A universal fluorescence-based toolkit for real-time quantification of DNA and RNA nuclease activity

Abstract

DNA and RNA nucleases play a critical role in a growing number of cellular processes ranging from DNA repair to immune surveillance. Nevertheless, many nucleases have unknown or poorly characterized activities. Elucidating nuclease substrate specificities and regulatory components can support a more definitive understanding of cellular mechanisms in physiology and disease. Using fluorescence-based methods, we have developed a quick, safe, reproducible, cost-effective, and real-time nuclease assay toolkit that could be used for small- and large- scale experimental assays. Additionally, these data can be analysed to determine each reaction's unique enzyme kinetics. We have designed a library of substrates that can be used to study catalytic rates, directionality, and substrate preferences. The assay is sensitive enough to detect kinetics of repair enzymes when confronted with DNA mismatches or DNA methylation sites. We have also extended this assay to consider analysing the kinetics of human single-strand DNA nuclease TREX2, DNA polymerases, and RNA:DNA nucleases, which are also involved in DNA repair and immune regulation, and have been associated with various disease conditions, including cancer and immune disorders.