Novel Probe for Quantitative Imaging of Gene Expression

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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.