The use of non-enzymatic ligation is widespread in the generation of synthetic DNA structures, the development of molecular sensing probes, and in the ligation of non-Watson-Crick base pairs, systems for which enzymes are less suited. Chemical ligation of DNA may also have been necessary in the chemical origins of life, prior to the emergence of enzymes. Nonetheless, chemical ligation reactions are often low yielding, and require week-long reaction times to approach completion. In this work, we elucidate the chemical principles that contribute to the low yields often encountered in non-enzymatic nucleic acid ligation reactions that utilize water soluble carbodiimides as the activating agent and propose principles for obtaining high yielding DNA ligation reactions. The systematic nature of this study reveals an interplay between template-substrate stability and the rates of the chemical steps of ligation which can cause a direct or inverse correlation between temperature and ligation rates. For example, oligonucleotides with low binding affinity can exhibit lower reaction rates with increasing temperature due to decreased equilibrium assembly of template and substrate, whereas more stable systems can exhibit high reaction rates with increasing temperature due to an enhanced rate of bond formation. Lastly, it is observed that the identity of base pairs flanking a nick site dictates initial ligation rates of the system. Altogether, our results demonstrate that high yielding ligating systems (i.e. greater than 95% ligated products) can be obtained when the template-substrate binding is optimized for a given reaction system, and when the reaction temperature is selected that allows full ligation within the lowest possible time, while also minimizing unproductive side reactions. The guidelines presented should prove to be of general use where non-enzymatic DNA ligation is needed.<br/>

Impact of substrate-template stability, temperature, and nick-site base pairs on non-enzymatic DNA ligation

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.