Click DNA ligation with deoxyribozyme

Deoxyribozymes that ligate DNA will expand the reaction scope of DNA catalysis and are useful in the construction of DNA nanostructures. Herein, we report the first efforts to isolate a novel class of DNA ligase deoxyribozymes from a random sequence DNA pool by in vitro selection. The identified deoxyribozymes catalyze the intermolecular linear DNA-DNA ligation via the formation of unnatural triazole linkages between a 5′ alkyne and a 3′ azide. One remarkable click-ligating deoxyribozyme, named CLDz2, ligated DNA with an observed rate constant (kobs) up to 2.7 × 10−2 h−1 at 10 mM Mn2+ (pH 7.0, 30°C), with up to 40% yield in overnight incubations. CLDz2 is predicated to have a four-way, junction-like structure comprised of four short duplexes, three hairpin loops, and two main interhelical unpaired elements. Comprehensive nucleotide covariation experiments suggest that CLDz2 should be generally applicable for click ligation of a wide range of 3′ azide DNAs. We further demonstrate a CLDz2-directed chemical ligation strategy for the synthesis of single-stranded monomeric circular DNA in high selectivity (97%), which can be used as a DNA template in rolling circle amplification.