Abstract
2-Thiouridine (s(2)U) 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 s(2)U:s(2)U 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 s(2)U:s(2)U base pair. The thermodynamic stability of the s(2)U:s(2)U base pair suggested that this self-pairing might lead to an increased error frequency during nonenzymatic RNA copying. However, competition experiments show that s(2)U:s(2)U base-pairing induces only a low level of misincorporation during nonenzymatic RNA template copying because the correct A:s(2)U base pair outcompetes the slightly weaker s(2)U:s(2)U base pair. In addition, even if an s(2)U 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 s(2)U. Our findings suggest that s(2)U may enhance the rate and extent of nonenzymatic copying with only a minimal cost in fidelity.