The goal: To prepare oligonucleotide probes that are coupled to fluorochromes to enter the cell and detect intracellular mRNAs in living cells with single copy sensitivity. This application is dedicated to increase in signal to noise ratio by factors exceeding 20 fold over existing technologies, focused mainly on background reduction approaches. 1. To synthesize fluorochrome coupled oligonucleotide probes using a phosphorothioate linkage to enter the cell and hybridize to RNA. We will then optimize the detection of the hybridized probes by initially using the site of highest signal in the nucleus: where the RNA is being transcribed. Various cellular models will be used with a dynamic range of four orders of magnitude; the highest can generate as many as 48,000 fluorochromes at the transcription site. We will then proceed to quantitatively test improvements in sensitivity by using models with progressively fewer targets. 2. To design a fluorochrome-quencher combination that will result in fluorescence only when the probe hybridizes to its target. We will design a library of chemical compounds to screen using a high-throughput probe fluorescence assay. 3. To design imaging hardware and software to filter out the background generated by unhybridized probes leaving only the hybridized fluorescent signals. 4. To multiplex the probes with different fluorochromes so that we will be able to detect multiple RNA species in the same cell. Ultimately, the approach can be formatted for use in living tissue. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM080247-02
Application #
7497047
Study Section
Special Emphasis Panel (ZRG1-BST-R (51))
Program Officer
Deatherage, James F
Project Start
2007-09-28
Project End
2011-08-31
Budget Start
2008-09-01
Budget End
2009-08-31
Support Year
2
Fiscal Year
2008
Total Cost
$248,522
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
110521739
City
Bronx
State
NY
Country
United States
Zip Code
10461
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Wang, Donghai; Pezo, Rossanna C; Corner, Georgia et al. (2010) Altered dynamics of intestinal cell maturation in Apc1638N/+ mice. Cancer Res 70:5348-57