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: 10329959
• 负责人: SANJAY TYAGI
• 金额: $ 54.62万
• 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 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10329959
• 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; single molecule; stem cells; success; targeted imaging; tissue/cell culture; transcription factor; treatment response; 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.

抽象的 细胞 RNA 可以作为多种疾病状态的强大生物标志物。最新动态 探针技术能够通过荧光原位检测单分子灵敏度的 RNA 杂交（sm-FISH）。该技术在临床诊断检测中具有广阔的前景 病原体和癌症。然而，其对病理切片成像的实用性目前有限，因为 为了获得单分子灵敏度，需要使用高倍物镜进行成像 它们的视野有限，一次只能观察几个细胞。相对较低的 当通过流式细胞术分析细胞时，这些信号的强度也限制了 sm-FISH 的实用性， 因为稀有细胞，例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单核苷酸变异。 我们正在开发新一代 sm-FISH，其中信号无需任何干扰即可放大 背景放大。在我们的方法中，我们将其称为严格靶标依赖性扩增 FISH （stamp-FISH），当一对二元探针与靶标结合时，隔离的序列就会暴露出来。这 然后暴露的序列作为杂交链式反应的引发剂，从而产生 仍然束缚在目标上的大的、高荧光 DNA 簇。我们获得了多达 10 倍的 信号放大，背景没有任何增强。此外，探针产生精致的 单核苷酸变异之间的区别。 我们将进一步开发这项技术，并将其应用于EGFR和BRAF点突变的检测 mRNA。目前，细胞内存在的目标 mRNA 拷贝的大约 15% 可以被 检测到。我们将使用旨在增加探针与探针结合的策略来提高这种效率。 目标 mRNA。将为 EGFR 和 BRAF 内的一组示例性突变开发探针组 基因，然后表达这些突变的细胞系和癌症组织将使用 这些探针组。我们还将开发多重检测方法来检测三种突变体和野生型 mRNA 同时地。此外，对于 T 细胞分析，我们建议多重检测 15 个 RNA 标记 T细胞。随着多重检测范围扩大到 15 个目标，我们将能够检测细胞亚型 趋化因子/细胞因子、转录因子或代谢因子的表达彼此不同 标记。这一发展将为这两个研究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。

项目成果

Interferon ε restricts Zika virus infection in the female reproductive tract.

干扰素γ限制女性生殖道中的寨卡病毒感染。

• DOI: 10.1101/2023.04.06.535968
• 发表时间: 2023
• 期刊: bioRxiv : the preprint server for biology
• 作者: Xu,Chuan;Wang,Annie;Ebraham,Laith;Sullivan,Liam;Tasker,Carley;Pizutelli,Vanessa;Couret,Jennifer;Hernandez,Cyril;Deb,PratikQ;Fritzky,Luke;Subbian,Selvakumar;Gao,Nan;Lo,Yungtai;Salvatore,Mirella;Rivera,Amariliz;Lemenze,Alexan
• 通讯作者: Lemenze,Alexan

SuperSelective primer pairs for sensitive detection of rare somatic mutations.

• DOI: 10.1038/s41598-021-00920-4
• 发表时间: 2021-11-17
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Kramer FR;Vargas DY
• 通讯作者: Vargas DY

Interferon ɛ restricts Zika virus infection in the female reproductive tract.

• DOI: 10.1093/pnasnexus/pgad350
• 发表时间: 2023-11
• 期刊: PNAS NEXUS
• 作者: Xu, Chuan;Wang, Annie;Ebraham, Laith;Sullivan, Liam;Tasker, Carley;Pizutelli, Vanessa;Couret, Jennifer;Hernandez, Cyril;Kolli, Priyanka;Deb, Pratik Q.;Fritzky, Luke;Subbian, Selvakumar;Gao, Nan;Lo, Yungtai;Salvatore, Mirella;Rivera, Amariliz;Lemenze, Alexander;Fitzgerald-Bocarsly, Patricia;Tyagi, Sanjay;Lu, Wuyuan;Beaulieu, Aimee;Chang, Theresa L.
• 通讯作者: Chang, Theresa L.

Fluorescence In Situ Imaging of Dendritic RNAs at Single-Molecule Resolution.

• DOI: 10.1002/cpns.79
• 发表时间: 2019-09-01
• 期刊: Current protocols in neuroscience
• 作者: Batish, Mona;Tyagi, Sanjay
• 通讯作者: Tyagi, Sanjay

Circular RNAs Represent a Novel Class of Human Cytomegalovirus Transcripts.

• DOI: 10.1128/spectrum.01106-22
• 发表时间: 2022-06-29
• 期刊: MICROBIOLOGY SPECTRUM
• 影响因子: 3.7
• 作者: Yang, Shaomin;Liu, Xiaolian;Wang, Mei;Cao, Di;Jaijyan, Dabbu Kumar;Enescu, Nicole;Liu, Jian;Wu, Songbin;Wang, Sashuang;Sun, Wuping;Xiao, Lizu;Gu, Alison;Li, Yaolan;Zhou, Hong;Tyagi, Sanjay;Wu, Jianguo;Tang, Qiyi;Zhu, Hua
• 通讯作者: Zhu, Hua