Download or read book Development of RNA Sensors for Cyclic Dinucleotide Second Messengers in Bacteria written by Colleen Ann Kellenberger and published by . This book was released on 2014 with total page 125 pages. Available in PDF, EPUB and Kindle. Book excerpt: Second messengers are intracellular signaling molecules that enable cells to respond to environmental changes by relaying signals received at the cell surface to enact downstream physiological effects. A recently expanded class of second messengers in bacteria are the cyclic dinucleotides, which include cyclic di-GMP (cdiG), cyclic di-AMP (cdiA), and 3',3' cyclic AMP-GMP (cAG). In bacteria, these messengers regulate processes ranging from chemotaxis and sporulation to antibiotic resistance and virulence. Recent studies have found that bacterial cyclic dinucleotides, as well as a mammalian 2',3' cAG homolog, trigger the innate immune response upon detection in the mammalian cell cytosol. However, many components and roles of cyclic dinucleotide signaling remain unidentified or poorly understood. A clear understanding of cyclic dinucleotide signal transduction would further the pursuit of these signaling pathways as therapeutic targets and the effects of using cyclic dinucleotides as small molecule adjuvants. Herein, novel strategies for cyclic dinucleotide detection are reported that use that natural ability of riboswitch RNAs to discriminate between these nucleotide-based messengers. First, a microfluidic electrophoretic mobility shift assay (μMSA) is presented that enables highly sensitive and rapid detection of cdiG in vitro. This method takes advantage of the ligand-induced conformational change of a natural cdiG GEMM-I class riboswitch aptamer, termed Vc2. This same aptamer was then converted to a fluorescent biosensor for live-cell imaging by fusing it to the Spinach aptamer, an RNA mimic of GFP. This Vc2-Spinach RNA-based biosensor senses low levels of endogenous cdiG in Escherichia coli and responds to increased levels of cdiG upon coexpression of a heterologous cdiG synthase. A similar Spinach-based strategy was used to develop a cdiA RNA-based biosensor through fusion to riboswitches from the recently reported ydaO class. This cdiA sensor is the first tool for live cell imaging of cdiA and provided the first demonstration of the Spinach system in Listeria monocytogenes, an important gram-positive intracellular pathogen. Finally, as no known biosensor existed for 3',3' cAG, rational engineering of a cdiG aptamer led to the discovery of a subclass of GEMM-I riboswitches in Geobacter species that is naturally specific for 3',3' cAG. Further analysis revealed that this novel riboswitch subclass, which represents the first known effector for this messenger, is important in regulating the process of extracellular electron transfer in Geobacter and these riboswitches were used to develop the first biosensor specific for 3',3' cAG. It is envisioned that the use of these cyclic dinucleotide-specific sensors can be used to further identify unknown components in the signal transduction of these important regulatory molecules.