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

Schematic illustration of Exo III detection using pre-sequenced DNB nanoarrays on the BGISEQ-500RS sequencer platform. Step-1: Initialization of DNB nanoarray. A pre-sequenced DNB nanoarray loaded with billions of fluorescently labeled DNBs was treated with excess amounts of Exo III to completely digest all synthesized strands from the DNBs, resulting in DNBs still attached to the flow cell in a way similar to fresh-loaded DNBs. Step-2: Formation of new DSC. The new DSC was formed by hybridizing a 25mer sequencing primer, followed by one cycle of sequencing using four different fluorescently labeled dNTPs in the presence of DNA polymerase. ESR1 was obtained after imaging. Step-3: Exo III activity assay. The newly formed DSC was digested with the test amount of Exo III. ESR2 was obtained after imaging. A high ESR2/ESR1 ratio indicates low Exo III activity, while a low ESR2/ESR1 ratio indicates high Exo III activity. [Display omitted] •DNA nanoball sequencing complex structure is used for measuring Exo III enzymatic activity.•DNBSEQ™ technology is utilized as a sensitive exonuclease III detection method by using the “used” DNB nanoarray.•The detection limit by using “used” DNB nanoarray on DNBSEQ™ technology is as low as 0.001 U/mL.•Our method achieves some advantages such as cost effective, superior sensitivity and simple in the experiment setting up. 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.