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

项目摘要 下一代测序平台已经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以下方面具有重要意义： 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中，可能对疾病的进展产生巨大的影响。 相反，癌细胞或病毒种群的多样性对癌症具有重要影响。 成功控制疾病。因此，在这些情况下的一个关键障碍是， 提供单细胞分析技术， 测序，以充分测量细胞的多样性。不幸的是，目前的单细胞 分析方法对于大的细胞群体是不可行的，太昂贵，和/或 需要大多数实验室无法提供的专用设备。为了解决这个问题，我们 我们已经设计了DNA折纸纳米结构，能够特异性地结合和保护两个 不同的mRNA转染细胞内，并使用新的分子方法促进了 限制mRNA的几何结构结合到DNA折纸，以产生双顺反子扩增子， 用于双端高通量下一代测序。重要的是，来自 在整个过程中，单个细胞保持物理连接，因此连接的序列来自 单个细胞。在这个建议中，我们开发了这种方法来量化的多样性， 淋巴细胞群中克隆分布的TCRα和TCRβ T细胞受体。我们有 表明我们可以高效率地检测多克隆T细胞群，分离DNA， 折纸纳米结构与来自转染的T细胞的结合TCR mRNA结合，并产生CDR 3 用于Illumina 2x250配对末端深度测序反应的扩增子，以获得连接的TCRα， 来自单个T细胞的TCRβ序列信息，而无需单细胞分选。我们 建议验证和使用开发的DNA折纸纳米结构，以提供第一个 小鼠的幼稚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， 对DNA折纸纳米结构探针序列的修饰，包括单细胞分析 其他不同的淋巴细胞群，包括其他T细胞亚群或抗体产生B 细胞，以及异质性肿瘤或不同微生物群落的单细胞分析。 此外，由于这种方法使用了大多数现代分子生物学中的设备， 实验室，它可以很容易地通过许多研究人员进行这些分析。

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.