A cyclic dinucleotide containing 2-aminopurine is a general fluorescent sensor for c-di-GMP and 3',3'-cGAMP.
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.
Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA. hsintim@umd.edu.
Benjamin T
BT
Roembke
Jie
J
Zhou
Yue
Y
Zheng
David
D
Sayre
Allan
A
Lizardo
Laurentee
L
Bernard
Herman O
HO
Sintim
D015075:2-Aminopurine / Q000378:metabolism*; D018441:Biofilms; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000737:chemistry*; D005453:Fluorescence; D015335:Molecular Probes / Q000737:chemistry; D009712:Nucleotides, Cyclic / Q000737:chemistry*; D015290:Second Messenger Systems; D015398:Signal Transduction
D015335:Molecular Probes; D009712:Nucleotides, Cyclic; C584311:cyclic guanosine monophosphate-adenosine monophosphate; D015075:2-Aminopurine; D006152:Cyclic GMP