Rapid Chemical Ligation of DNA and <i>Acyclic</i> Threoninol Nucleic Acid (<i>a</i>TNA) for Effective Nonenzymatic Primer Extension
Hikari Okita, Shuto Kondo, Keiji Murayama, Hiroyuki Asanuma
Abstract
High Resolution Image Download MS PowerPoint Slide Previously, nonenzymatic primer extension reaction of acyclic l -threoninol nucleic acid (L- a TNA) was achieved in the presence of N -cyanoimidazole (CNIm) and Mn 2+; however, the reaction conditions were not optimized and a mechanistic insight was not sufficient. Herein, we report investigation of the kinetics and reaction mechanism of the chemical ligation of L- a TNA to L- a TNA and of DNA to DNA. We found that Cd 2+, Ni 2+, and Co 2+ accelerated ligation of both L- a TNA and DNA and that the rate-determining step was activation of the phosphate group. The activation was enhanced by duplex formation between a phosphorylated L- a TNA fragment and template, resulting in unexpectedly more effective L- a TNA ligation than DNA ligation. Under optimized conditions, an 8-mer L- a TNA primer could be elongated by ligation to L- a TNA trimers to produce a 29-mer full-length oligomer with 60% yield within 2 h at 4 °C. This highly effective chemical ligation system will allow construction of artificial genomes, robust DNA nanostructures, and xeno nucleic acids for use in selection methods. Our findings also shed light on the possible pre-RNA world.