Development of photoreceptor-aptamer systems for the spatiotemporal control of gene expression

Abstract

Gene expression regulation is a fundamental process underlying numerous biological mechanisms, from development and homeostasis to cellular responses and disease progression. Advances in the ability to control gene expression with high temporal and spatial precision have driven significant progress in synthetic and developmental biology. Although traditional methods using chemical or heat inducers have laid the groundwork for this field, they come with limitations that have prompted the need for development of tools which allow for control of gene expression with spatiotemporal precision. Optogenetics has emerged as a transformative approach, offering precise, non-invasive, and reversible control of gene activity using light-sensitive proteins.<br /> This dissertation introduces the development of the flyPAL system, an optogenetic tool specifically designed for transcriptional activation in Drosophila melanogaster. The flyPAL platform integrates the spatial specificity provided by the GAL4-UAS system with the temporal control facilitated by the CRISPRa-PAL system. The core component of this system is the photoreceptor protein PAL (PAS-ANTAR-LOV), which undergoes reversible conformational changes in response to blue light and binds with high affinity to an RNA aptamer (53.19) in a light-dependent manner. By leveraging this interaction, flyPAL enables blue light-mediated, tissue-specific regulation of gene expression, allowing for precise spatial and temporal control. The efficacy of the flyPAL system was validated through experiments utilizing a fluorescent reporter gene (mRFP), demonstrating light-dependent and reversible gene expression in Drosophila Schneider 2 cells and in vivo in third-instar larvae. These findings establish flyPAL as an optoribogenetic tool for gene regulation studies, with potential to enhance investigations into dynamic biological processes and disease models.<br /> Additionally, this research explores the use of another blue-light responsive photoreceptor protein, NdPAL1. NdPAL1 is a homolog of PAL and it undergoes reversible conformational changes in response to blue light, as PAL. NdPAL1 features a faster dark recovery rate compared to PAL, making it particularly well-suited for applications requiring rapid and reversible control, such as in neuroscience. An Opto- SELEX strategy was adapted to identify RNA aptamers that selectively bind to the light-activated state of NdPAL1, leading to the discovery of the TN10.2.21 aptamer. Functional studies demonstrated that NdPAL1 can perform light-switching in mammalian cells, highlighting its potential for real-time gene regulation in cellular environments.<br/> Overall, this dissertation introduces flyPAL, an optoribogenetic tool for gene regulation in Drosophila, and demonstrates the development and optimization of RNA aptamers that bind to NdPAL1 in a light-dependent manner in vitro.