(Supplied by the Applicant): This application addresses broad Challenge Area (06) Enabling Technologies and specific Challenge Topic 06-GM-102* .Chemist/biologist collaborations facilitating tool development. The objective of this grant is to develop fluorescent chemical tags that both have high photon counts and are cell permeable and soluble to enable single-molecule detection in cells. Single-molecule imaging can now be carried out in vitro and has provided a breakthrough for understanding the molecular mechanisms of complex biological assemblies because it allows the dynamics of individual complexes to be directly observed--but in vivo single-molecule imaging has only been carried out by a handful of leading experts in the field. In our opinion a major barrier to routine single-molecule imaging in cells is the difficulty of labeling proteins selectively in cells with suitable fluorophores. The fluorescent proteins do not have high enough photon counts to be readily detected with single molecule resolution in cells, and the organic fluorophores with high photon counts developed for in vitro biophysics do not behave well in the cell.

Public Health Relevance

Here, we propose that chemical tags including our commercial TMP-tag can provide the combination of genetic encoding and an organic fluorophore label needed for single-molecule imaging in cells. We have assembled an interdisciplinary team of a chemical biologist expert in chemical tags (PI Cornish), a physical chemist skilled in fluorescence microscopy (co-PI Kaufman), a leading biochemist studying the mechanism of the spliceosome reaction (co-PI Moore), and a cell biologist who is a pioneer in the application of state-of-the-art microscopy to cell motility (co-PI Sheetz) to meet this objective. We propose to meet this objective by (1) challenging the chemical tags for single-molecule imaging of the spliceosome in yeast cell extracts, (2) developing fluorescent chemical tags suitable for single-molecule imaging in cells, and (3) developing chemical tags with specialized properties for high-resolution imaging.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
NIH Challenge Grants and Partnerships Program (RC1)
Project #
1RC1GM091804-01
Application #
7819771
Study Section
Special Emphasis Panel (ZRG1-SBIB-V (58))
Program Officer
Lewis, Catherine D
Project Start
2009-09-30
Project End
2011-08-31
Budget Start
2009-09-30
Budget End
2010-08-31
Support Year
1
Fiscal Year
2009
Total Cost
$427,867
Indirect Cost
Name
Columbia University (N.Y.)
Department
Chemistry
Type
Other Domestic Higher Education
DUNS #
049179401
City
New York
State
NY
Country
United States
Zip Code
10027
Chen, Zhixing; Cornish, Virginia W; Min, Wei (2013) Chemical tags: inspiration for advanced imaging techniques. Curr Opin Chem Biol 17:637-43
Jing, Chaoran; Cornish, Virginia W (2013) Design, synthesis, and application of the trimethoprim-based chemical tag for live-cell imaging. Curr Protoc Chem Biol 5:131-55
Jing, Chaoran; Cornish, Virginia W (2013) A fluorogenic TMP-tag for high signal-to-background intracellular live cell imaging. ACS Chem Biol 8:1704-12
Anzalone, Andrew V; Wang, Tracy Y; Chen, Zhixing et al. (2013) A common diaryl ether intermediate for the gram-scale synthesis of oxazine and xanthene fluorophores. Angew Chem Int Ed Engl 52:650-4
Gatzogiannis, Evangelos; Chen, Zhixing; Wei, Lu et al. (2012) Mapping protein-specific micro-environments in live cells by fluorescence lifetime imaging of a hybrid genetic-chemical molecular rotor tag. Chem Commun (Camb) 48:8694-6
Chen, Zhixing; Jing, Chaoran; Gallagher, Sarah S et al. (2012) Second-generation covalent TMP-tag for live cell imaging. J Am Chem Soc 134:13692-9
Yang, Ya-li; Sun, Charles; Wilhelm, Matthew E et al. (2011) Influence of chondroitin sulfate and hyaluronic acid on structure, mechanical properties, and glioma invasion of collagen I gels. Biomaterials 32:7932-40
Hoskins, Aaron A; Friedman, Larry J; Gallagher, Sarah S et al. (2011) Ordered and dynamic assembly of single spliceosomes. Science 331:1289-95
Mackowiak, Stephan A; Kaufman, Laura J (2011) When the Heterogeneous Appears Homogeneous: Discrepant Measures of Heterogeneity in Single Molecule Observables. J Phys Chem Lett 2:438-442
Jing, Chaoran; Cornish, Virginia W (2011) Chemical tags for labeling proteins inside living cells. Acc Chem Res 44:784-92

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