Novel Probe for Quantitative Imaging of Gene Expression

我们建议开发一种分子成像探针，将提供定量信息， mRNA表达水平的空间和时间分辨率。具体来说，一种基于谷胱甘肽的探针 将被设计成形成茎环结构，并将在一端用“报告”荧光团标记， 另一个是猝灭剂，类似于分子信标;然而，寡核苷酸也将被标记， 与第二光学上不同的“参比”染料/纳米颗粒，其将被选择为使得其未猝灭 而不管探针的构造如何。标记的中性抗生物素蛋白和量子点将 测试它们作为参比染料的适用性。我们假设， 与常规分子信标相比，新型探针将包括（1）监测转染的能力 由于存在未猝灭的参比染料，因此效率降低。这将减少假阴性， 允许在未转染的细胞和具有低水平基因表达的细胞之间进行区分。（二） 通过比率成像（即，报告荧光/参考荧光）消除以下因素影响的能力 仪器和实验可变性。(3)定量比较基因变异的能力 样品之间、单个样品内的细胞之间以及甚至亚细胞之间的表达水平 （4）定量基因表达的能力 由于报告分子和参比染料之间的共价键， 确保它们表现出等同的细胞内寿命和共定位模式。(5)运用能力 量子点/中性抗生物素蛋白作为连接靶向剂的平台， 显像(6)由于“报告”染料的猝灭， 猝灭剂分子和“参比”染料。为了评估这些特征，我们将研究两个主要的 在拟议的研究目标：1）我们将设计，合成和表征的'定量' 分子信标（QMB）的信号背景和检测下限（体外和体内） 和2）我们将评估QMB定量乳腺癌中内源性mRNA表达的能力 细胞实时据设想，这里提出的方法将使我们的研究取得重大进展。 它可以帮助我们了解人类健康和疾病，并可能成为一种强大的诊断工具。

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.