PROBES FOR MULTIPLEXING SINGLE RNA MOLECULE DETECTIION IN LIVING CELLS

描述(申请人提供)：目标：制备与荧光染料偶联的寡核苷酸探针进入细胞，并以单拷贝敏感性检测活细胞内的mRNAs。这项应用致力于将信噪比提高超过现有技术的20倍，主要集中在背景减少方法上。1.以硫代硫代修饰的方式合成荧光偶联寡核苷酸探针，进入细胞并与RNA杂交。然后，我们将首先使用细胞核中信号最高的位置：RNA被转录的位置，来优化杂交探针的检测。不同的细胞模型将被使用，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，我们将通过使用目标逐渐减少的模型来定量测试敏感度的改善。2.设计一种荧光猝灭剂组合，只有当探针与其目标杂交时才会产生荧光。我们将设计一个化合物文库，使用高通量探针荧光分析进行筛选。3.设计成像硬件和软件，滤除非杂交探针产生的背景，只留下杂交荧光信号。4.用不同的荧光染料对探针进行复合，使我们能够在同一细胞中检测多个RNA物种。最终，这种方法可以格式化，用于活组织。

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.