High Fidelity Enzyme‐Free Primer Extension with an Ethynylpyridone Thymidine Analog

High fidelity base pairing is important for the transmission of genetic information. Weak base pairs can lower fidelity, complicating sequencing, amplification and replication of DNA. Thymidine 5′‐monophosphate (TMP) is the most weakly pairing nucleotide among the canonical deoxynucleotides, causing high errors rates in enzyme‐free primer extension. Here we report the synthesis of an ethynylpyridone C‐nucleoside analog of 3′‐amino‐2′,3′‐dideoxythymidine monophosphate and its incorporation in a growing strand by enzyme‐free primer extension. The ethynylpyridone C‐nucleotide accelerates extension more than five‐fold, reduces misincorporation and readily displaces TMP in competition experiments. The results bode well for the use of the C‐nucleoside as replacements for thymidine in practical applications.