A Highly Sensitive Anion Exchange Chromatography Method for Measuring cGAS Activity in vitro

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Cyclic GMP-AMP synthase (cGAS) is a pattern recognition receptor (PRR) that senses
double stranded DNA (dsDNA) in the cytosol and this leads to the activation of stimulator of interferon
genes (STING) via the secondary messenger 2’3’-cyclic GMP-AMP (2’3’-cGAMP). STING then recruits
TANK binding kinase 1 (TBK-1) and this complex can phosphorylate and activate interferon regulatory
factor 3 (IRF3) leading to the induction of type I interferons and other antiviral genes. The cGAS:DNA
complex catalyzes the synthesis of 2’3’-cGAMP and the purpose of the protocol presented here is to
measure the in vitro activity of purified cGAS in the presence of dsDNA. The protocol was developed to
elucidate the relationship between dsDNA length and the level of cGAS activity. The method involves
an in vitro reaction with low concentrations of cGAS and dsDNA followed by quantification of the reaction
product using anion exchange chromatography. The low concentrations of cGAS and dsDNA and the
high sensitivity of this assay is a key advantage when comparing different DNA fragments’ ability to
activate cGAS.
OriginalsprogEngelsk
TidsskriftBio-protocol
Vol/bind8
Nummer20
ISSN2331-8325
DOI
StatusUdgivet - okt. 2018

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