Characterizing gene regulation with single molecule sensitive probes

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DESCRIPTION (provided by applicant): Deregulation of gene expression contributes to aberrant phenotypes and behaviors of cancer cells. Acquiring a new profile of expressed proteins and their subsequent activation enable cancer cells to re-enter the cell cycle, or give them survival and migratory advantages over those of the "normal cells". Alterations in cis-acting sequences, RNA binding proteins, or in upstream signaling pathways affect the stability and/or translational efficiency of mRNAs encoding proto-oncogenes, cytokines, cell cycle regulators and other regulatory proteins to promote tumorigenesis and cancer progression, especially during cellular stress induced by hypoxia or reactive oxygen species (ROS). In addition, these environments can also induce stress granule formation, a consequence of translational suppression and eIF21 phosphorylation, which can inhibit apoptosis. In this application we will focus on developing technology to characterize the deregulation of gene expression by: 1) evaluating the transcriptional activity of cancer cells by assessing mRNAs already transcribed, and 2) by characterizing mRNA/RNA binding protein interactions in the cellular/tissue milieu. To do this we will further develop probes to image low copy number native mRNAs in both fixed and living cells, and develop a novel method for detecting RNA-protein interactions with single interaction sensitivity. Recently we have developed a new probe consisting of four high-affinity, nuclease resistant, linear nucleic acids, labeled with multiple, high quantum-yield fluorophores linked together by streptavidin, via the biotin-streptavidin linkage. This design results in a multivalent, single RNA sensitive and versatile imaging probe. Because these probes contain a protein at their core, streptavidin, it is our contention that when combined with antibody-based proximity ligation assays (PLAs) and multicolor fluorescence microscopy, single RNA-protein interactions can be detected in fixed tissue or post live-cell hybridization, fixed cell samples. Proximity ligation assays utilize oligonucleotide-linked antibodies, ligation, rolling circle amplification (RCA) and fluorescence detection to achieve single interaction sensitivity. Last, once these interactions are identified, fluorescent proteins will be developed for the relevant RNA binding proteins to allow for the monitoring of these interactions in live cells during cancer relevant processes. Thus, through the development of this technology, we will be able to characterize these events in cellular models of prostate tumor progression, both fixed and live, and in clinical tumor samples. From this characterization we hope to identify new targets for therapeutic development and possibly gain insight into mechanisms that may make tumor cells refractory to chemo and radiation therapy.