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Molecular & Cellular Analysis Technologies
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We propose to develop a molecular imaging probe that will provide quantitative information on theexpression level of mRNA with spatial and temporal resolution. Specifically, an oligonucleotide-based probewill be designed to form a stem-loop structure and will be labeled with a 'reporter' fluorophore at one end anda quencher at the other, analogous to a molecular beacon; however, the oligonucleotide will also be labeledwith a second optically distinct 'reference' dye/nanoparticle, which will be selected such that it is unquenchedregardless of the conformation of the probe. Fluorescently labeled neutravidin and quantum dots will betested for their suitability in serving as the reference dye. We hypothesize that beneficial features of thisnovel probe compared with conventional molecular beacons will include (1) the ability to monitor transfectionefficiency due to the presence of the unquenched reference dye. This will reduce false-negatives byallowing 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 ofinstrumental and experimental variability. (3) The ability to quantitatively compare variations in geneexpression levels between samples, between cells within individual samples, and even between sub-cellularcompartments by using the reference dye as a point of reference (4) The ability to quantify gene expressionwith spatial and temporal resolution since the covalent linkage between the reporter and reference dyeensures they exhibit an equivalent intracellular lifetime and co-localization pattern. (5) The ability to use thequantum dot/neutravidin as a platform to attach targeting agents, opening up the possibility for in vivoimaging. (6) The possibility of an improved signal-to-background due to quenching of the 'reporter' dye byboth the quencher molecule and the 'reference' dye. To evaluate these features we will pursue two majoraims 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 cancercells in real-time. It is envisioned that the approach proposed here will allow significant advancements in ourunderstanding of human health and disease and could potentially prove to be a powerful diagnostic tool.