CAREER: Photoactivatable Nucleic Acids for Control of Molecular and Cellular Processes

这个项目的目标是展示一种基于光的诱导技术来控制核酸的生物活性。这项技术侧重于光笼化，即关键核酸功能上的封闭基的共价连接和随后的光化学裂解。笼化的RNA寡核苷酸在暴露于光之前是生物惰性的，这使得能够在空间和时间上控制它们与蛋白质的相互作用。阻止核酸生物活性直到光照的笼化策略取决于在适当的位置附着足够的笼状分子以使其失活的能力，以及将它们光解以恢复反应的能力。我们将通过使用可以结合到标准合成器中的修改的核酸构建块来演示光活性笼形基团与寡核苷酸的位置特异性结合。在细胞培养和斑马鱼胚胎中，通过熔化曲线分析和基因沉默活性，将评估失活的光笼寡核苷酸对杂交的阻断作用。这种活性的恢复将在UVA(~365 nm)光和双光子激发下实现。这项研究的一部分将是探索细胞培养中光激活剂量的影响。我们将通过细胞存活率和细胞凋亡分析来评估曝光强度、持续时间、脉冲/连续模式等的调制方式。通过细胞培养和斑马鱼胚胎发育中的GFP来测量光笼中RNA干扰(RNAi)的阻断和恢复活性。用光控制笼式siRNA细胞中的基因沉默，将为功能基因组学和发育研究提供新的工具。将制定一项综合教育方案，包括实验室部分和提供侧重于光学和分子方法的研究生课程。将开发和提供关于这些主题的闭环系统学习模块。课程的服务学习活动将根据研究计划的基本原理开发外展模块。

The goal of this project is to demonstrate a light-based induction technique to control the bioactivity of nucleic acids. This technique focuses on photocaging, the covalent attachment and subsequent photochemical cleavage of blocking groups on key nucleic acid functionalities.Caged RNA oligonucleotides are biologically inert until exposed to light, which enables control over their interactions with proteins both spatially and temporally. The caging strategy to block nucleic acid bioactivity until light exposure is dependent upon both the ability to attach enough cage molecules at appropriate sites for inactivation, and also to photolyze them to restoreactivity. We will demonstrate site-specific incorporation of a photoactive caging group into an oligonucleotide by use of modified nucleic acid building blocks that can be incorporated into standard synthesizers. The inactive photocaged oligoribonucleotides will be evaluated for blockade of hybridization through melt curve analysis and gene silencing activity in cell culture and zebrafish embryos. Restoration of this activity will be achieved with UVA (~365nm) light and also 2-photon excitation. Part of this study will be to explore the effects of photoactivating doses in cell culture. Modulation of exposure regimes such as intensity, duration,pulsed/continuous patterns will be evaluated through cell viability and apoptosis assays.Blockade and restoration of RNA interference (RNAi) activity of photocaged siRNAs will be measured through GFP in cell cultures and developing zebrafish embryos. Control over gene silencing with light in cells with caged siRNAs will enable new tools for functional genomics and developmental studies. An integrated educational program consisting of a laboratory component and graduate offering of classes focusing on optical and molecular methods will be developed. Closed-loop learning modules on these topics will be developed and made available. Service-learning activities for the classes will develop outreach modules based on the fundamentals of the research program.