Quick Click: The DNA-Templated Ligation of 3'-O-Propargyl- and 5'-Azide-Modified Strands Is as Rapid as and More Selective than Ligase

Abstract

The copper(I)-mediated azide-alkyne cycloaddition (CuAAC) of 3'-propargyl ether and 5'-azide oligonucleotides is a particularly promising ligation system because it results in triazole linkages that effectively mimic the phosphate-sugar backbone of DNA, leading to unprecedented tolerance of the ligated strands by polymerases. However, for a chemical ligation strategy to be a viable alternative to enzymatic systems, it must be equally as rapid, as discriminating, and as easy to use. We found that the DNA-templated reaction with these modifications was rapid under aerobic conditions, with nearly quantitative conversion in 5 min, resulting in a k_obs value of 1.1 min^-1 , comparable with that measured in an enzymatic ligation system by using the highest commercially available concentration of T4 DNA ligase. Moreover, the CuAAC reaction also exhibited greater selectivity in discriminating C:A or C:T mismatches from the C:G match than that of T4 DNA ligase at 29 °C; a temperature slightly below the perfect nicked duplex dissociation temperature, but above that of the mismatched duplexes. These results suggest that the CuAAC reaction of 3'-propargyl ether and 5'-azide-terminated oligonucleotides represents a complementary alternative to T4 DNA ligase, with similar reaction rates, ease of setup and even enhanced selectivity for certain mismatches.

Keywords: DNA; click chemistry; copper; oligonucleotides; template synthesis.