Enhanced Nonenzymatic RNA Copying with 2-Aminoimidazole Activated Nucleotides

Abstract

Achieving efficient nonenzymatic replication of RNA is an important step toward the synthesis of self-replicating protocells that may mimic early forms of life. Despite recent progress, the nonenzymatic copying of templates containing mixed sequences remains slow and inefficient. Here we demonstrate that activating nucleotides with 2-aminoimidazole results in superior reaction kinetics and improved yields of primer extension reaction products. This new leaving group significantly accelerates monomer addition as well as trimer-assisted RNA primer extension, allowing efficient copying of a variety of short RNA templates with mixed sequences.

Figure 1

Development of 2-aminoimidazole as a superior leaving group for nonenzymatic primer extension. Top row: the template (cyan)-dependent reaction of 2-MI activated C monomers (red) with the primer (green) was used to probe the effect of adjacent leaving groups (LG, black) on the reaction rate. Curved arrow indicates the site of reaction. Varying the leaving group (1a–1k) influenced the rates by up to 200 times, with 2-aminoimidazole (1j) giving the fastest rate. Reaction conditions: 1.5 μM primer, 5 μM template, 200 mM HEPES pH 8.0, 50 mM MgCl₂, and 20 mM monomers. All experiments were performed in triplicate. Blue: mean observed rate in h^–1; standard error of the mean (s.e.m.) in the last digit is shown in parentheses. Magenta: pK_a of the leaving group.

Figure 2

Efficient nonenzymatic RNA copying using phosphoroimidazolides with 2-AI leaving group. The time course of primer extension reaction using phosphoroimidazolides 1j-pG and 1a-pG were assayed using polyacrylamide gel electrophoresis (PAGE). Positions of primer and +1 to +4 products are indicated. Reaction conditions as described in Figure 1.

Figure 3

Efficient one-pot extension of primers by four nucleotides. (a) Electrophoretogram of extension products after different reaction times, as assayed by PAGE. Positions of primer and +1 to +7 products are indicated. Reaction conditions: 1.2 μM primer (green), 1.5 μM template (cyan), 200 mM HEPES pH 8.0, 50 mM MgCl₂, 0.5 mM trimer (black), and 10 mM monomer (red). (b) Overlay of extracted ion chromatograms of residual primer (P) and extension products after 1 day (+1 to +7) observed by LC-MS. Observed ions for the peak labeled +3* are consistent with a product having misincorporated s²U in place of A at the +3 position. Observed ions for the peak +7^† are consistent with either the correct +7 product or an alternative double mutant product (see Table S1).

Figure 4

Efficient one-pot nonenzymatic copying of various short RNA templates. (a) Copying AA dinucleotide templates using 2-AI activated U monomers and trimers. (b) Copying AA dinucleotide templates using 2-AI activated s²U monomers and trimers. (c) Extending primers by UAGC using 2-AI activated A, C, G, and U monomers and trimers. (d) Extending primers by the sequence AUGAGGC using 2-AI activated A, C, G, and U monomers and trimers. The extension products were assayed by PAGE. Positions of primers and extended products are indicated. Reaction conditions as described in Figure 3.