Deconstructing the molecular basis of normal physiology and disease requires an ability to control gene function with genomic, spatial, and temporal specificity. Functional genomic studies have typically utilized homologous recombination, RNA interference, mRNA/cDNA overexpression, or other biological methods, yet these technologies are increasingly limiting as we strive to understand more complex in vivo systems. For example, applying these methods to specific cell populations is hindered by our nascent knowledge of cis- regulatory elements, and they can be unwieldy for targeting combinations of genes. Their kinetic requirements (e.g., rates of Cre recombinase expression, genome editing, RNA degradation, and protein depletion) also diminish the temporal precision with which they can be applied. Light-gated technologies can address these limitations by allowing the optical targeting of multiple genes in specific tissues within seconds. Accordingly, our laboratories and other research groups have devised several strategies for caging morpholino oligonucleotides (MOs), building upon the extensive use of these synthetic antisense reagents in ascidians, sea urchins, zebrafish, frogs, and other animals that develop ex utero. Current caged MOs (cMOs) include hairpin, cyclic, duplex, or nucleobase-modified probes, yet each of these technologies has drawbacks: (1) hairpin and duplex reagents utilize inhibitory oligonucleotides that can increase their cytotoxicity; (2) hairpin, cyclic, and duplex reagents have varying degrees of leakiness; and (3) multiple caged nucleobases are required to completely block MO function, limiting photoactivation efficiency. To overcome these challenges and develop a universal approach for MO photo control, we are developing a new class of cMOs that adopt single- or double-lariat conformations. Each of these novel structures utilizes a single light-cleavable tether to achieve a terminus-to-backbone (Specific Aim 1) or terminus-to-base (Specific Aim 2) linkage, and the resulting oligonucleotide curvature and/or nucleobase functionalization will prevent RNA binding. Linker photolysis will then release these constraints to allow efficient MO/RNA hybridization. We will explore different conjugation sites within the MO oligonucleotide and various linker structures to optimize lariat cMO function, guided by in vitro assays of RNA function and well- characterized zebrafish models. We will also evaluate different caging chromophores for multi-wavelength activation and establish combinations that allow simultaneous or sequential gene knockdowns (Specific Aim 3). We will then use lariat cMOs to uncover how pancreatic and duodenal homeobox factor 1 (pdx1) and motor neuron and pancreas homeobox factor 1 (mnx1) cooperatively regulate endocrine pancreas development. These studies integrate our laboratories' expertise in optochemical probes and zebrafish models, and the resulting technologies will advance our understanding of in vivo biology at the molecular and systems levels.
Deciphering the genetic programs that regulate organismal biology requires an ability to control gene function with spatial and temporal precision. The new light-activatable technologies described in this application will enable conditional and combinatorial gene knockdowns that are not possible with current reverse-genetic methods. These optochemical tools will enhance the utility of zebrafish and other model organisms in biomedical research and reveal the dynamic molecular mechanisms that contribute to normal physiology and disease.
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