We propose to develop a molecular imaging probe that will provide quantitative information on the? expression level of mRNA with spatial and temporal resolution. Specifically, an oligonucleotide-based probe? will be designed to form a stem-loop structure and will be labeled with a 'reporter' fluorophore at one end and? a quencher at the other, analogous to a molecular beacon; however, the oligonucleotide will also be labeled? with a second optically distinct 'reference' dye/nanoparticle, which will be selected such that it is unquenched? regardless of the conformation of the probe. Fluorescently labeled neutravidin and quantum dots will be? tested for their suitability in serving as the reference dye. We hypothesize that beneficial features of this? novel probe compared with conventional molecular beacons will include (1) the ability to monitor transfection? efficiency due to the presence of the unquenched reference dye. This will reduce false-negatives by? allowing for the differentiation between untransfected cells and cells with low levels of gene expression. (2)? The ability to remove via ratiometric imaging (i.e. reporter fluorscence/reference fluorescence) the impact of? instrumental and experimental variability. (3) The ability to quantitatively compare variations in gene? expression levels between samples, between cells within individual samples, and even between sub-cellular? compartments by using the reference dye as a point of reference (4) The ability to quantify gene expression? with spatial and temporal resolution since the covalent linkage between the reporter and reference dye? ensures they exhibit an equivalent intracellular lifetime and co-localization pattern. (5) The ability to use the? quantum dot/neutravidin as a platform to attach targeting agents, opening up the possibility for in vivo? imaging. (6) The possibility of an improved signal-to-background due to quenching of the 'reporter' dye by? both the quencher molecule and the 'reference' dye. To evaluate these features we will pursue two major? aims during the proposed research: 1) We will design, synthesize and characterize the 'quantitative'? molecular beacon (QMB) in terms of its signal-to-background and lower detection limit (in vitro and in vivo)? and 2) we will evaluate the ability of the QMBs to quantify endogenous mRNA expression in breast cancer? cells in real-time. It is envisioned that the approach proposed here will allow significant advancements in our? understanding of human health and disease and could potentially prove to be a powerful diagnostic tool.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21CA116102-02
Application #
7295673
Study Section
Special Emphasis Panel (ZCA1-SRRB-C (M1))
Program Officer
Li, Jerry
Project Start
2006-09-26
Project End
2009-08-31
Budget Start
2007-09-01
Budget End
2009-08-31
Support Year
2
Fiscal Year
2007
Total Cost
$161,429
Indirect Cost
Name
University of Pennsylvania
Department
Biomedical Engineering
Type
Schools of Engineering
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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Song, Yang; Zhang, Xuemei; Huang, Lingyan et al. (2014) Real-time imaging of single engineered RNA transcripts in living cells using ratiometric bimolecular beacons. J Vis Exp :e51544
Zhang, Xuemei; Song, Yang; Shah, Akash Y et al. (2013) Quantitative assessment of ratiometric bimolecular beacons as a tool for imaging single engineered RNA transcripts and measuring gene expression in living cells. Nucleic Acids Res 41:e152
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Chen, Antony K; Davydenko, Olga; Behlke, Mark A et al. (2010) Ratiometric bimolecular beacons for the sensitive detection of RNA in single living cells. Nucleic Acids Res 38:e148
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Lu, Jing; Tsourkas, Andrew (2009) Imaging individual microRNAs in single mammalian cells in situ. Nucleic Acids Res 37:e100
Chen, Antony K; Behlke, Mark A; Tsourkas, Andrew (2009) Sub-cellular trafficking and functionality of 2'-O-methyl and 2'-O-methyl-phosphorothioate molecular beacons. Nucleic Acids Res 37:e149
Chen, Antony K; Cheng, Zhiliang; Behlke, Mark A et al. (2008) Assessing the sensitivity of commercially available fluorophores to the intracellular environment. Anal Chem 80:7437-44

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