Novel Probe for Quantitative Imaging of Gene Expression

我们建议开发一种分子成像探针，它将提供关于 MRNA的表达水平具有时间和空间分辨率。具体地说，基于寡核苷酸的探针 将被设计成形成茎-环结构，并将在一端贴上‘报告者’荧光团的标签 另一端的猝灭剂，类似于分子灯塔；然而，寡核苷酸也将被标记 具有第二光学上不同的参考染料/纳米颗粒，该染料/纳米颗粒将被选择为不猝灭 不管探测器的构象如何。荧光标记的中性亲和素和量子点将被 测试了它们作为参考染料的适宜性。我们假设这一点的有益特征 与传统的分子信标相比，新的探针将包括：(1)监测转染率 由于存在未猝灭的参考染料而产生的效率。这将通过以下方式减少假阴性 允许未转基因细胞和低水平基因表达的细胞之间的分化。(2) 通过比率成像(即报告荧光/参考荧光)消除 仪器变异性和实验变异性。(3)定量比较基因变异的能力 样本之间、单个样本内的细胞之间、甚至亚细胞之间的表达水平 通过使用参比染料作为参照点的隔间(4)量化基因表达的能力 由于报告和参考染料之间的共价键，因此具有空间和时间分辨率 确保它们表现出同等的细胞内寿命和共定位模式。(5)使用 量子点/中性亲和素作为连接靶向试剂的平台，为体内研究打开了可能性 成像。(6)由于“报告者”染料被猝灭而改善信号对背景的可能性 猝灭剂分子和“参比”染料。为了评估这些功能，我们将遵循两个主要原则 拟议研究的目标：1)我们将设计、合成和表征“定量” 分子信标(QMB)的背景信号和检测下限(体外和体内) 2)我们将评估QMBs定量检测乳腺癌内源性mrna表达的能力。 实时的细胞。据设想，这里提出的方法将使我们的 对人类健康和疾病的了解，并可能被证明是一个强大的诊断工具。

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.