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

用于原位和流式细胞术应用的无背景扩增单分子 FISH

• 批准号: 10082444
• 负责人: SANJAY TYAGI
• 金额: $ 55.73万
• 依托单位: RBHS-NEW JERSEY MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-02-16 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10082444
• 关键词: Address; Alleles; BRAF gene; Binding; Biological Assay; Biological Markers; CD28 gene; Cancerous; Cell Line; Cells; Classification; Clinical; Clinical Pathology; Code; Color; DNA; DNA Probes; Detection; Development; Discrimination; Disease; Drug resistance; EGFR gene; Epidermal Growth Factor Receptor; Fishes; Flow Cytometry; Fluorescent in Situ Hybridization; Gene Fusion; Generations; Genes; Genetic Transcription; Human; Image; Immunologic Memory; In Situ; In Situ Hybridization; Infection; Left; Length; Malignant Neoplasms; Malignant neoplasm of lung; Maps; Melanoma Cell; Messenger RNA; Metabolic Marker; Methods; Microscopy; Molecular; Mutate; Mutation; Mutation Detection; Neoplasm Circulating Cells; Nucleotides; Pathogen detection; Pathologic; Peripheral; Peripheral Blood Mononuclear Cell; Point Mutation; RNA; RNA Binding; RNA marker; RNA-Binding Proteins; Reaction; Reporting; Research; Signal Transduction; Site; Structure; Syncope; T-Lymphocyte; T-Lymphocyte Subsets; TNFRSF6 gene; Technology; Time; Tissue Microarray; Tissue Stains; Tissues; Treatment Failure; Vaccination; Variant; Visualization; arm; base; cancer therapy; cell transformation; chemokine; circular RNA; clinical diagnostics; combinatorial; cytokine; design; digital; drug sensitivity; experimental study; image processing; improved; locked nucleic acid; molecular marker; multiplex assay; multiplex detection; mutant; pathogenic virus; peripheral blood; protein crosslink; response; single molecule; stem cells; success; targeted imaging; tissue/cell culture; transcription factor; tumor; viral detection

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.

摘要 细胞RNA可以作为多种疾病状态的强大生物标志物。最近的事态发展 在探针技术中，能够通过荧光原位检测具有单分子灵敏度的RNA 杂交（sm-FISH）。该技术在临床诊断中具有很大的前景，用于检测 病原体和癌症。然而，其用于成像病理切片的效用目前是有限的，因为 为了获得单分子的灵敏度，成像需要用高放大倍数的物镜来进行 视野有限，一次只能观察几个细胞。相对较低 这些信号的强度也限制了当通过流式细胞术分析细胞时sm-FISH的应用， 因为表达低水平mRNA的稀有细胞，如免疫记忆细胞或干细胞， 标记，无法检测。另一个限制是不能检测到单核苷酸变异。 我们正在开发新一代的sm-FISH，其中信号被放大，而没有任何 背景放大。在我们的方法中，我们称之为严格的靶依赖性扩增FISH 在stamp-FISH（stamp-FISH）中，当一对二元探针与靶标结合时，隔离的序列被暴露。的 然后，暴露的序列充当杂交链反应的引发剂，这导致杂交链反应的产生。 大的，高荧光的DNA簇，仍然拴在目标上。我们获得的收益高达10倍 在背景中没有任何增强的信号放大。此外，探针产生精致的 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EGFR和BRAF点突变的检测 mRNA。目前，存在于细胞内的靶mRNA的约15%的拷贝可以被复制。 检测到我们将使用旨在增加探针与蛋白质结合的策略来提高这种效率。 靶向mRNA。将针对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组织，将使用 这些探针组。我们还将开发多重检测，将检测三个突变体和野生型mRNA 同步此外，对于T细胞分析，我们建议在多个细胞中检测15种RNA标记物。 T细胞。随着多重范围扩大到15个目标，我们将能够检测细胞的亚型 它们通过表达趋化因子/细胞因子、转录因子或代谢因子而彼此不同。 标记。这一发展将为两个研究领域的不同领域创造强大的新分析可能性。 和临床环境，因此将具有广泛的用途。