Engineering New Methods To Investigate Cyclic Dinucleotides

Download or read book Engineering New Methods to Investigate Cyclic Dinucleotides written by Alex J. Pollock and published by . This book was released on 2021 with total page 112 pages. Available in PDF, EPUB and Kindle. Book excerpt: Nucleotide second messengers are nucleotide-based small molecules which orchestrate cellular responses to internal cues and adaptation to environmental conditions. These messengers are ubiquitous throughout life and their flux coordinates diverse processes via binding response regulator proteins or riboswitches to induce post-translational, translational, or transcriptional responses. Some of the best studied nucleotide second messengers include cAMP which regulates carbon source utilization, (p)ppGpp which regulates biosynthetic metabolism in response to nutrient limitation, and 3′3′-c-di-GMP which regulates motility and biofilm production. Nucleotide second messengers are hypothesized to be ubiquitous because they are rapidly diffusible and can be quickly synthesized from and hydrolyzed into common substrates making them amenable to effective cellular coordination. This work focuses on the recently discovered nucleotide second messengers 3′3′-c-di-AMP and 2′3′-cGAMP which are respectively an essential bacterial molecule regulating virulence, cell wall homeostasis, and osmotic regulation and a mammalian innate immune signaling molecule signaling mis-localized double stranded DNA. These second messengers are synthesized by the condensation of two ATPs or an ATP and a GTP molecule, respectively, and have been the subject of increasing academic and clinical interest in the last decade. Despite significant advances in these fields, quantitative, single cell, and kinetic tools to detect these important nucleotide second messengers are lacking. This dearth has resulted in many important investigations being intractable. In response, we have generated FRET biosensors for both 2′3′-cGAMP and 3′3′-c-di-AMP. These adaptable biosensors allow for entirely new perspectives of bacterial and metazoan cyclic-di-nucleotide signaling. Additionally, we have also developed a luminescence-based coupled enzyme assay to facilitate higher-throughput population level quantification of 3′3′-c-di-AMP. We note that the recently discovered bacteriophage-defense signaling molecule, 3′3′-cGAMP can also be detected using this coupled enzyme assay but utility will be limited until a high affinity binding protein is found and adapted to extract 3′3′-cGAMP from complex samples. The tools developed in this work will facilitate diverse investigations of immune signaling and bacterial regulation with implications for immunotherapy, autoimmune disease, viral and bacterial restriction, and vaccine development.