Ultrasensitive quantification and comprehensive evaluation of nicking endonuclease efficiency

Area of Science:

• Biotechnology
• Molecular Biology
• Enzymology

Background:

• Sequence-specific nicking endonucleases (NEs) are vital for biotechnology, cleaving only one DNA strand.
• Fundamental understanding of NE reaction efficiencies is limited by assay method sensitivity.

Purpose of the Study:

• To develop an ultrasensitive method for quantifying nicking endonuclease efficiencies.
• To compare the efficiencies of various NEs, including canonical and programmable types.

Main Methods:

• Utilized a single-molecule counting approach for ultrasensitive quantification.
• Employed sequence-optimized DNA substrates for parallel measurements.
• Enabled digital quantification of nicking efficiency via fluorescent protein synthesis in droplet reactors.

Main Results:

• Established a novel, ultrasensitive assay for NE nicking efficiency.
• Demonstrated NEs achieve a median nicking efficiency of 99.6%.
• Showed NEs generally outperform Type II restriction enzymes in efficiency and consistency.

Conclusions:

• The developed method provides a comprehensive view of NE reaction efficiencies.
• NEs exhibit superior performance compared to Type II restriction enzymes.
• This study offers tools and insights for advancing enzyme-based biotechnology and biological research involving NEs.