Regulating Gene Expression With Light-Activated Oligonucleotides
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.