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 #
5R01NS064516-02
Application #
7827952
Study Section
Molecular Neurogenetics Study Section (MNG)
Program Officer
Mamounas, Laura
Project Start
2009-05-15
Project End
2014-04-30
Budget Start
2010-05-01
Budget End
2011-04-30
Support Year
2
Fiscal Year
2010
Total Cost
$292,793
Indirect Cost
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
Warner, Katherine Deigan; Sjeklo?a, Ljiljana; Song, Wenjiao et al. (2017) A homodimer interface without base pairs in an RNA mimic of red fluorescent protein. Nat Chem Biol 13:1195-1201
Song, Wenjiao; Filonov, Grigory S; Kim, Hyaeyeong et al. (2017) Imaging RNA polymerase III transcription using a photostable RNA-fluorophore complex. Nat Chem Biol 13:1187-1194
Filonov, Grigory S; Jaffrey, Samie R (2016) Preface. Methods Enzymol 572:xv-xvi
Litke, J L; You, M; Jaffrey, S R (2016) Developing Fluorogenic Riboswitches for Imaging Metabolite Concentration Dynamics in Bacterial Cells. Methods Enzymol 572:315-33
Chakraborty, Kasturi; Veetil, Aneesh T; Jaffrey, Samie R et al. (2016) Nucleic Acid-Based Nanodevices in Biological Imaging. Annu Rev Biochem 85:349-73
Yang, Wang-Yong; He, Fang; Strack, Rita L et al. (2016) Small Molecule Recognition and Tools to Study Modulation of r(CGG)(exp) in Fragile X-Associated Tremor Ataxia Syndrome. ACS Chem Biol 11:2456-65
Laptenok, S P; Conyard, J; Page, P C Bulman et al. (2016) Photoacid Behaviour in a Fluorinated Green Fluorescent Protein Chromophore: Ultrafast Formation of Anion and Zwitterion States.†. Chem Sci 7:5747-5752
Filonov, Grigory S; Jaffrey, Samie R (2016) RNA Imaging with Dimeric Broccoli in Live Bacterial and Mammalian Cells. Curr Protoc Chem Biol 8:1-28
Svensen, Nina; Jaffrey, Samie R (2016) Fluorescent RNA Aptamers as a Tool to Study RNA-Modifying Enzymes. Cell Chem Biol 23:415-25
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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