Research Introduction

    The biological functions of RNA molecules are just beginning to be discovered and explored. Despite these exciting observations assigning biological functions, structural motifs, and molecular interactions of RNAs inside the cellular context has been heretofore extremely challenging. Technological innovations are needed to further understand the role of RNA molecules in regulating basic biological function. Further, there is a need to expand the biochemistry toolkit to understand how large groups of RNAs are working in parallel inside living cells. Our goal as a lab is to merge seemingly disparate disciplines: chemistry & genomics, to gain novel insights into RNA structure and function. We always begin our projects with particular biological questions in mind. We use these questions to start our "catalytic cycle of discovery". We then design small molecules that will help us start to answer our questions. Biochemical approaches are then used to test our molecules and identify the best probe or conditions for in cell analyses. We then merge the chemical reactivity of our probes with deep sequencing to obtain a systems level analysis of RNA biology. Ultimately, we aim to uncover novel biological insights that lead to new interesting lines of research to dig deeper into RNA biology and function.

How can we track RNA expression within complex tissues?

   RNA expression profiling is a critical first step towards discovering and characterizing the RNA content of a cell. These studies are mostly limited to cell culture of a single cell type. However, cells as they exist in their natural environment reside in heterogenous populations. Profiling RNA expression derived from a single cell type within a complex structure or network is an extremely difficult task. Furthermore, identifying the nascent RNA pool from specific cell types is incredibly challenging.

   Stringent chemical methods to profile RNA expression within discrete cellular populations remains a key challenge in biology. To address this issue we have worked to design, synthesize, and test a suite of caged nucleoside metabolic intermediates and demonstrate their ability to be metabolically incorporated into RNA. Incorporation can be profiled and imaged using bioorthogonal chemistry. We anticipate that this platform will provide the community with a much-needed chemical toolset for cell-type specific profiling of whole transcriptomes derived from complex biological systems.

   We are now working on expanding the chemical repetoir for metabolic labeling of RNA and also are in pursuit of novel nucleoside-enzyme pairs to expand the scope of cell-specific metabolic labeling of RNA. Finally, we are working to transition the tools we have developed in cell culture experiments into more complex tissue environments inside living animals.

How does RNA structure control biology?

   Critical to understanding RNA function is a full description of its structure. Chemical methods to measure RNA structure have been part of the RNA biologists toolkit for several decades. We have focused our efforts on the design and implementation of novel chemical probes for measuring two aspects of RNA structure in cells. First, SHAPE, which measures RNA flexibility and single-strandedness in RNA. Second, LASER, which measures solvent accesibility at the C-8 position of purines. Following on our reagent discoveries we are utilizing them for analyses of large groups of RNA structures as they function inside the native environment of the cell. We aim to intersect our observations with orthogonal datasets such as RNA-protein interactions, ribosome profiling, synthesis and decay, and localization to extract general principles for how the structure of RNA molecules controls their function.

Where are RNAs localized inside cells?

   We have been working to develop a novel chemical-genetic method for assaying RNA localization within living cells. RNA localization is critical for normal physiology as well as the onset of cancer and neurodegenerative disorders. Despite its importance there is a real lack of chemical methods to directly assay RNA localization with high resolution in living cells. Our novel approach relies on in situ oxidation by singlet oxygen generated from spatially confined fluorophores. We have demonstrated that our novel method can identify RNA molecules localized within specific cellular compartments. Future plans are to systematically track RNA localization within many subcellular compartments with the long term vision of construcing a "Global Positioning System (GPS)" for RNA. We anticipate that this platform will provide the community with a much-needed methodology for tracking RNA localization within living cells, and sets the stage for systematic large scale analysis of RNA localization in living systems.