Impact of substrate-template stability, temperature, phosphate location, and nick-site base pairs on non-enzymatic DNA ligation: Defining parameters for optimization of ligation rates and yields with carbodiimide activation

Non-enzymatic, chemical ligation is an important tool for the generation of synthetic DNA structures, which are used for a wide range of applications. Surprisingly, reported chemical ligation yields range from 30% to 95% for the same chemical activating agent and comparable DNA structures. We report a systematic study of DNA ligation using a well-defined bimolecular test system and water-soluble carbodiimide (EDC) as a phosphate-activating agent. Our results reveal interplay between template-substrate stability and the rates of the chemical steps of ligation, which can cause yields to increase or decrease with increasing temperature. Phosphate location at the nick site also exhibits a strong influence on ligation rates and yields, with a 3’ phosphate providing yields near 100% after 24 hours for particularly favourable reaction conditions, while comparable reactions with the phosphate on the 5’ position of the nick site only reach 40% ligation even after 48 hours. Ligation rates are also shown to be sensitive to the identity of base pairs flanking a nick site, with some varying by more than three-fold. Finally, DNA substrate modification by EDC can, in some cases, make long reaction times and repeated addition of EDC an ineffective strategy for increasing ligation yields.