Reusable and sensitive exonuclease III activity detection on DNB nanoarrays based on cPAS sequencing technology

Area of Science:

• Biochemistry
• Molecular Biology
• Assay Development

Background:

• Exonuclease III (ExoIII) is a crucial enzyme in DNA repair and metabolism.
• Sensitive and selective detection methods for ExoIII are essential for various biological and diagnostic applications.
• Existing methods for ExoIII detection often lack sensitivity, selectivity, or are complex and costly.

Purpose of the Study:

• To develop a highly sensitive and selective assay for detecting exonuclease III activity.
• To leverage the DNA nanoball (DNB) nanoarray technology from the BGISEQ-500 sequencing kit for enzyme detection.
• To evaluate the performance characteristics of the novel ExoIII assay.

Main Methods:

• Utilized a DNB nanoarray, formed by billions of DNBs on a sequencing flow cell.
• Employed a 3'-end recessed double-stranded DNA (dsDNA) structure as a specific substrate for ExoIII.
• Integrated the ExoIII assay with reagents from the BGISEQ-500 sequencing kit, requiring only one additional sequencing cycle.

Main Results:

• Achieved a highly sensitive detection limit for ExoIII as low as 0.001 U/mL.
• Demonstrated superior selectivity of the assay for ExoIII over other nucleases (exonuclease I, RecJf, nuclease P1).
• Showcased the reusability of the DNB nanoarray for the ExoIII assay for at least five cycles.

Conclusions:

• The DNB nanoarray-based method provides a sensitive, selective, and reusable platform for ExoIII detection.
• This novel assay offers a low-cost and simple alternative to existing enzyme detection methods.
• The integration with sequencing kit technology simplifies the assay setup and execution.