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 DNA and RNA substrates that can be used to study resecting nucleases and nickases, conferring the ability to ascertain substrate preference and enzyme directionality. 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.

Role of EXO1 nuclease activity in genome maintenance, the immune response and tumor suppression in Exo1D173A mice

“…EXO1 catalytic function is important for survival and tumor suppression DNA mismatches and DSBs are considered the most abundant and harmful of all genotoxic lesions, respectively. Failure to adequately repair these lesions can have severe adverse effects on organisms, including genomic instability and induction of tumorigenesis(36)(37)(38). To study the long-term effects of EXO1 nuclease inactivation on survival and cancer susceptibility,…”

Functionathon: a manual data mining workflow to generate functional hypotheses for uncharacterized human proteins and its application by undergraduate students

“…The nuclease activity and substrate preferences of the recombinant human C7orf25 protein produced in Escherichia coli can be studied using a fluorogenic assay ( 62 ).…”

Label-Free Screening of SARS-CoV-2 NSP14 Exonuclease Activity Using SAMDI Mass Spectrometry

“…The SAMDI-MS assay offers a label-free alternative toward measuring nuclease activities, including radioactivity 7,8,17 and fluorescence assays. 18 Eliminating the need for radionuclides offers a solution for handling the hazardous material, along with waste and safety concerns. Without a fluorescent reporter, the SAMDI-MS assay eliminates false positives that result from optical interference of library compounds.…”

“…Alternatively, fluorescent assays have emerged that use intercalation dyes that are removed upon nuclease activity, leading to a loss of signal. 18 While the assays offer a high-throughput readout and convenient reagents, these assays are prone to high rates of false-positive and/or false-negative results due to optical interference of library compounds.…”