Programmable metafluorophores for quantitative multiplex in situ imaging of mRNA

描述（由申请人提供）：转录程序在动物发育过程中指定和详细说明细胞身份，因为单个细胞会产生组成成年动物的数百种细胞类型。因此，基因表达的时间、地点、水平和分子环境对其功能至关重要。成像技术目前处于独特的位置，可以同时以亚细胞分辨率捕获所有这些特征。原位杂交是将靶mRNA与互补核酸探针杂交，是观察完整胚胎和组织样本中mRNA表达的主要方法。然而，目前的mRNA原位杂交检测方法并没有充分利用成像的潜力来捕获关于许多基因在空间和时间上表达的定量信息。在这里，我们建议开发一种基于“超荧光团”概念的mRNA原位杂交荧光检测方法：一种可编程的DNA支架，将定义数量的荧光团靶向mRNA。与目前的方法相比，这种方法将改善信噪比特性，将是定量的，将能够同时检测许多基因，并且在成本，规模和可用数据的时间方面更加可行。

DESCRIPTION (provided by applicant): Transcriptional programs specify and elaborate cell identity during animal development, as a single cell gives rise to the hundreds of cell types that comprise the adult animal. The time, place, level and molecular context in which a gene is expressed are therefore critical for its function. Imaging techniques are currently in a unique position to capture all of these features simultaneously with sub-cellular resolution. In situ hybridization, where a target mRNA is hybridized to a complementary nucleic acid probe, is the primary method used to visualize mRNA expression in intact embryos and tissue samples. However, current detection methods for mRNA in situ hybridization do not take full advantage of the potential of imaging to capture quantitative information about the expression of many genes over space and time. Here, we propose to develop a fluorescent detection method for mRNA in situ hybridization based on the concept of a "metafluorophore": a programmable DNA scaffold to target defined numbers of fluorophores to mRNA. Compared to current methods, this approach will improve signal to noise characteristics, will be quantitative, will be able to detecting many genes simultaneously and will be more feasible - in terms of cost, scale and time to usable data. PUBLIC HEALTH RELEVANCE: All cells in the body have the same genome, but acquire different capabilities by expressing subsets of their genes. This proposal develops technology to measure the level of expression of many genes in individual cells using fluorescent imaging, and applies it to understand how gene expression varies during development.