An interdisciplinary team of biological, chemical, and physical scientists from Stanford University and Kent State University will embark upon a high-risk/high-potential-payoff program to design, develop and apply unique fluorophores to explore protein localization in cells, down to the single-molecule level. This will involve (i) design, synthesis, characterization and optimization of a new class of fluorescent tags for biological imaging, (ii) development of innovative cellular targeting technologies for these new fluorophores to determine location and co-location of proteins, and (iii) demonstration of methods for detection of coordinated of protein location (or mislocation in mutant strains) and gene expression to explore the regulatory function of spatial positioning in bacteria. Item (iii) is particularly important, in that the new fluorophores and targeting protocols will be challenged by a specific need and will be tested directly on a key model biological system. The members of the research team have recently demonstrated optical detection of fluorescent single molecules in living cells using both extrinsic fluorescent antigenic peptides as well as green fluorescent protein mutant fusions. In this application, the new fluorophores to be designed, synthesized, and applied are based on a class of fluorophores with exceptional fluorescence properties which already allow detection at the single-copy level. As single-molecule labels, these molecules provide more than an order of magnitude improvement in photophysical properties compared to green fluorescent protein mutants, thus the ability to detect single molecules in cells should be improved by more than an order of magnitude, opening a new frontier in the observation of dynamical events in living cells. In addition to single-molecule experiments, the new reporter fluorophores generated by this project will enable advances in time-lapse fluorescence microscopy by virtue of increased signal-to-noise and photobleaching resistance, properties that will allow lower concentrations of labels to be used in cellular fluorescence investigations.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Exploratory Grants (P20)
Project #
3P20HG003638-04S1
Application #
7676442
Study Section
Special Emphasis Panel (ZGM1-CMB-9 (CI))
Program Officer
Felsenfeld, Adam
Project Start
2004-08-01
Project End
2008-12-31
Budget Start
2007-08-01
Budget End
2008-12-31
Support Year
4
Fiscal Year
2008
Total Cost
$75,756
Indirect Cost
Name
Stanford University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Conley, Nicholas R; Dragulescu-Andrasi, Anca; Rao, Jianghong et al. (2012) A selenium analogue of firefly D-luciferin with red-shifted bioluminescence emission. Angew Chem Int Ed Engl 51:3350-3
Biteen, Julie S; Shapiro, Lucy; Moerner, W E (2011) Exploring protein superstructures and dynamics in live bacterial cells using single-molecule and superresolution imaging. Methods Mol Biol 783:139-58
Thompson, Michael A; Biteen, Julie S; Lord, Samuel J et al. (2010) Molecules and methods for super-resolution imaging. Methods Enzymol 475:27-59
Biteen, Julie S; Moerner, W E (2010) Single-molecule and superresolution imaging in live bacteria cells. Cold Spring Harb Perspect Biol 2:a000448
Lord, Samuel J; Conley, Nicholas R; Lee, Hsiao-Lu D et al. (2009) DCDHF fluorophores for single-molecule imaging in cells. Chemphyschem 10:55-65
Lu, Zhikuan; Liu, Na; Lord, Samuel J et al. (2009) Bright, Red Single-Molecule Emitters: Synthesis and Properties of Environmentally Sensitive Dicyanomethylenedihydrofuran (DCDHF) Fluorophores with Bisaromatic Conjugation. Chem Mater 21:797
Conley, Nicholas R; Biteen, Julie S; Moerner, W E (2008) Cy3-Cy5 covalent heterodimers for single-molecule photoswitching. J Phys Chem B 112:11878-80
Bowman, Grant R; Comolli, Luis R; Zhu, Jian et al. (2008) A polymeric protein anchors the chromosomal origin/ParB complex at a bacterial cell pole. Cell 134:945-55
Lee, H-L; Dubikovskaya, E A; Hwang, H et al. (2008) Single-molecule motions of oligoarginine transporter conjugates on the plasma membrane of Chinese hamster ovary cells. J Am Chem Soc 130:9364-70
Pomerantz, Andrea K; Moerner, W E; Kool, Eric T (2008) Visualization of long human telomere mimics by single-molecule fluorescence imaging. J Phys Chem B 112:13184-7

Showing the most recent 10 out of 22 publications