title: An RNA-Based Fluorescent Biosensor for High-Throughput Analysis of the cGAS-cGAMP-STING Pathway.
abstract: In mammalian cells, the second messenger (2'-5',3'-5') cyclic guanosine monophosphate-adenosine monophosphate (2',3'-cGAMP), is produced by the cytosolic DNA sensor cGAMP synthase (cGAS), and subsequently bound by the stimulator of interferon genes (STING) to trigger interferon response. Thus, the cGAS-cGAMP-STING pathway plays a critical role in pathogen detection, as well as pathophysiological conditions including cancer and autoimmune disorders. However, studying and targeting this immune signaling pathway has been challenging due to the absence of tools for high-throughput analysis. We have engineered an RNA-based fluorescent biosensor that responds to 2',3'-cGAMP. The resulting "mix-and-go" cGAS activity assay shows excellent statistical reliability as a high-throughput screening (HTS) assay and distinguishes between direct and indirect cGAS inhibitors. Furthermore, the biosensor enables quantitation of 2',3'-cGAMP in mammalian cell lysates. We envision this biosensor-based assay as a resource to study the cGAS-cGAMP-STING pathway in the context of infectious diseases, cancer immunotherapy, and autoimmune diseases.
authors.affiliation: Department of Chemistry, University of California, Berkeley, CA 94720, USA.
authors.forename: Debojit
authors.initials: D
authors.lastname: Bose
authors.affiliation: Department of Chemistry, University of California, Berkeley, CA 94720, USA.
authors.forename: Yichi
authors.initials: Y
authors.lastname: Su
authors.affiliation: Department of Molecular & Cell Biology, University of California, Berkeley, CA 94720, USA.
authors.forename: Assaf
authors.initials: A
authors.lastname: Marcus
authors.affiliation: Department of Molecular & Cell Biology, University of California, Berkeley, CA 94720, USA.
authors.forename: David H
authors.initials: DH
authors.lastname: Raulet
authors.affiliation: Department of Chemistry, University of California, Berkeley, CA 94720, USA; Department of Molecular & Cell Biology, University of California, Berkeley, CA 94720, USA. Electronic address: mingch@berkeley.edu.
authors.forename: Ming C
authors.initials: MC
authors.lastname: Hammond
keywords: GEMM-II; STING; Spinach aptamer; cGAMP; cGAS; cyclic AMP-GMP; cyclic GMP-AMP; cyclic dinucleotide; high-throughput screen; riboswitch