A pyrophosphatase-coupled assay to monitor the NTase activity of cGAS

Richard Hooy, Jungsan Sohn

Research output: Chapter in Book/Report/Conference proceedingChapter

3 Scopus citations


Cyclic GMP-AMP synthase, cGAS, converts ATP and GTP into a cyclic dinucleotide second messenger, cyclic GMP-AMP or cGAMP, through its enzymatic, nucleotidyl transferase (NTase) activity. Although many methods are available to directly measure cGAMP production, these assays often have high cost of implementation and/or experimental limitations. This chapter details how to implement an alternative approach that is relatively inexpensive, accurate and medium-throughput. The assay measures cGAS NTase activity by quantifying pyrophosphate production, a byproduct of the cGAS reaction. A coupling enzyme, pyrophosphatase, catalyzes the hydrolysis of pyrophosphate into inorganic phosphate, which enables facile detection of cGAS activity through conventional phosphomolybdate-malachite green absorbance methodology. This method is amenable for conventional steady-state kinetic measurements as well as high-throughput compound screening.

Original languageEnglish (US)
Title of host publicationDNA Sensors and Inflammasomes
EditorsJungsan Sohn
PublisherAcademic Press Inc.
Number of pages10
ISBN (Print)9780128183595
StatePublished - 2019

Publication series

NameMethods in Enzymology
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988


  • Nucleotidyl transferase (NTase)
  • PPase
  • Pyrophosphatase
  • Steady-state
  • cGAS

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology


Dive into the research topics of 'A pyrophosphatase-coupled assay to monitor the NTase activity of cGAS'. Together they form a unique fingerprint.

Cite this