Rapid Chemical Ligation of DNA and Acyclic Threoninol Nucleic Acid (aTNA) for Effective Nonenzymatic Primer Extension

Previously, nonenzymatic primer extension reaction of acyclic l-threoninol nucleic acid (L-aTNA) wasachieved in the presence of N-cyanoimidazole (CNIm)and Mn2+; however, the reaction conditions were not optimizedand a mechanistic insight was not sufficient. Herein, we report investigationof the kinetics and reaction mechanism of the chemical ligation ofL-aTNA to L-aTNA and of DNA to DNA.We found that Cd2+, Ni2+, and Co2+ accelerated ligation of both L-aTNA and DNA and that the rate-determining step was activationof the phosphate group. The activation was enhanced by duplex formationbetween a phosphorylated L-aTNA fragment and template,resulting in unexpectedly more effective L-aTNA ligationthan DNA ligation. Under optimized conditions, an 8-mer L-aTNA primer could be elongated by ligation to L-aTNA trimers to produce a 29-mer full-length oligomer with60% yield within 2 h at 4 & DEG;C. This highly effective chemicalligation system will allow construction of artificial genomes, robustDNA nanostructures, and xeno nucleic acids for use in selection methods.Our findings also shed light on the possible pre-RNA world.