Spontaneous formation and base pairing of plausible prebiotic nucleotides in water

Abstract

The RNA World hypothesis presupposes that abiotic reactions originally produced nucleotides, the monomers of RNA and universal constituents of metabolism. However, compatible prebiotic reactions for the synthesis of complementary (that is, base pairing) nucleotides and mechanisms for their mutual selection within a complex chemical environment have not been reported. Here we show that two plausible prebiotic heterocycles, melamine and barbituric acid, form glycosidic linkages with ribose and ribose-5-phosphate in water to produce nucleosides and nucleotides in good yields. Even without purification, these nucleotides base pair in aqueous solution to create linear supramolecular assemblies containing thousands of ordered nucleotides. Nucleotide anomerization and supramolecular assemblies favour the biologically relevant β-anomer form of these ribonucleotides, revealing abiotic mechanisms by which nucleotide structure and configuration could have been originally favoured. These findings indicate that nucleotide formation and selection may have been robust processes on the prebiotic Earth, if other nucleobases preceded those of extant life.

Figure 1. Spontaneous formation of nucleotides by barbituric acid (BA) and melamine in water.

(a) Chemical structures of the four canonical nucleobases of RNA shown in their Watson–Crick base pairs, and BA with melamine in an analogous Watson–Crick-like base pair. The R group on all nucleobases, which is H for the free nucleobases, indicates the position of ribose attachment through a glycosidic bond on the canonical bases and for melamine and BA in the current work. (b) Chemical structures of the two C-nucleotide anomers of BA-ribosyl-monophosphate (C-BMP) and ¹H NMR spectrum of a BA+R5P crude reaction mixture revealing the formation of α-C-BMP and β-C-BMP. (c) Chemical structures of the two anomers of melamine-ribosyl-monophosphate (MMP) and the ¹H NMR spectrum of a melamine+R5P crude reaction mixture revealing the formation of α-MMP and β-MMP. The anomeric proton resonances for each nucleotide are labelled, and those for R5P are marked with † indicating α-R5P, and ‡ indicating β-R5P. Relative integrated intensities of the nucleotide anomeric resonances show that, for these two reactions, the total C-BMP yield was 82%, and the total MMP yield was 55%. The HOD peaks have been removed from the NMR spectra for clarity. See the Methods for reaction details.

Figure 2. NMR characterization of C-BMP nucleotides.

(a) Chemical structure of α-C-BMP and β-C-BMP with arrows indicating through-space proton–proton magnetization transfer (ROE) as shown in c. (b) Heteronuclear single-quantum correlation (HSQC) and heteronuclear multiple bond correlation (HMBC) spectra showing ¹H-¹³C couplings for α-C-BMP and β-C-BMP. ¹H-¹³C correlation observed between H1′ and C5 of β-C-BMP and H1′-C4/C6 of α-C-BMP and β-C-BMP in the HMBC as well as the absence of H5-C5 correlations in the HSQC support the C-nucleoside assignment. α-Anomer cross peaks are shown in red, β-anomer cross peaks in blue and overlapping cross peaks in purple. ¹H Correlation spectroscopy (COSY) spectrum of a mixture of C-BMP nucleotides is provided in the Supplementary Information. (c) ¹H NMR and 1D ROE spectra of a solution containing a 1:2 ratio of α-C-BMP to β-C-BMP. (Top) ¹H NMR spectrum with resonance assignments as indicated in a. (Middle) Irradiation of the H1′ of α-C-BMP results in through space magnetization transfer to the H2′ and H3′ of α-C-BMP. (Bottom) Irradiation of the H1′ of β-C-BMP results in through space magnetization transfer to the H4′ of β-C-BMP. * Indicates TOCSY transfer from β-H1′ to β-H2′ and β-H3′.

Figure 3. NMR characterization of MMP nucleotides.

(a) Chemical structure of α-MMP and β-MMP with arrows indicating through-space proton–proton magnetization transfer as shown in c. (b) HSQC and HMBC spectra showing ¹H-¹³C couplings for α-MMP and β-MMP. ¹H-¹³C correlation observed in the HMBC (H1′–C5) of α-MMP and β-MMP show coupling between ribose and melamine. α-Anomer cross peaks are shown in red, β-anomer cross peaks in blue and overlapping cross peaks in purple. ¹H COSY spectrum of a mixture of MMP nucleotides is provided in the Supplementary Information. (c) ¹H NMR and 1D ROE spectra of a solution containing an approximately equal concentration of α-MMP to β-MMP. (Top) ¹H NMR spectrum with resonance assignments as indicated in a. (Middle) Irradiation of the H1′ of α-MMP results in through space magnetization transfer to the H3′ of α-MMP. (Bottom) Irradiation of the H1′ of β-MMP results in through space magnetization transfer to the H2′ and H4′ of β-MMP. * Indicates TOCSY transfer from β-H1′ to β-H3′.

Figure 4. Nucleotides assemble into supramolecular polymers.

(a) CD spectra of melamine-R5P and BA-R5P crude reaction mixtures, separate and combined at 5 °C. Black curve is mixture of melamine-R5P and BA-R5P crude reaction mixtures; red curve is BA-R5P crude reaction mixture; blue curve is melamine-R5P crude reaction mixture. Ultraviolet spectra associated with CD spectra are provided in Supplementary Fig. 11. (b–e) AFM topographic images of (b) mixture of melamine-R5P and BA-R5P crude reaction mixtures, (c) mixture of purified C-BMP and MMP, (d) purified C-BMP mixed with melamine, (e) purified MMP mixed with BA. Inset in b shows height measurements of blue line in image. Scale bar in b is 300 nm, and all AFM images are shown at the same magnification. (f) Chemical structures of MMP and C-BMP and their association into 2 nm wide stacked hexads. The green R and green spheres indicate R5P. The similarity of AMP to MMP and UMP to C-BMP, and the inability of these canonical nucleosides to assemble in aqueous solution is also illustrated. All solutions contained 1 M NaCl.

Figure 5. MMP assembly and anomerization in the presence of BA.

(a) Plot of NMR visible resonance intensity of unassembled α-MMP and β-MMP as a function of temperature in a solution containing 50 mM MMP and BA. (b) Plot showing fraction of both MMP anomers assembled at various temperatures (plot generated by subtracting data shown in a from measured total concentration of α-MMP and β-MMP in each sample). (c) Plot showing the change in anomeric ratio (by percent) of β-MMP as a function of time in solutions containing both MMP and BA, or MMP alone. Samples were maintained at 5 °C during the experiment and diluted just prior to analysis to disassemble MMP in order to enable quantification of the total MMP in solution by NMR. (d) Schematic showing preferential assembly (stacked hexads) and anomerization. All solutions contained 0.3 M NaCl.