Figure 3

Stereochemical coding dictated by a stem-terminal sequence. Time courses showing the mixed anhydride aminoacyl-transfer kinetics with varied trinucleotide stem sequences according to the top selectivity score (A) l-Ala; (B) l-Leu; (C) l-Leu and l-Val; (D) l-Pro; (E) Gly, (A–E) all with 1 equiv of donor transferred at 10 °C; (F) 1 equiv of tetramer donor for l-Ala at 10 °C; (G) 1 equiv of trimer donor for l-Ala at 10 °C; (H) 1 or 3 equiv of trimer donor for l-Ala at 16 °C. T_M, melting temperature; T_R, temperature at which the aminoacyl-transfer reaction was conducted. Solid lines represent the best fits from nonlinear regression analyses. Conditions: both oligos (100 μM), NaCl (100 mM), MgCl₂ (5 mM), HEPES (50 mM, pH 6.8).

Figure 4

Representative structures for l-Ala (A, B) and d-Ala systems (C, D), selected from the energy landscape databases. Comparing panels A and C suggests an explanation for the observed stereoselectivity. For l-Ala (A), the Burgi-Dunitz trajectory is freely accessible (black arrow). In contrast, the trajectory is blocked in d-Ala (C). For panels B and D, the top base pair in the stem is frayed in both cases, giving more flexible structures. The aminoacyl moiety is highlighted in green, and the top pair of bases in the stem is highlighted in orange.