A cyclic dinucleotide containing 2-aminopurine is a general fluorescent sensor for c-di-GMP and 3′,3′-cGAMP

Abstract

Cyclic dinucleotides have emerged as second messengers that regulate diverse processes in bacteria, as well as regulating the production of type I interferons in metazoans. Fluorescent sensors for these important second messengers are highly sought-after for high-throughput inhibitor discovery, yet most sensors reported to date are not amenable for high-throughput screening purposes. Herein, we demonstrate that a new analog, 3′,3′-cG(d2AP)MP, which is a 2-aminopurine (2AP)-containing cyclic dinucleotide, self-associates in the presence of Mn²⁺ with an association constant of 120 000 M⁻¹. 3′3′-cG(d2AP)MP can also form a heterodimer with cGAMP, activator of immune regulator, STING, or the bacterial biofilm regulator, c-di-GMP in the presence of Mn(II). Upon dimer formation, the fluorescence of 3′,3′-cG(d2AP)MP is quenched and this provides a convenient method to monitor the enzymatic processing of both DGC and PDE enzymes, opening up several opportunities for the discovery of inhibitors of nucleotide signaling.