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

Emily C Sheppard,Sally Rogers,Nicholas J Harmer,Richard Chahwan

doi:10.1038/s41598-019-45356-z

Emily C Sheppard, Sally Rogers + Show 2 more

Open Access

https://doi.org/10.1038/s41598-019-45356-z

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Journal: Scientific Reports	Publication Date: Jun 20, 2019
Citations: 8	License type: open-access

Affiliation: University of Exeter, University of Zurich

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 co-factors can support a more definitive understanding of cellular mechanisms in physiology and disease. Using fluorescence-based methods, we present a quick, safe, cost-effective, and real-time versatile nuclease assay, which uniquely studies nuclease enzyme kinetics. In conjunction with a substrate library we can now analyse nuclease 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 our analysis to study the kinetics of human single-strand DNA nuclease TREX2, DNA polymerases, RNA, and RNA:DNA nucleases. These nucleases are involved in DNA repair, immune regulation, and have been associated with various diseases, including cancer and immune disorders.

Highlights

DNA and RNA nucleases are a hallmark of a growing number of cell signalling cascades including the DNA damage response and immune diversification
Radiolabelled oligonucleotides are gradually being replaced with fluorescent nucleic acid stains such as DAPI15 and other commercially-available dyes including, but not limited to, Midori Green, SYBR Green I and Acridine Orange[16]
PicoGreen (PG) is a commercially available dye that emits a fluorescent signal upon intercalation with double-stranded DNA, emitting a fluorescent signal 1,000-fold stronger compared to when it is free in solution

Summary

Introduction

DNA and RNA nucleases are a hallmark of a growing number of cell signalling cascades including the DNA damage response and immune diversification. The complex roles DNA and RNA nucleases play in DNA repair pathways underpin several premature ageing-, immune-, and tumour- related syndromes All of these can result from aberrations in the structural and/or catalytic functions of DNA and RNA nucleases (reviewed in[1,2]). There are several yet uncharacterised proteins harbouring predicted nuclease domains in mammalian genomes[5,6,7,8] Even with their myriad and complex functions, DNA/RNA nucleases can be broadly defined by their substrate specificity, directionality of resection, and processivity. Methods involving graphene oxide surfaces[20], electrochemical redox reactions[21,22,23], complexing of DNA with a polycationic polymer[24], or immobilising nucleotides on magnetic beads[25] have been developed The full potential of PG and QI in real-time visualisation of reaction progression remains limited[38]

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