Unusual Base Pair between Two 2-Thiouridines and Its Implication for Nonenzymatic RNA Copying

Abstract

2-Thiouridine (s²U) is a nucleobase modification that confers enhanced efficiency and fidelity both on modern tRNA codon translation and on nonenzymatic and ribozyme-catalyzed RNA copying. We have discovered an unusual base pair between two 2-thiouridines that stabilizes an RNA duplex to a degree that is comparable to that of a native A:U base pair. High-resolution crystal structures indicate similar base-pairing geometry and stacking interactions in duplexes containing s²U:s²U compared to those with U:U pairs. Notably, the C═O···H-N hydrogen bond in the U:U pair is replaced with a C═S···H-N hydrogen bond in the s²U:s²U base pair. The thermodynamic stability of the s²U:s²U base pair suggested that this self-pairing might lead to an increased error frequency during nonenzymatic RNA copying. However, competition experiments show that s²U:s²U base-pairing induces only a low level of misincorporation during nonenzymatic RNA template copying because the correct A:s²U base pair outcompetes the slightly weaker s²U:s²U base pair. In addition, even if an s²U is incorrectly incorporated, the addition of the next base is greatly hindered. This strong stalling effect would further increase the effective fidelity of nonenzymatic RNA copying with s²U. Our findings suggest that s²U may enhance the rate and extent of nonenzymatic copying with only a minimal cost in fidelity.

Figure 1

Structures of the UU1 and SS1 duplexes with two separated U:U or s²U:s²U base pairs. (A) Sequence for UU1 and SS1 (with s²U modified at the red position). (B) Superposition of the overall structures for UU1 (yellow) and SS1 (magenta). (C) Comparison of the U:U pair in UU1 (yellow) and the s²U:s²U pair in SS1 (magenta). (D) U:U base pair in UU1. (E) s²U:s²U base pair in SS1.The meshes indicate that 2F_o-F_c omit maps contoured at 1.5 σ.

Figure 2

Structures of the UU2 and SS2 duplexes with two adjacent U:U or s²U:s²U base pairs. (A) Sequence for UU2 and SS2 (with s²U modified at the red positions). (B) Superposition of the overall structures for UU2 (yellow) and SS2 (magenta). (C) Comparison of the U8:U9′ pair in UU2 (yellow) and the s²U8:s²U9′ pair in SS2 (magenta). (D) Comparison of the U9:U8′ pair in UU2 (yellow) and the s²U9:s²U8′ pair in SS2 (magenta). (E) U:U base pair in UU2. U9:U8′ and U8:U9′ are the same due to symmetry. (F) s²U9:s²U8′ base pair in SS2. (G) s²U8:s²U9′ base pair in SS2. Meshes indicate that 2F_o-F_c omit maps contoured at 1.5 σ. (H) Sphere structure for UU2. (I) Surface structure for UU2. O2 atoms of the U:U pairs are displayed in green. (J) Sphere structure for SS2. (K) Surface structure for SS2. S2 atoms of the s²U:s²U pairs are displayed in yellow.

Figure 3

Nonenzymatic primer extension rates with base-pairing between s²U, U, A, C, and G. (A) Schematic representation of nonenzymatic primer extension with an activated mononucleotide (*N) and an activated trinucleotide helper. (B) Pseudo-first-order reaction rate constants (h^–1) of primer extension with different *N across different templates: N = U, s²U, A, C, G and X = U, A, s²U, G, C. All reactions were performed at room temperature with 1.5 μM primer, 2.5 μM template, 20 mM *N, 0.5 mM activated helper, 100 mM MgCl₂, and 200 mM Tris-HCl pH 8.0. Standard errors (N ≥ 3) are reported at the appropriate significant digit in parentheses.

Figure 4

Kinetics of primer extension with s²U in either the substrate or the template. (A) Nonenzymatic primer extension with a 2-thiouridine imidazolium-bridged dinucleotide (s²U*s²U) in a primer-template-blocker complex. (i) Schematic representation. (ii) Michaelis–Menten curves for primer extension reactions with s²U*s²U across different templates. (iii) Kinetic parameters for extension with s²U*s²U on different templates. Part of the data was adapted from Figure S6 in ref (17) with permission under a Creative Commons Attribution 4.0 International License. Copyright 2022 Ding et al.; Published by Oxford University Press on behalf of Nucleic Acids Research. (B) Nonenzymatic primer extension with different bridged dinucleotides (N*Ns) on an s²Us²U template. (i) Schematic representation. (ii) Michaelis–Menten curves for primer extension reactions with different N*N substrates on the s²Us²U template. See also Figure S6. (iii) Kinetic parameters for extension with N*N on the s²Us²U template. All reactions were performed at room temperature with 1.5 μM primer, 2.5 μM template, 3.5 μM blocker, 100 mM MgCl₂, and 200 mM Tris-HCl 8.0. Standard errors (N ≥ 3) are reported at the appropriate significant digit in parentheses.

Figure 5

Competition between different activated mononucleotides on a template containing 2-thiouridine for nonenzymatic primer extension. (A) Schematic representation of the nonenzymatic primer extension with activated monomer mix (*s²U, *A, *C, and *G) and *GAC. (B) Quantification of the extension products through LC-MS. (i) A representative overlay of extracted ion chromatograms of residual FAM-primer and extension products observed by LC-MS from one of the trails. (ii) Quantitative analysis of the LC-MS. The + A, + AG, and + AGAC extensions are combined because all of them are the correct extension products through Watson–Crick base pairs. (C) PAGE gel analysis of the same extension reaction as in B(i). All +1 extensions overlap each other. All reactions were performed for 10 min with 20 mM total *N (5 mM *s²U, *A, *C, and *G), 0.5 mM *GAC, 100 mM MgCl₂, and 200 mM Tris-HCl 8.0.

Figure 6

Stalling factors comparison between s²U and U. (A) Schematic representation of nonenzymatic primer extension of G*G with a primer that contained (N₁) at the 3′ end, paired to T₁ at the template. (B) Michaelis–Menten curves plotted for + G extension across different base pairs at the 3′ end of the primer. (C) Kinetic parameters for G*G from the Michaelis–Menten fitting and the stalling factors.