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

本计画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。该技术的重点是光笼化，共价连接和随后的关键核酸功能上的封闭基团的光化学切割。笼状RNA寡核苷酸在暴露于光之前是生物惰性的，这使得能够控制它们与蛋白质的空间和时间相互作用。阻断核酸生物活性直至暴露于光的笼化策略依赖于在适当位点连接足够笼分子以灭活的能力以及光解它们以恢复活性的能力。我们将证明通过使用可以并入标准合成仪的修饰的核酸构建块将光敏笼化基团位点特异性并入寡核苷酸中。将通过熔解曲线分析和细胞培养物和斑马鱼胚胎中的基因沉默活性来评价无活性的光笼化寡核糖核苷酸的杂交阻断。该活性的恢复将通过UVA（~365nm）光以及双光子激发来实现。本研究的一部分将探索光活化剂量在细胞培养中的作用。将通过细胞活力和细胞凋亡测定来评估暴露方案的调节，例如强度、持续时间、脉冲/连续模式。将通过GFP在细胞培养物和发育中的斑马鱼胚胎中测量光笼化siRNA的RNA干扰（RNAi）活性的阻断和恢复。在具有笼状siRNA的细胞中用光来控制基因沉默将为功能基因组学和发育研究提供新的工具。将开发一个综合教育计划，包括实验室组成部分和研究生课程，重点是光学和分子方法。将开发和提供关于这些专题的闭环学习单元。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.