Oligoarginine peptides slow strand annealing and assist non-enzymatic RNA replication

Abstract

The non-enzymatic replication of RNA is thought to have been a critical process required for the origin of life. One unsolved difficulty with non-enzymatic RNA replication is that template-directed copying of RNA results in a double-stranded product. After strand separation, rapid strand reannealing outcompetes slow non-enzymatic template copying, which renders multiple rounds of RNA replication impossible. Here we show that oligoarginine peptides slow the annealing of complementary oligoribonucleotides by up to several thousand-fold; however, short primers and activated monomers can still bind to template strands, and template-directed primer extension can still occur, all within a phase-separated condensed state, or coacervate. Furthermore, we show that within this phase, partial template copying occurs even in the presence of full-length complementary strands. This method to enable further rounds of replication suggests one mechanism by which short non-coded peptides could have enhanced early cellular fitness, and potentially explains how longer coded peptides, that is, proteins, came to prominence in modern biology.

Figure 1

The reannealing problem and a proposed solution. a) Complete template-directed primer extension results in a full-length duplex (newly synthesized strand in maroon, original template in gray) (1). After strand separation by heating (2), subsequent cooling results in rapid reannealing of the newly synthesized complementary strand to the template strand (1); this prevents primer-template binding, outcompeting the slow process of nonenzymatic RNA polymerization (3) thereby preventing further rounds of RNA replication. b) RNA-binding oligoarginine peptides (green) inhibit strand annealing and promote further rounds of nonenzymatic replication. After an RNA duplex is formed (1), the strands are separated by heating (2). Subsequent cooling allows the peptide to bind to the separated complementary strands but not to the shorter RNA primers. This selectivity prevents reannealing of the full-length replicated strands, allowing each strand to act as a template, to which shorter primers can then bind (3). The nonenzymatic polymerization reaction is free to proceed, resulting in a complete replication cycle that would not be possible without the peptide.

Figure 2

RNA-peptide binding measured by fluorescence anisotropy and circular dichroism. Experiments were performed in annealing buffer (Methods). Error bars indicate ± one SEM. a) Fluorescence anisotropy of a 2-aminopurine (2AP)-containing RNA 15-mer (5′-CC(2AP)GUCAGUCUACGC-3′, 10 μM) in the presence of three oligoarginine peptides of different lengths (R5, R7, and R10NH₂; NH₂: C-terminal amide) over increasing peptide concentrations. Higher peptide concentrations and greater peptide length lead to increased fluorescence anisotropy. b) Fluorescence anisotropy of 2AP-containing RNAs of increasing lengths (7-mer, 10-mer, and 15-mer, 10 μM; see Methods for sequences) with increasing R10NH₂ concentrations. Longer RNAs show a greater increase in anisotropy. c) Circular dichroism (CD) traces of an RNA 15-mer (5′-CCAGUCAGUCUACGC-3′, 5 μM) with increasing concentrations of R10NH₂. The initial spectrum is characteristic of an A-type helical conformation. The molar circular dichroism (Δε) of the 270 nm peak decreases with increasing peptide concentration (up to 25 μM).

Figure 3

RNA annealing rates in the presence of peptides. Initial second-order annealing half-lives (t_1/2) were obtained for an RNA 15-mer (5′-GCGUAGACUGACUGG-3′) and its 2AP-containing complement (5′-CC(2AP)GUCAGUCUACGC-3′) in annealing buffer (Methods) at RNA concentrations of 1 μM (See Supplementary Methods for fitting parameters and Supplementary Table S1 for a list of all conditions tested). Error bars indicate ± one SEM. a) Kinetic traces showing the annealing of the two RNA 15-mers in the presence or absence of 100 μM R5, R7, R9, or R10NH₂. b) t_1/2 for RNA 15-mers with increasing concentrations of R5, R7, R9, and R10NH₂. c) t_1/2 for RNA of different lengths (7-mers to 15-mers, on x-axis; see Supplementary Methods for sequences) annealing to the 2AP-containing 15-mer with or without 100 μM R10NH₂. Longer RNAs exhibit slower annealing kinetics in the presence of peptide.

Figure 4

Nonenzymatic RNA polymerization. Reactions performed with 10 mM MgCl₂, 250 mM Na-HEPES pH 8, and 10 mM 2-MeImpG. a) Polyacrylamide gel of nonenzymatic additions of 2-MeImpG to a 5′-cyanine 3 (Cy3)-labeled primer (5′-Cy3-CAGACUGG-3′, 2 μM) on a C4 template (5′-AACCCCCCAGUCAGUC-3′, 2.5 μM) with or without 1 mM R10NH₂. Bolded cytosines represent 2-MeImpG binding sites on the template. b) log of the fraction of unreacted primer for gel lanes in a vs. time, with (red) or without (black) R10NH₂. The slope of the lines (linear fit, R² = 0.99) represents the pseudo-first-order rate constant, k_obs, in h⁻¹ for the respective reactions. With 1 mM R10NH₂, k_obs = 0.084(5) h⁻¹. With no peptide, k_obs = 0.110(5) h⁻¹. SEM in parentheses. n = 5. c) Polyacrylamide gel of an overnight nonenzymatic primer extension experiment with 1.2 μM primer and 1.25 μM template (incubated with or without 1.5 mM R10NH₂), after addition of 0 μM or 2 μM complementary strand to the template (5′-GACUGACUGGGGGGUU-3′) separately incubated with or without 1.5 mM R10NH₂. d) Post-replication round of nonenzymatic primer extension. The template (gray) and its complement (red) were annealed, then peptide, monomer, and primer (blue) were added and the mixture was heated briefly to 95 °C. After cooling to 4 °C to allow primer-template binding, the system was allowed to warm to room temperature. e) Polyacrylamide gel of the overnight primer extension experiment described in d with 0.95 μM primer, 1 μM template, and with or without 1.2 μM complementary strand and 1.5 mM R10NH₂, respectively.

Figure 5

Condensed phase of RNA. Scale bars, 10 microns. a) Confocal fluorescence microscopy image (488 nm excitation, 525 nm emission) of a 5′-6-carboxyfluorescein (FAM)-labeled RNA 15-mer (5′-FAM-CCAGUCAGUCUACGC-3′, 5 μM) with 1 mM R10NH₂ in annealing buffer (Methods). b) Image of the sample from a but with an added complementary RNA 15-mer (5′-GCGUAGACUGACUGG-3′, 5 μM). c) Confocal fluorescence microscopy image (561 nm excitation, 595 nm emission) of a 5′-Cy3-labeled RNA 8-mer (5′-Cy3-CAGACUGG-3′, 10 μM) with a complementary RNA 15-mer (5′-CCAGUCAGUCUACGC-3′, 10 μM) with 1 mM R10NH₂, 100 mM Tris-Cl pH 8, and 10 mM MgCl₂. d) Nonenzymatic primer extension experiment after 4 hours with the Cy3-labeled RNA primer (1.25 μM) and template (1.4 μM) from Fig. 4, 10 mM MeImpG, 10 mM MgCl₂, 250 mM Na-HEPES pH 8, and with or without 1 mM R10NH₂. A sample containing peptide was immediately centrifuged and the supernatant was isolated from the condensed-phase pellet; the reaction was then allowed to proceed separately in the supernatant and in the pellet.