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

Louisiana State University, Baton Rouge LA

Investigators

Abstract

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 restore activity. 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.