Enhanced base pairing and replication efficiency of thiothymidines, expanded-size variants of thymidine

Abstract

Here we test the steric effects of added size on DNA base pairing and replication by the use of thiocarbonyl group replacements for natural nucleoside thymidine. The 2-thioT (2S) and 4-thioT (4S) nucleosides were reported previously, but their pairing specificity and replication fidelity were unknown. We find that 2S pairs with higher specificity than thymidine, and both 2S and 4S nucleoside triphosphates are replicated with higher efficiency than natural dTTP. The results indicate possible biotechnological uses for these analogues and have implications in the ongoing use of thionucleobases in cancer treatment.

Figure 1

Structures of 2-thiothymidine (2S) and 4-thiothymidine (4S), with unmodified thymidine shown at right for comparison. Space-filling models of the bases alone are shown below each structure.

Figure 2

Histogram showing pairing stability (as T_m) and selectivity of 2S and 4S placed opposite each of the natural bases in a 12 base pair DNA duplex. The sequence is dAAGAAXGAAAAG · dCTTTTCYTTCTT, where X is 2S, 4S, or T, and Y is each of the four natural bases as shown. Conditions: 2 M DNA, 100 mM NaCl, 10 mM MgCl₂, 10 mM phosphate (pH 7.0). Uncertainty in T_m values is less than ± 0.5 °C.