Stereoselectivity of Aminoacyl-RNA Loop-Closing Ligation

Abstract

The origin of amino acid homochirality remains an unresolved question in the origin of life. The requirement of enantiopure nucleotides for nonenzymatic RNA copying strongly suggests that the homochirality of nucleotides and RNA arose early. However, this leaves open the question of whether and how homochiral RNA subsequently imposes biological homochirality on other metabolites, including amino acids. Previous studies have reported moderate stereoselectivity for various aminoacyl-RNA transfer reactions. Here, we examine aminoacyl-RNA loop-closing ligation, a reaction that "captures" aminoacylated RNA in a stable phosphoramidate product, such that the amino acid bridges two nucleotides in the RNA backbone. We find that the rate of this reaction is much higher for RNA aminoacylated with L-amino acids than for RNA aminoacylated with D-amino acids. We present an RNA sequence that nearly exclusively captures L-amino acids in loop-closing ligation. Finally, we demonstrate that ligation of aminoacyl-L-RNA results in an inverse stereoselectivity for D-amino acids. The observed stereochemical link between D-RNA and L-amino acids in the synthesis of RNA stem-loops containing bridging amino acids constitutes a stereoselective structure-building process. We suggest that this process led to a selection for the evolution of aminoacyl-RNA synthetase ribozymes that were selective for L-amino acids, thereby setting the stage for the subsequent evolution of homochiral peptides and, ultimately, protein synthesis.

1. Outline of the Reactions and RNA Architectures Studied in this Work.

1

Reaction Schemes and Time Courses for aminoacyl-RNA loop-closing ligation and hydrolysis. (A) Left: reaction scheme for aminoacyl-RNA loop-closing ligation using an imidazole-activated capture strand. Right: time course of the ligation reaction. (B, C) Left: reaction schemes for aminoacyl-RNA hydrolysis under duplex and single-stranded conditions. Right: time courses of the hydrolysis reaction. Reaction rates are presented in Tables , S1 and S2, and gel images are shown in Figure S1. All reactions were conducted at 0 °C in 5 mM MgCl₂, 100 μM Na₂EDTA, 100 mM imidazole, pH 8.0, and 5 μM RNA oligonucleotides. Shaded regions envelope the 95% prediction interval as determined from the kinetic model.

2

Aminoacyl-RNA loop-closing ligation for RNA architecture 1 with four different amino acids. Top: RNA architecture. Bottom: time course of loop-closing ligations. The % ligated product was determined as in Figure from gel images. All reactions were conducted in three technical replicates at 0 °C in 5 mM MgCl₂, 100 μM Na₂EDTA, and 100 mM imidazole, pH 8.0, with 5 μM RNA oligonucleotides. Shaded regions envelope the 95% prediction interval as determined from the kinetic model. See also Figure S3 for hydrolysis timecourses.

3

Aminoacyl-RNA ligation rates for three different RNA architectures and four different amino acids. Top: schematics for RNA architectures 1, 2, and 3. Bottom: observed rates for aminoacyl-RNA loop-closing ligation were for the three RNA architectures with the amino acids alanine, leucine, lysine, and proline. Ligation reactions were conducted in triplicate at 0 °C in 5 mM MgCl₂, 100 μM Na₂EDTA, 100 mM imidazole, pH 8.0, with 5 μM RNA oligonucleotides. Dark green bars: RNA aminoacylated with L-amino acids; yellow bars: RNA aminoacylated with D-amino acids. Ligation rates were derived from kinetic models using ligation yields and experimental rates for aminoacyl hydrolysis (see Kinetic Analysis section in the Materials and Methods). All L- and D- pairwise comparisons were statistically significant, with L-aminoacyl loop-closing ligation rates higher than D- in all cases. Error bars and significance were estimated as detailed in the Statistical Analysis section in the Materials and Methods.

4

Loop-closing ligation with aminoacylated L-RNA. Rate constants for loop-closing ligation with D-RNA (left) and L-RNA (right). The D- and L- versions of RNA 1 exhibit high stereoselectivity for l- and d-lysine, respectively. Inversion of stereoselectivity is seen for all three D- vs L-RNAs. Each reaction was conducted in three technical replicates at 0 °C in 5 mM MgCl₂, 100 μM Na₂EDTA, 100 mM imidazole, and pH 8.0 with 5 μM RNA oligonucleotides. Dark green bars: RNA aminoacylated with L-amino acids; yellow bars: RNA aminoacylated with D-amino acids. Error bars and significance (p < 0.05) were estimated using the Monte Carlo method as detailed in the Statistical Analysis section in the Materials and Methods.