Background free amplified single-molecule FISH for in situ and flow cytometric applications

摘要 细胞RNA可以作为多种疾病状态的强大生物标志物。最近的发展 In探针技术使荧光原位检测具有单分子敏感性的RNA成为可能 杂交(sm鱼)。这项技术在临床诊断中具有很大的前景。 病原体和癌症。然而，它对病理切片成像的实用性目前是有限的，因为 为了获得单分子灵敏度，需要使用高倍率物镜进行成像 它们的视野有限，一次只能观察几个细胞。相对较低的 当用流式细胞术分析细胞时，这些信号的强度也限制了SM-FISH的使用， 因为罕见的细胞，如免疫记忆细胞或干细胞，表达低水平的mRNA 标记，无法检测到。另一个限制是无法检测到单核苷酸变异。 我们正在开发新一代sm-Fish，在这种鱼中，信号被放大而不需要任何 背景放大。在我们的方法中，我们称之为严格依赖靶标的扩增鱼 (STAMP-FISH)，当一对二元探针与目标结合时，隔离的序列就会暴露出来。这个 暴露的序列然后作为杂交连锁反应的启动子，这导致产生 大的，高荧光的DNA簇，仍然拴在目标上。我们获得了高达10倍的 在没有任何背景增强的情况下放大信号。此外，探头产生了精致的 单核苷酸变异之间的区别。 我们将进一步发展这项技术，并将其应用于检测EGFR和BRAF的点突变 MRNAs。目前，存在于细胞内的大约15%的靶mRNA拷贝可以是 检测到。我们将使用旨在增加探针与 靶向mRNAs。将为EGFR和BRAF内的一组示例性突变开发探针组 基因，然后是表达这些突变的细胞系和癌症组织，将使用 这些探测仪。我们还将开发多种检测方法，以检测三个突变体和野生型mrna。 同时。此外，对于T细胞图谱，我们建议在多路传输中检测15个RNA标记 T细胞。随着多路传输范围扩大到15个目标，我们将能够检测到细胞的亚型 由于趋化因子/细胞因子、转录因子或代谢因子的表达而彼此不同 记号笔。这一发展将为这两个研究的不同领域创造强大的新的分析可能性 和临床环境，因此将具有广泛的实用价值。

Abstract Cellular RNAs can serve as powerful biomarkers of diverse disease states. Recent developments in probe technology enable the detection of RNAs with single-molecule sensitivity by fluorescence in situ hybridization (sm-FISH). This technology holds great promise in clinical diagnostics for the detection of pathogens and cancers. However, its utility for imaging pathological sections is presently limited, because in order to obtain single-molecule sensitivity, imaging needs to be performed with high magnification objectives that have a limited field of view, permitting the observation of only a few cells at a time. The relatively low intensity of these signals also limits the utility of sm-FISH when the cells are analyzed by flow cytometry, because rare cells, such as immunological memory cells or stem cells, which express a low level of mRNA markers, cannot be detected. A further limitation is that single nucleotide variations cannot be detected. We are developing a new generation of sm-FISH in which the signals is amplified without any amplification of background. In our approach, that we refer to as strictly target-dependent amplified FISH (stamp-FISH), when a pair of binary probes bind to the target a sequestered sequence is exposed. The exposed sequence then serves as an initiator of a hybridization chain reaction, which leads to creation of a large, highly fluorescent DNA cluster that remains tethered at the target. We obtain as much as 10-fold amplification of signal without any enhancement in background. Furthermore, the probes yield exquisite discrimination between single nucleotide variations. We will develop this technology further and apply it for detection of point mutations in EGFR and BRAF mRNAs. Currently, about fifteen percent of the copies of target mRNA that are present within the cells can be detected. We will improve this efficiency using strategies aimed at increasing the binding of probes to the target mRNAs. Probe sets will be developed for an exemplary set of mutations within the EGFR and BRAF genes and then cell lines and cancer tissues, in which these mutations are expressed, will be imaged using these probe sets. We will also develop multiplex assays that will detect three mutants and the wild-type mRNA simultaneously. In addition, for T cell profiling we are proposing to detect 15 RNA markers in multiplex in T cells. With this expansion of the multiplexing range to 15 targets, we will be able to detect subtypes of cells that differ from each other by the expression of chemokine/cytokines, transcription factors, or metabolic markers. This development will create powerful new analytical possibilities for diverse fields in both research and clinical settings and thus will be of broad utility.