DNA Origami Nanostructures for Single-Cell Multi-gene Analysis without Single Cell Sorting

用于单细胞多基因分析的 DNA 折纸纳米结构，无需单细胞分选

• 批准号: 9302285
• 负责人: Joseph N Blattman
• 金额: $ 18.79万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-22 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9302285
• 关键词: Address; Adopted; Antibodies; Antigens; B-Lymphocytes; Binding; Biological Process; Biotin; Cell Separation; Cells; Complementary DNA; Coupled; DNA; Developmental Biology; Disease Progression; Engineering; Equilibrium; Equipment; Genes; Geometry; Human; Immune; Immune response; Immune system; Immunology; Individual; Laboratories; Ligation; Link; Lymphocyte; Maintenance; Malignant Neoplasms; Measures; Messenger RNA; Methods; Modernization; Modification; Molecular; Molecular Biology; Mus; Nanostructures; Outcome; Population; Population Heterogeneity; Process; Reaction; Research Personnel; Reverse Transcription; T-Cell Receptor; T-Lymphocyte; T-Lymphocyte Subsets; Techniques; Technology; Testing; Transfection; Transgenic Organisms; Validation; Virus; alpha-beta T-Cell Receptor; cancer cell; combat; cost; deep sequencing; design; disorder control; high standard; immunological diversity; internal control; microbial community; next generation sequencing; novel; nuclease; oncology; pathogen; precursor cell; response; sequencing platform; single cell analysis; single cell sequencing; tumor; virtual

Project Summary Next generation sequencing platforms have revolutionized modern approaches for understanding a wide variety of biological processes, including immune responses and cancer. However, the diversity of the cells involved in these processes has important implications for understanding biologic outcomes. For instance, the diversity of T cell receptors on lymphocytes during responses to virus or cancer can have dramatic effects on disease progression. Conversely, the diversity of cancer cells or virus populations has important implications for successful control of disease. Therefore, a critical hurdle in these situations is the ability to provide single-cell analysis techniques coupled with high-throughput next generation sequencing, to adequately measure the diversity of cells. Unfortunately, current single-cell analysis approaches are either unfeasible for large cell populations, too expensive, and/or require specialized equipment that is not available to most labs. To address this problem, we have engineered DNA origami nanostructures that are able to specifically bind and protect two different mRNA within transfected cells, and use novel molecular approaches facilitated by the constrained geometry of the mRNA bound to DNA origami to generate bi-cistronic amplicons for use in paired-end high-throughput next generation sequencing. Importantly, the mRNA from individual cells remain physically linked throughout this process, so linked sequences are from individual cells. In this proposal we develop this approach for quantitating the diversity of clonally-distributed TCRα and TCRβ T cell receptors in lymphocyte populations. We have shown that we can transfect polyclonal populations of T cells with high efficiency, isolate DNA origami nanostructures with bound TCR mRNA from transfected T cells, and generate CDR3 amplicons for Illumina 2x250 paired-end deep sequencing reactions to obtain linked TCRα and TCRβ sequence information from individual T cells without the need for single cell sorting. We propose to validate and use the developed DNA origami nanostructures to provide the first estimate of total TCR diversity in the naïve T cell repertoires of mice. This technology will be useful for downstream application to a wide variety of biologic processes, by relatively simple modifications to the DNA origami nanostructure probe sequences, including single-cell analysis of other diverse lymphocyte populations, including other T cell subsets or antibody producing B cells, as well as single cells analysis of heterogeneous tumors or diverse microbial communities. Moreover, because this approach utilizes equipment found in most modern molecular biology laboratories, it can be easily adopted by many researchers for these analyses.

项目概要 下一代测序平台彻底改变了现代方法 了解各种生物过程，包括免疫反应和癌症。 然而，参与这些过程的细胞的多样性对 了解生物学结果。例如，淋巴细胞上 T 细胞受体的多样性 在对病毒或癌症的反应过程中，可能会对疾病进展产生巨大影响。 相反，癌细胞或病毒群体的多样性对 成功控制疾病。因此，在这些情况下的一个关键障碍是能够 提供单细胞分析技术与高通量下一代 测序，以充分测量细胞的多样性。不幸的是，目前的单细胞 分析方法要么对于大量细胞群不可行，要么太昂贵，和/或 需要大多数实验室无法使用的专用设备。为了解决这个问题，我们 设计了 DNA 折纸纳米结构，能够特异性结合和保护两个 转染细胞内的不同 mRNA，并使用由 限制几何形状的 mRNA 与 DNA 折纸结合，生成双顺反子扩增子 用于配对末端高通量下一代测序。重要的是，mRNA来自 各个细胞在整个过程中保持物理连接，因此连接的序列来自 单个细胞。在本提案中，我们开发了这种方法来量化多样性 淋巴细胞群体中克隆分布的 TCRα 和 TCRβ T 细胞受体。我们有 表明我们可以高效转染 T 细胞多克隆群体，分离 DNA 折纸纳米结构与来自转染 T 细胞的 TCR mRNA 结合，并生成 CDR3 用于 Illumina 2x250 双端深度测序反应的扩增子，以获得连接的 TCRα 和 来自单个 T 细胞的 TCRβ 序列信息，无需进行单细胞分选。我们 提议验证并使用开发的 DNA 折纸纳米结构来提供第一个 小鼠初始 T 细胞库中总 TCR 多样性的估计。这项技术将 通过相对简单的方法，可用于下游应用到各种生物过程 DNA折纸纳米结构探针序列的修改，包括单细胞分析 其他不同的淋巴细胞群，包括其他 T 细胞亚群或产生抗体的 B 细胞，以及异质肿瘤或不同微生物群落的单细胞分析。 此外，因为这种方法利用了大多数现代分子生物学中的设备 实验室，许多研究人员可以轻松采用它进行这些分析。