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

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.