Replacing uridine with 2-thiouridine enhances the rate and fidelity of nonenzymatic RNA primer extension

Abstract

The nonenzymatic replication of RNA oligonucleotides is thought to have played a key role in the origin of life prior to the evolution of ribozyme-catalyzed RNA replication. Although the copying of oligo-C templates by 2-methylimidazole-activated G monomers can be quite efficient, the copying of mixed sequence templates, especially those containing A and U, is particularly slow and error-prone. The greater thermodynamic stability of the 2-thio-U(s(2)U):A base pair, relative to the canonical U:A base pair, suggests that replacing U with s(2)U might enhance the rate and fidelity of the nonenzymatic copying of RNA templates. Here we report that this single atom substitution in the activated monomer improves both the kinetics and the fidelity of nonenzymatic primer extension on mixed-sequence RNA templates. In addition, the mean lengths of primer extension products obtained with s(2)U is greater than those obtained with U, augmenting the potential for nonenzymatic replication of heritable function-rich sequences. We suggest that noncanonical nucleotides such as s(2)U may have played a role during the infancy of the RNA world by facilitating the nonenzymatic replication of genomic RNA oligonucleotides.

Figure 1

Template-directed primer extension system. (a) Primer extension reaction scheme. A 5′-Cy5-tagged RNA primer anneals to a complementary template. 2-MeImpX analogues form Watson–Crick base pairs on a complementary template and participate in template-directed primer extension. (b) Structure of thiolated uracil and thymine nucleobases in 2-methylimidazole-activated nucleotides.

Figure 2

Kinetic studies of primer extension reactions. Pseudo-first-order rates were determined from the extent of primer disappearance as a function of time, and the resultant observed rates were determined as a function of activated nucleotide concentration to give k_max. Reaction conditions: 200 mM HEPES pH 7.0, 0.5 μM primer P1, 1.5 μM template, on ice. Buffer 1 (blue): 1.0 M NaCl, 200 mM MgCl₂. Buffer 2 (red): 100 mM MgCl₂. (a) Primer extension reaction on template T1 (A₆) with 2-MeImpU* (U* = U, s²U, or s²T). (b) Primer extension reaction on templates T2 (U₆), T3 (s²U₆), or T4 (s²T₆) with 2-MeImpA. (c) Primer extension reaction on template T8 (AC₅) with 2-MeImpU* (U* = U, s²U, and s²T) and 40 mM 2-MeImpG. (d) Primer extension reaction on template T5 (UC₅), T6 (s²UC₅), and T7 (s²TC₅) with 2-MeImpA and 40 mM 2-MeImpG.

Figure 3

Schematic representation of MiSeq library assembly protocol. (A) Primer extension reaction. (B) Biotinylated-primer/streptavidin bead association. (C) Template removal. (D) Biotinylated primer/streptavidin dissociation. (E) 3′ Adaptor ligation. (F) Primer hybridization. (G) Reverse transcription: First strand cDNA synthesis. (H) PCR enrichment.

Figure 4

Sequence analysis of products of nonenzymatic primer-extension. (a) Top: Primer-extension was carried out on 2.0 μM template T9 (AGAGAG) in the presence of 40 mM 2-MeImpC and 50 mM 2-MeImpU* (U* = U, s²U, or s²T) on ice for 7 days. Bottom: Products were sequenced, and the sequence reads binned according to the number of nucleotides added to the primer; Y = C or U*. Reaction conditions: 2.0 μM primer P2, 200 mM HEPES pH 7.0, 100 mM MgCl₂. (b) Top: Primer-extension was carried out on 2.0 μM template T5 (UC₅), T6 (s²UC₅), or T7 (s²TC₅) in the presence of 40 mM 2-MeImpG and 50 mM 2-MeImpA on ice for 7 days. Bottom: Products were sequenced and the sequence reads binned according to the number of nucleotides added to the primer; R = A or G.

Figure 5

Fidelity of primer-extension reactions. Sequence reads obtained from the products of primer-extension on template T9 (AGAGAG), as described in Figure 4, were sorted into bins according to the number and identity of nucleotides added to the primer. (a) Left: products extended by at least two nucleotides, with correct incorporation of U* at position 1, were sorted according to whether the correct nucleotide C or the incorrect nucleotide U* was incorporated at position 2. Right: products extended by at least two nucleotides, with incorrect incorporation of C at position 1, were sorted according to whether the correct nucleotide C or the incorrect nucleotide U* was incorporated at position 2. (b) Left: products extended by at least three nucleotides, with correct incorporation at positions 1 and 2, were sorted according to whether the correct nucleotide U* or the incorrect nucleotide C was incorporated at position 3. Right: products extended by at least three nucleotides, with incorrect incorporation of U* at position 2, were sorted according to whether the correct nucleotide U*or the incorrect nucleotide C was incorporated at position 3. Misincorporated nucleotides are represented by red.