The zebrafish (Danio rerio) has become a powerful model for investigating the genetics of human disease and development; however, there is currently no mechanism for dissecting embryonic gene function in a switchable, spatio-temporal manner. The overall goal of this project is to develop multiple mechanisms for spatio-temporal control of gene and transgene expression in zebrafish via light irradiation. The specific intent is to utilize small photo-activatable (photo-caged) and photo-switchable organic compounds to tightly regulate ribozyme and riboswitch function using UV light. These RNA based methods will be utilized to control gene and transgene expression within the developing zebrafish embryo. Our strategies are based on: 1) the ability to synthesize photo-activatable and photo-switchable compounds, 2) the ability to select allosteric ribozymes in vitro whose activity relies on the presence of specific organic compounds, 3) the ability to select short RNA sequences (RNA aptamers) in vitro that bind selectively organic compounds, and 4) the ability to combine 1-3 to create light-responsive switches for activation and deactivation of gene expression in zebrafish.
The Specific Aims are to: 1. Develop systems for photochemical control of RNA function. We will synthesize non-toxic, cell permeable, photo-responsive small organic molecules and in vitro select ribozymes that can be activated by these molecules in conjunction with UV light. Moreover, RNA aptamers will be evolved in a way that their binding to these molecules can be modulated with UV light. 2. Develop systems for photochemically mediated gene regulation. Specifically we will demonstrate spatio- temporal control of gene knock-down in zebrafish embryos in an irreversible and a reversible manner using irradiation with UV light. 3. Develop systems for photochemically mediated transgene regulation. Specifically, we will demonstrate spatio-temporal control of transgene expression in zebrafish embryos in an irreversible and a reversible manner using irradiation with UV light. All developed components (e.g. photoresponsive small organic molecules, allosteric ribozymes, RNA. aptamers, expression plasmids, etc.) and protocols (e.g. for organic synthesis, UV irradiation, etc.) will be made accessible to the scientific community via a dedicated web-page. ? ? ?
| Hemphill, James; Liu, Qingyang; Uprety, Rajendra et al. (2015) Conditional control of alternative splicing through light-triggered splice-switching oligonucleotides. J Am Chem Soc 137:3656-62 |
| Govan, Jeane M; Young, Douglas D; Lively, Mark O et al. (2015) Optically Triggered Immune Response through Photocaged Oligonucleotides. Tetrahedron Lett 56:3639-3642 |
| Walsh, Steven; Gardner, Laura; Deiters, Alexander et al. (2014) Intracellular light-activation of riboswitch activity. Chembiochem 15:1346-51 |
| Hemphill, James; Govan, Jeane; Uprety, Rajendra et al. (2014) Site-specific promoter caging enables optochemical gene activation in cells and animals. J Am Chem Soc 136:7152-8 |
| Engelke, Hanna; Chou, Chungjung; Uprety, Rajendra et al. (2014) Control of protein function through optochemical translocation. ACS Synth Biol 3:731-6 |
| Karginov, Andrei V; Hahn, Klaus M; Deiters, Alexander (2014) Optochemical activation of kinase function in live cells. Methods Mol Biol 1148:31-43 |
| Liu, Qingyang; Deiters, Alexander (2014) Optochemical control of deoxyoligonucleotide function via a nucleobase-caging approach. Acc Chem Res 47:45-55 |
| Yamazoe, Sayumi; Liu, Qingyang; McQuade, Lindsey E et al. (2014) Sequential gene silencing using wavelength-selective caged morpholino oligonucleotides. Angew Chem Int Ed Engl 53:10114-8 |
| Govan, Jeane M; Uprety, Rajendra; Thomas, Meryl et al. (2013) Cellular delivery and photochemical activation of antisense agents through a nucleobase caging strategy. ACS Chem Biol 8:2272-82 |
| Govan, Jeane M; Young, Douglas D; Lusic, Hrvoje et al. (2013) Optochemical control of RNA interference in mammalian cells. Nucleic Acids Res 41:10518-28 |
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