Biophysical and Biochemical Applications of Isomorphic Fluorescent Cyclic Dinucleotide Analogs

As cyclic dinucleotides’ impact on pathogenic bacteria and eukaryotic cells is gradually being recognized, shedding light on the enzymatic pathways involved in its production, degradation and recognition is of significance. Fluorescence-based techniques have been playing key roles in modern biochemical and biophysical research due to their high sensitivity and informativeness. In order to take advantage of fluorescence-based techniques, we developed isomorphic emissive cyclic dinucleotide analogs using previously developed fluorescent nucleoside analogs in the Tor lab.Instead of going through the challenging chemical synthesis of cyclic dinucleotide analogs, we decided to exploit bacterial dinucleotide cyclases, DncV and DisA, to convert emissive nucleoside triphosphates into corresponding cyclic dinucleotides (CDNs). Rewardingly, DncV and DisA were able to synthesize emissive cyclic dinucleotides with thGTP, tzGTP, thATP and tzATP with yields comparable to that of native NTPs. Using DncV and DisA, a library of emissive CDNs were successfully synthesized.To demonstrate the power and utility of these fluorescent CDN analogs, we sought to use them to monitor the enzyme-mediated conversion of CDNs, which is an important process in CDN signaling in cells. The quantum yields of the emissive CDNs were found to be significantly lower than that of corresponding nucleosides, which is caused by fluorescence quenching by adjacent nucleobases in the CDNs. We thus utilized the quenching effect to monitor DncV-mediated CDN synthesis with steady state fluorescence spectroscopy. By carefully analyzing the emission spectra of the reaction, we were able to derive reaction rate constants comparable to those calculated from HPLC analysis. It is a great advantage to monitor such a process with fluorescence, as traditional methods such as radioisotope labeling or HPLC cannot be used for real- time monitoring, and the experimental procedures are often time-consuming. We speculate that such a fluorescence-based method of monitoring ...