Griepenburg, Julianne C

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  • Publication
    Regulating Gene Expression With Light-Activated Oligonucleotides
    (2014-01-01) Griepenburg, Julianne C
    The work in this thesis identifies new photochemical approaches to gain high spatiotemporal control over molecular structure and function, for broad applications in materials and biological science. "Caged" compounds provide a method for temporarily blocking function until acted upon by an external trigger, typically near-UV light. To enable multiplexing studies, three new biomolecular caging strategies were developed that can be activated with various wavelengths of near-UV or visible light. The first method, an oligonucleotide hairpin structure incorporating one or two nitrobenzyl photolinkers, was applied to a miRNA antagomir and used to "turn off" let-7 miRNA in zebrafish embryos with 365 nm light. To achieve bidirectional control over miRNA, a circular construct was designed for the ability to "turn on" the release of exogenous miRNA into zebrafish embryos with 365 nm light. A second oligonucleotide caging method, using a ruthenium-based photolinker (RuBEP), was designed to extend photoactivation to the visible spectrum, with additional potential for two-photon activation. RuBEP was used to cage antisense morpholinos through circularization via a Cu(I)-mediated [3+2] Huisgen cycloaddition reaction. RuBEP-caged morpholinos were photoactivated to "turn on" antisense activity and successfully knocked down zebrafish chd and ntl genes with 450 nm light, with limited background activity prior to irradiation. A third method of caging was based on encapsulation within photoresponsive nano-polymersomes. Self-assembly of nano-polymersomes was optimized to generate visible-light-responsive vesicles that incorporate a porphyrin dimer in the hydrophobic membrane. These nanovesicles were shown to encapsulate a variety of cargo, including 25mer oligonucleotides, a small molecule fluorescent dye, and two biologically relevant metal ions, Zn2+ and Ca2+. The photoresponsiveness of the system was modulated with light wavelength, irradiation time, and the presence of dextran in the aqueous core.