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. 2025 May;5(5):e70140.
doi: 10.1002/cpz1.70140.

Highly Effective Chemical Ligation of DNA and l-aTNA

Hikari Okita  1   2 , Keiji Murayama  1 , Hiroyuki Asanuma  1
Affiliations

Highly Effective Chemical Ligation of DNA and l-aTNA

Hikari Okita et al. Curr Protoc. 2025 May.

Abstract

Chemical ligation of nucleic acids is a significant strategy for the establishment of a variety of functional biological tools and other applications. However, the conventional methodology has been suffering from low reaction efficiency or generation of unnatural structures at the ligation site. This article describes an effective chemical ligation method that connects the hydroxyl group and the monophosphate group at the nick site on the template, generating a natural phosphodiester bond. The method uses only N-cyanoimidazole (CNIm) and divalent metal cations to achieve chemical ligation in a template-directed manner, which can be applied to the ligations of not only DNA but also artificial nucleic acids such as acyclic l-threoninol nucleic acid (l-aTNA). Quantitative ligation is available within 10 min for l-aTNA fragments on an l-aTNA template, and within 2 hr for DNA fragments on a DNA template under optimized conditions. The effective chemical ligation system reported here will enable the development of biotechnology and nanotechnology, including exploration of l-aTNA aptamer via in vitro selection, chemical synthesis of genome-sized DNA and l-aTNA, functionalization of nanostructure, and creation of an l-aTNA-based artificial life in the future. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Chemical ligation using CNIm and divalent metal cation Basic Protocol 2: Imaging analysis of ligation reaction Basic Protocol 3: Mass spectrometry of ligation products Support Protocol: Kinetic analysis.

Keywords: DNA; N‐cyanoimidazole; artificial nucleic acid; chemical ligation; l‐aTNA; template‐directed synthesis.

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References

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