The proposed light-activated oligonucleotides will make it possible to turn genes """"""""off"""""""" or """"""""on"""""""" in living cells and zebrafish with very high spatiotemporal resolution, and probe the precise functions of different proteins using a state-of-the-art UV confocal microscope. This will build on successful in vivo experiments in the PI's lab using light-activated antisense oligonucleotides.
In Aim 1, we will develop two complementary methods for down-regulating gene expression with light using caged phosphorothioated DNA (S- DNA) to recruit RNase H or caged peptide nucleic acid (PNA) to sterically block the ribosome. Antisense (18-25-mer) S-DNA and PNA strands will be conjugated to a complementary oligonucleotide of variable length (8-16 nt) and composition (S-DNA, 2'-OMe RNA, PNA, LNA) via a photoactive linker. These conjugates will be optimized with in vitro assays to achieve at least 10-fold down-regulation of protein.
In Aim 2, related RNA """"""""bandages"""""""" will be developed by attaching two short (6-12mer) 2'-OMe RNA strands via a photocleavable linker. Binding the RNA bandage to a target mRNA sequence will block ribosomal translation until photocleavage occurs. The thermodynamic stability of the RNA bandages relative to the individual 2'-OMe RNA strands will be optimized using in vitro assays to achieve a 10-fold upregulation of protein.
In Aim 3, fluorescent reporters will be developed that allow real-time monitoring of oligonucleotide photoactivation in living specimens. Strategies for down-regulating gene expression will be tested and optimized in leukemia cells (against c-myb), zebrafish (against chordin), and neurons (against GluR2). Several additional genes, including GFP, will be upregulated as proof-of-concept in these biological systems. This project will improve human health by creating tools for probing the function of important proteins in cancer cells, zebrafish, and neurons. Possible clinical applications include light-activated, less toxic gene therapies for human leukemia. Additional studies will explore how the spatial control of protein translation in zebrafish is important in hindbrain development and in dendrites is important for memory formation. We propose to develop light-activated oligonucleotides for down- and up-regulating gene expression with ultraviolet light, at high spatial and temporal resolution. These constructs will be tested and optimized in blood cells, zebrafish embryos, and neurons. ? ? ?
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