In situ assay imaging nuclear RNA exosome activity for cancer studies Abstract The goal of this project is the initial development and demonstration of a new molecular technology which offers highly novel measurement and targeting capabilities potentially transformative for cancer research. This innovative approach will enable a new type assessment of molecular mechanisms of RNA turnover, essential for cancer biology. The project will introduce the first in situ technology capable of labeling the RNA degrading activity of nuclear RNA exosome. RNA exosome (not to be confused with the unrelated vesicular exosomes) is the major enzymatic complex controlling RNA metabolism in cells. It is essential for life. Its fundamental function is to keep cells in the proliferating state. An overactive exosome complex leads to higher rates of cellular proliferation and is implicated in cancer development and progression. It is also a key molecular target of anticancer therapies. Nuclear RNA exosome activity is critical in assessments of tumor cell stress and cell death propensity, and in evaluating cancer response to therapies. In spite of the high utility of an assay labeling RNA exosome activity in situ, in fixed cells and tissue sections, presently there is no such imaging technology. The process is currently studied by using bulk biochemical approaches which have limited value in heterogeneous tissue samples. In this project we will overcome this limitation and will develop the first assay for labeling activity of nuclear RNA exosome in the fixed tissue section format. The project will demonstrate the core functional capabilities of the new molecular imaging technology with wide applicability in cancer studies. Specific Aims of the proposal are: 1. To develop the first approach for specific labeling of nuclear RNA exosome activity in the fixed tissue section format. The approach will permit visualization of nuclear exosome activity by using the innovative capped hybrid RNA probe. 2. To test and validate the core functional capabilities of the newly developed in situ labeling technique in tissue sections from models with activated and normal nuclear RNA exosome activity including glioblastoma. To optimize the new method’s specificity, sensitivity and assure the robust reliability of detection.

Narrative Statement The goal of the proposed research is to introduce a new enabling technology for biomedical imaging. This will produce a new and useful histological assay with broad application in the development of more efficient therapies and for molecular research in cancer, as well as stroke, heart disease and other pathologies.