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

Abstract

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.