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 Mn(2+) 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.
Highlights
Cyclic dinucleotides (Fig. 1) have piqued the interests of scientists of different disciplines in the last few years due to the increasingly diverse processes that they regulate in both prokaryotes and eukaryotes
We hypothesized that the two nucleobases of a 2-aminopurine (2AP) analog of 30,30-cGAMP (30,30-cG(d2AP)MP, 1) cannot stack with each other because the distance between the nucleobases of a 30,30-cyclic dinucleotide is B6.7 Å,31 which is too far for effective orbital overlap for p–p stacking interaction
At low concentrations of 30,30-cG(d2AP)MP, when it will mainly exist in the monomeric form, it would form heterodimers with other natural cyclic dinucleotides and the heterodimerization could be detected via fluorescence reduction
Summary
Cyclic dinucleotides (Fig. 1) have piqued the interests of scientists of different disciplines in the last few years due to the increasingly diverse processes that they regulate in both prokaryotes and eukaryotes. Having established that 30,30-cG(d2AP)MP can report the presence of c-di-GMP and 30,30-cGAMP, we proceeded to determine the association/dissociation constants of this cyclic dinucleotide (see Fig. 5).
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