The main goal of this project is to develop a simple, yet powerful and versatile technology for detection and imaging of epigenetic histone modifications and histone modifying enzymes in living cells. A full understanding of how various histone modifications and the responsible modifying enzymes function require precise knowledge of their localization, movement, and dynamics in a living cell. Current technologies lack the sensitivity and the versatility required to track these targets in their native environment. To achieve these goals, we propose to construct multivalent RNA aptamers comprised of three main features;i) a peptide/protein targeting aptamer, ii) multiple copies of an aptamers that bind and enhance fluorescence of weakly fluorescing analogs of normally highly-fluorescent small molecules (referred to as fluorescent molecule analogs (FMAs)), and iii) a core multi-way RNA junction that combines the previous types of aptamers in a compact structure. We propose to express such multivalent aptamers in cells or whole animals. The MPFAs will also bind FMAs exogenously supplied to cells. Since the FMA binding aptamers will have been selected to enhance dramatically the quantum yield of the FMA molecule upon binding, the MPFAs will render the target visible for detection with fluorescent microscopy. Different combinations of peptide/protein-targeting and FMA-binding aptamers with spectrally separable FMAs will be utilized for multiplex imaging of multiple proteins within the same cell. A key advantage of this approach is that it does not depend on covalent attachment of the imaging moiety, which can interfere with the localization and function of the target protein. The multivalent aptamers can be expressed just prior to imaging and therefore cause little interference with the function of the target protein or the general physiology of the cell. The designed MPFAs will then be tested carefully for in vivo imaging of the select set of histone modification and modifying enzyme targets in Drosophila salivary gland cells and diploid cells. Sensitivity of the proposed technology will be compared to existing detection/imaging methods. The ultimate goal in detection sensitivity is the single-molecule in vivo detection/imaging using MPFAs and exogenously supplied FMAs. The project brings together principle investigators that have complementary expertise in chemistry;design, synthesis and characterization of fluorescent molecules and their derivatives (Lin Lab), in molecular/cell biology;gene and chromatin regulation, genetics and reverse- genetics, RNA aptamer selections, and design and expression of multivalent RNAs (Lis Lab), in applied and engineering physics;design and fabrication of micro- and nano-fluidic mechano-electronic devices (Craighead Lab), in biomedical engineering;fluorescence confocal and multi-photon microscopy and photophysical characterization of FMAs (Zipfel Lab) and a proven record of productive collaborations.
This application titled In vivo Detection and Imaging of Epigenetic Histone Modifications and Modifying Enzymes Using Multivalent RNA Aptamers addresses the RFA-RM-09-016: Developing Technologies for Improved In Vivo Epigenetic Imaging or Analysis. The project is expected to have a major impact on medical and life sciences research by developing a sensitive multivalent RNA aptamer-based detection/imaging technology that allows multiplex imaging of two or more epigenetic histone modifications and/or modifying enzymes in vivo. This novel powerful and versatile technology will complement the existing reagents and technologies, overcome many of the technical limitations of existing imaging technologies, and thus provide further insights and a more comprehensive understanding of the epigenetic regulation of histone modifications that relate to human health and diseases.
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