Header

UZH-Logo

Maintenance Infos

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


Sheppard, Emily C; Rogers, Sally; Harmer, Nicholas J; Chahwan, Richard (2019). A universal fluorescence-based toolkit for real-time quantification of DNA and RNA nuclease activity. bioRxiv 548628.

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.

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.

Statistics

Citations

Dimensions.ai Metrics

Altmetrics

Downloads

2 downloads since deposited on 05 Mar 2019
2 downloads since 12 months
Detailed statistics

Additional indexing

Item Type:Working Paper
Communities & Collections:04 Faculty of Medicine > Institute of Experimental Immunology
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:14 February 2019
Deposited On:05 Mar 2019 15:18
Last Modified:27 Mar 2019 15:22
Series Name:bioRxiv
OA Status:Hybrid
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1101/548628

Download

Download PDF  'A universal fluorescence-based toolkit for real-time quantification of DNA and RNA nuclease activity'.
Preview
Content: Published Version
Filetype: PDF
Size: 32MB
View at publisher