Comparison of multiple factors that impact the efficiency of non-enzymatic DNA ligation: Key parameters for maximizing ligation rates and yields with carbodiimide activation.

We report a systematic study of DNA ligation using a well-defined bimolecular test system and a water-soluble carbodiimide (EDC) as a phosphate-activating agent. Our results emphasize the interplay between template-substrate complex stability and the rates of the chemical steps of ligation, with 3´ phosphate substrates providing yields near 100% after 24 hours for particularly favorable reaction conditions. Ligation rates are also shown to be sensitive to the identity of base pairs flanking a nick site, with as much as three-fold variation. Finally, the observation that DNA substrates are modified by EDC at rates that can be comparable with ligation rates emphasizes the importance of considering side reactions when designing protocols to maximize ligation yields. PMID: 32705742 [PubMed - as supplied by publisher]
Source: Chembiochem - Category: Biochemistry Authors: Tags: Chembiochem Source Type: research