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

In this article we describe a sensitive exonuclease III detection method using a DNB nanoarray from a BGISEQ-500 sequencing kit and demonstrate a detection limit as low as 0.001 U/mL. The flow cell of the sequencing kit was loaded with billions of DNA nanoballs (DNBs) to form the DNB nanoarray and initially used for massively parallel sequencing. The 3'-end recessed dsDNA structure formed by sequencing was shown to be a perfect substrate for exonuclease III, but not for other nucleases such as exonuclease I, RecJf and nuclease P1. We developed an exonuclease III assay using the DNB nanoarray, together with other reagents within the BGISEQ-500 sequencing kit, which only required one additional cycle of sequencing. The DNB nanoarray can be reused for the exonuclease III assay at least five times. This method demonstrated superior sensitivity, selectivity, and reusability compared with other assay methods and is accompanied by low cost and simple setup.