A method for detection of endogenous unmodified RNA in live bacterial cells
Toran, Paul T.
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RNAs are dynamic molecules that orchestrate a breadth of processes for prokaryotes and eukaryotes in both the nucleus and cytoplasm. These processes include regulation of transcription, translation, and post-translational modifications. It has been demonstrated that the spatio-temporal localization of different RNAs is an important factor in development and the correct localization of some RNAs is crucial for proper development, structural organization and function of the cell. In vivo detection of endogenous RNAs is a challenging task because of low RNA concentrations in live cells, its transient character, limited accessibility for molecular probes, and sensitivity to modification. Methods that study RNA localization utilize either hybridization techniques with pre-labeled probes in fixed cells or require modifications to target RNAs in living cells potentially altering their in vivo behavior. The goal of this project is to design and explore a new non-invasive technique that is capable of detecting unmodified endogenous RNA in living cells. This method utilizes a combination of protein complementation and split aptamer probe technology to fluorescently detect unmodified endogenous RNA in living cells. Experiments show that this approach is capable of detecting a full-length β-globin mRNA in a sequence-dependent manner in live E. coli cells. Most importantly, fluorescent detection of the endogenous inducible phosphate stress response mRNA pstC IS successfully demonstrated. This fluorescent signal Is dependent on the concentration of pstC mRNA. Furthermore, the fluorescent signal revealS a punctate localization of pstC mRNA, which dOES not overlap with nucleoid DNA. This work holds the potential for the next generation of molecular tools for basic RNA research, clinical diagnostics and genetic therapeutics.