The objective of the proposed research is to expand and develop a novel and simple method for imaging RNA and small molecules in living cells. Unlike green fluorescent protein (GFP), which can be used to monitor proteins in cells, there are currently no analogous simple and straightforward approaches to track RNA movement in living cells. Currently available approaches have critical limitations that have precluded their widespread use. We have engineered both an RNA receptor and a small molecule ligand that interact to form a fluorescent complex. The RNA "switches on" the fluorescence of an otherwise nonfluorescent small molecule. We have characterized this RNA-fluorophore complex, optimized its sequence to improve its fluorescence properties, and shown that it can be used to monitor RNAs in living cells. Additionally, we have shown that the RNA aptamer can be fused to other aptamers to generate allosterically regulated fluorescent analyte sensors. In order to develop our system into a widely-used, simple and sensitive technique that would permit the study of the cell biology of RNA, the specific aims of this application are: (1) To develop a palette of RNA-fluorophore complexes;(2) To optimize the fluorescence properties of RNA-fluorophore complexes. In this aim, we will use affinity maturation and other techniques to increase the fluorescence of the RNA-fluorophore complexes;(3) To use RNA- fluorophore complexes to visualize mRNAs in cells and mRNA trafficking in axons. In this aim, we describe experiments to optimize the fluorescence of RNA-fluorophore complexes in cells and to monitor mRNA trafficking and mRNA degradation during axon turning;(4) To develop fluorescent sensors from RNA-fluorophore complexes. We present a simple and generalizable approach for generating fluorescent analyte sensors and using them in cells. Together, the experiments in these four aims will result in a versatile RNA imaging and fluorescent sensor technology that is simpler and more specific than any other methodology that is currently available.

Public Health Relevance

RNA is increasingly recognized as being a critical regulator of cellular function;however, there are currently no simple and straightforward approaches to track RNA movement in living cells. The proposed experiments will result in a highly powerful RNA imaging and fluorescent sensor technology that is simpler and more specific than any other methodology that is currently available. This will provide a molecular toolkit that will substantially enhance our ability to study the role of RNA in health and disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS064516-04
Application #
8254408
Study Section
Molecular Neurogenetics Study Section (MNG)
Program Officer
Mamounas, Laura
Project Start
2009-05-15
Project End
2014-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
4
Fiscal Year
2012
Total Cost
$423,151
Indirect Cost
$170,316
Name
Weill Medical College of Cornell University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
060217502
City
New York
State
NY
Country
United States
Zip Code
10065
Svensen, Nina; Peersen, Olve B; Jaffrey, Samie R (2016) Peptide Synthesis on a Next-Generation DNA Sequencing Platform. Chembiochem 17:1628-35
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Svensen, Nina; Jaffrey, Samie R (2016) Fluorescent RNA Aptamers as a Tool to Study RNA-Modifying Enzymes. Cell Chem Biol 23:415-25
Strack, Rita L; Jaffrey, Samie R (2015) Live-cell imaging of mammalian RNAs with Spinach2. Methods Enzymol 550:129-46
Filonov, Grigory S; Kam, Christina W; Song, Wenjiao et al. (2015) In-gel imaging of RNA processing using broccoli reveals optimal aptamer expression strategies. Chem Biol 22:649-60
You, Mingxu; Litke, Jacob L; Jaffrey, Samie R (2015) Imaging metabolite dynamics in living cells using a Spinach-based riboswitch. Proc Natl Acad Sci U S A 112:E2756-65
You, Mingxu; Jaffrey, Samie R (2015) Structure and Mechanism of RNA Mimics of Green Fluorescent Protein. Annu Rev Biophys 44:187-206
Lu, Zhipeng; Filonov, Grigory S; Noto, John J et al. (2015) Metazoan tRNA introns generate stable circular RNAs in vivo. RNA 21:1554-65
Warner, Katherine Deigan; Chen, Michael C; Song, Wenjiao et al. (2014) Structural basis for activity of highly efficient RNA mimics of green fluorescent protein. Nat Struct Mol Biol 21:658-63
Strack, Rita L; Song, Wenjiao; Jaffrey, Samie R (2014) Using Spinach-based sensors for fluorescence imaging of intracellular metabolites and proteins in living bacteria. Nat Protoc 9:146-55

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