Measuring Real-time DNA/RNA Nuclease Activity through Fluorescence

Abstract

DNA and RNA nucleases are wide-ranging enzymes, taking part in broad cellular processes from DNA repair to immune response control. Growing interest in the mechanisms and activities of newly discovered nucleases inspired us to share the detailed protocol of our nuclease assay ( Sheppard et al., 2019 ). This easy and inexpensive method can provide data that enables understanding of the molecular mechanism for novel or tested nucleases, from substrate preference and cofactors involved to catalytic rate of reaction.

Structural and biochemical characterization of the mitomycin C repair exonuclease MrfB

“…We used a PicoGreen fluorescence assay to quantitively assess exonuclease activity on several dsDNA substrates (30) (Figure S2). MrfB variants were first analyzed with a blunt 80 bp dsDNA substrate that was blocked on one side with biotin-streptavidin, so that only one free 3’ end was available (Figure S2b).…”

“…Timepoints were taken every 90 s for 2 h at 30°C on a Tecan Infinite M1000 plate reader with a 3 s shake before each read (483 nm excitation, 530 nm emission). The data were processed in GraphPad Prism version 10.0.3 as described in the Bio-protocol, using known dsDNA substrate lengths of 80, 60, 40, and 20 bp to create a calibration curve (30). A negative control of a dsDNA 80 bp substrate with blocks on all 4 ends and wild-type MrfB was used to correct for photobleaching and background by dividing the data by the average reads from the negative control (Figure S2).…”

“…PicoGreen exonuclease assay. The PicoGreen (PG) assay was adapted from a published Bioprotocol and uses PG to monitor exonuclease activity on dsDNA (30). The PG dye in DMSO was stored at -20°C in 5 μL aliquots.…”

Structural and biochemical characterization of the mitomycin C repair exonuclease MrfB

“…The PicoGreen (PG) assay was adapted from a published Bio-protocol and uses PG to monitor exonuclease activity on dsDNA ( 30 ). The PG dye in DMSO was stored at –20°C in 5 μl aliquots.…”

“…Timepoints were taken every 90 s for 2 h at 30°C on a Tecan Infinite M1000 plate reader with a 3 s shake before each read (483 nm excitation, 530 nm emission). The data were processed in GraphPad Prism version 10.0.3 as described in the Bio-protocol, using known dsDNA substrate lengths of 80, 60, 40, and 20 bp to create a calibration curve ( 30 ). A negative control of a dsDNA 80 bp substrate with blocks on all four ends and wild-type MrfB was used to correct for photobleaching and background by dividing the data by the average reads from the negative control ( Supplementary Figure S2 ).…”