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

Cyclic dinucleoties, such as cGAMP, c-di-GMP and c-di-AMP, are fascinating second messengers with diverse roles in both prokaryotes and eukaryotes. Consequently there is a need for simple and inexpensive methods for profiling these compounds in biological media, monitoring their synthesis or degradation by enzymes and for identifying inhibitors of proteins that metabolize or bind to these dinucleotides. Since 2011, when we reported the first simple method to detect c-di-GMP (S. Nakayama, I. Kelsey, J. Wang, K. Roelofs, B. Stefane, Y. Luo, V. T. Lee and H. O. Sintim, , 2011, , 4856) or in 2014 when we revealed another surprisingly simple assay to detect c-di-AMP (J. Zhou, D. A. Sayre, Y. Zheng, H. Szmacinski and H. O. Sintim, , 2014, , 2412), there have been efforts to develop assays to detect cyclic dinucleotides by others. However a unified and simple assay, which can be used for all cyclic dinucleotides is lacking. Here, we investigate STING binding by various fluorescein-labeled c-di-GMP, c-di-AMP and cGAMP, using fluorescent polarization (FP). Fluorescein-labeled c-di-GMP (F-c-di-GMP) was found to be the best binder of STING. This probe could be displaced by unlabeled cGAMP, c-di-AMP or c-di-GMP and hence it is a universal probe, which can be used to monitor all three dinucleotides. HPLC analysis was used to validate the new F-c-di-GMP-based FP assay.