Development of cGAMP-Luc, a sensitive and precise coupled enzyme assay to measure cGAMP in complex biological samples

Abstract

2′,5′/3′,5′-cGMP-AMP (cGAMP) is a second messenger produced in response to cytosolic dsDNA that activates the stimulator of interferon genes (STING) pathway. We recently discovered that cGAMP is exported by cancer cells and that this extracellular signal is an immunotransmitter key to tumor detection and elimination by the innate immune system. The enhancement of extracellular cGAMP levels therefore holds great promise for managing cancer. However, there is still much more to understand about the basic biology of cGAMP before its full therapeutic potential can be realized. To answer these questions, we must be able to detect and quantitate cGAMP with an assay that is high-throughput, sensitive, and precise. Existing assays fall short of these needs. Here, we describe the development of cGAMP-Luc, a coupled enzyme assay that relies on the degradation of cGAMP to AMP by ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) and an optimized assay for the detection of AMP by luciferase. We also developed STING-CAP, a STING-mediated method to concentrate and purify cGAMP from any type of biological sample. We conclude that cGAMP-Luc is an economical high-throughput assay that matches the accuracy of and surpasses the detection limit of MS, the current gold standard of cGAMP quantitation. We propose that cGAMP-Luc is a powerful tool that may enable discoveries that advance insights into extracellular cGAMP levels in healthy and diseased tissues, such as cancer.

A STING-based biosensor affords broad cyclic dinucleotide detection within single living eukaryotic cells

“…2d ) 48 . Together, these findings suggest that recombinant BioSTING can be used as a bulk cell extract 2ʼ3ʼ-cGAMP detection method; although, other sensitive methods for end-point 2ʼ3ʼ-cGAMP detection exist 22 , 48 .…”

“…Current in vitro methods for monitoring CDN production employ sensitive EIA, mass spectrometry, and cGAMP-luc endpoint measures, as well as less sensitive continuous tools, including a cGAMP RNA biosensor and an indirect pyrophosphate release assay 3 , 4 , 6 , 22 – 25 . Given the limitations of current CDN detection methods, the ease of recombinant BioSTING production, and its capacity to directly report on a variety of CDNs at biologically relevant concentrations, we employed BioSTING to monitor in vitro CDN synthase activity and inhibition to demonstrate its utility for kinetic characterization and feasibility for high throughput screening related to these enzymes.…”

“…These tools, however, are limited to a qualitative, binary localization readout. In lieu of these reporter assays, methods to directly measure cyclic dinucleotides, including mass spectrometry, enzyme immunoassays (EIA), ENPP1-based luciferase assays (cGAMP-luc), and RNA-based biosensors have been developed 3 , 4 , 6 , 13 , 22 – 25 . While these assays are specific, they range in their sensitivity and only provide bulk endpoint measurements following destruction of the biological sample.…”

Regulation and inhibition of the DNA sensor cGAS

“…ii cGAMP-Luc, an assay that uses recombinant ENPP1 to cleave cGAMP and detects the resulting AMP indirectly by phosphorylation to ATP and measurement with Cell-Titer Glo (Promega). Suitable for high throughput studies (Mardjuki et al, 2020).…”

A STING-based fluorescent polarization assay for monitoring activities of cyclic dinucleotide metabolizing enzymes

“…ELISA kits that utilize antibodies to detect cGAMP are commercially available but are expensive. Other methods such as RNA fluorescent c-di-GMP or c-di-AMP or cGAMP sensors, [32][33][34] pyrophosphatase-coupled assay, 35 cGAMP-luciferase assay 36 and BioSTING assay, 37 which utilizes FRET, have been described for monitoring cyclic dinucleotides, highlighting the high interests in identifying convenient methods to monitor the aforementioned critical enzymes in the cGAS-STING pathway. Whiles these prior developed assays/ biosensors have facilitated CDN research, we found that these methods are not ideal for our medium-to-high throughput screening campaigns for inhibitors of CDN metabolizing enzymes.…”