Ultrasensitive quantification and comprehensive evaluation of nicking endonuclease efficiency

Sequence-specific nicking endonucleases (NEs) behave similarly to restriction endonucleases (REs) on dsDNA but cleave only one strand rather than both. This unique property made NEs indispensable for certain applications, with profound impacts on enzyme-based biotechnology. Despite the essential roles of NEs, fundamental investigations of their general reaction efficiencies have been limited due to the lack of sensitive and straightforward assay methods. This study applies single-molecule counting approach to enable ultrasensitive quantification of the nicking efficiencies of various NEs, including canonical and programmable ones. A DNA substrate was sequence-optimized to allow parallel measurements across different enzymes. A single nick in either strand led to subsequent substrate degradation. Unnicked single DNA molecules can serve as templates to initiate fluorescent protein synthesis in massively parallel droplet reactors for detection, thus enabling digital quantification of the unnicked DNA fraction as well as nicking efficiency. None of the NEs achieved complete consumption of the substrate DNA. The NEs currently available showed a median nicking efficiency of 99.6%. Overall, NEs outperformed Type II REs, as evidenced by higher efficiencies on average and a narrower efficiency distribution. The accumulated dataset further allowed population-level statistical analysis, attributing the efficiency differences between NEs and REs to their basic scission properties. The comprehensive measurements offered the first overall view of the DNA-nicking enzymes in terms of reaction efficiencies. The approach established and the knowledge gained in this study may provide tools and insights for future research, whether to expand the repertoire of enzymes or to interrogate biological phenomena involving NEs.