PROBES FOR MULTIPLEXING SINGLE RNA MOLECULE DETECTIION IN LIVING CELLS

描述（由申请人提供）：目的：制备与荧光染料偶联的寡核苷酸探针，以进入细胞并以单拷贝灵敏度检测活细胞中的细胞内mrna。该应用程序致力于将信噪比提高到现有技术的20倍以上，主要集中在背景还原方法上。1. 利用硫代磷酸键合成荧光染料偶联寡核苷酸探针进入细胞并与RNA杂交。然后，我们将优化杂交探针的检测，首先使用细胞核中最高信号的位点：RNA正在转录的地方。将使用各种细胞模型，动态范围为四个数量级；最高的可以在转录位点产生多达48,000个荧光染料。然后，我们将使用目标逐渐减少的模型，对灵敏度的改进进行定量测试。2. 设计一种荧光染料-猝灭剂组合，仅当探针与目标杂交时才产生荧光。我们将设计一个化学化合物库，使用高通量探针荧光法进行筛选。3. 设计成像硬件和软件，滤除非杂交探针产生的背景，只留下杂交的荧光信号。4. 用不同的荧光染料多重使用探针，这样我们就能在同一细胞中检测到多种RNA。最终，该方法可以格式化用于活体组织。

DESCRIPTION (provided by applicant): The goal: To prepare oligonucleotide probes that are coupled to fluorochromes to enter the cell and detect intracellular mRNAs in living cells with single copy sensitivity. This application is dedicated to increase in signal to noise ratio by factors exceeding 20 fold over existing technologies, focused mainly on background reduction approaches. 1. To synthesize fluorochrome coupled oligonucleotide probes using a phosphorothioate linkage to enter the cell and hybridize to RNA. We will then optimize the detection of the hybridized probes by initially using the site of highest signal in the nucleus: where the RNA is being transcribed. Various cellular models will be used with a dynamic range of four orders of magnitude; the highest can generate as many as 48,000 fluorochromes at the transcription site. We will then proceed to quantitatively test improvements in sensitivity by using models with progressively fewer targets. 2. To design a fluorochrome-quencher combination that will result in fluorescence only when the probe hybridizes to its target. We will design a library of chemical compounds to screen using a high-throughput probe fluorescence assay. 3. To design imaging hardware and software to filter out the background generated by unhybridized probes leaving only the hybridized fluorescent signals. 4. To multiplex the probes with different fluorochromes so that we will be able to detect multiple RNA species in the same cell. Ultimately, the approach can be formatted for use in living tissue.