Multiplexed Reiterative Immunofluorescence Analyses via Engineered DNA Circuitry

描述（由申请人提供）：通过免疫组织学分析评价细胞和组织中分子标志物水平的空间分布构成了人类疾病（包括癌症）的诊断、预后和临床管理的重要组成部分。然而，免疫组织学方法仍然受到一个事实的限制，即只有少数分子标记物可以在一个单一的标本上进行检查。考虑到临床标本的大小可能很小，而提供信息的分子标志物的数量可能很大，因此对单个样本实际进行的分子和细胞分析的类型和数量经常受到影响。这些问题限制了目前通过分子标记物分析个性化癌症临床管理的努力。此外，目前需要利用多个组织切片或抽吸活检来检查多种标志物，这限制了充分表征单个稀有细胞和细胞小生境的能力。该项目将通过开发一种新的多路复用和重复性免疫荧光成像方法来克服这些问题，称为DNA催化分子生物标记物成像和扩增（DC-MBIA）。DC-MBIA采用来自DNA纳米技术领域的原理，使得（1）多个分子探针的选择性荧光团标记（例如，独特的DNA偶联抗体，其指导携带荧光团的DNA复合物的核苷酸序列特异性反应），（2）在分子标记物的局部附近的荧光信号的化学计量放大，和（3）通过异常温和的处理条件从样品中去除荧光团。以这种方式，DC-MBIA允许荧光团再利用在一个单一的标本;相同类型的荧光染料分子可以选择性地交换分子标记之间，因此，不同的荧光通道的显微镜现在可以多次使用图像的几组分子标记，即使感兴趣的标记是在低水平。在建立必要的基础设施以促进这一进步的同时，该项目将评估和优化DC-MBIA的协议，以促进多重和重复性标记分析。在此，短期可行性目标是证明与当前技术相比，可以在单个标本上检查的分子标记物的数量至少增加四倍（即，几十个标记成像，视线分辨几百个）。 公共卫生相关性：拟议项目将创建一种新的分子探针技术，允许在一次临床活检中检查大量分子生物标志物，从而提高生物标本在癌症早期检测和临床管理中的实用性。此外，拟议的技术将克服目前的技术障碍，禁止在单个标本中表征稀有细胞和低丰度标记物，这反过来将导致更好地了解肿瘤内发生的分子和细胞水平的变化，并有助于未来发现新的靶向癌症标记物。

DESCRIPTION (provided by applicant): The evaluation of the spatial distributions of molecular marker levels in cells and tissues via immunohistological analyses constitutes a vital component of the diagnosis, prognosis and clinical management of human diseases including cancer. Nevertheless, immunohistological methods remain substantially restricted by the fact that only a few molecular markers can be examined on a single specimen. Considering that the size of clinical specimens can be small and that the number of informative molecular markers can be large, the types and number of molecular and cellular analyses that are actually performed on individual samples are frequently compromised. These issues limit current efforts to personalize the clinical management of cancer via molecular marker analyses. Furthermore, the present need to utilize multiple tissue sections or aspiration biopsies to examine multiple markers limits the ability to fully characterize individual rare cells and cellular niches. This project will surmount these problems by developing a new multiplexed and reiterative immunofluorescence imaging method called DNA-Catalyzed Molecular Biomarker Imaging and Amplification (DC-MBIA). Employing principles from the field of DNA-nanotechnology, DC-MBIA enables (1) the selective fluorophore labeling of multiple molecular probes (e.g., unique DNA-conjugated antibodies that direct nucleotide sequence-specific reactions of fluorophore-bearing DNA-complexes), (2) the stoichiometric amplification of fluorescent signals in the local proximity of a molecular marker, and (3) the removal of fluorophores from a sample via exceptionally-mild processing conditions. In this way, DC-MBIA permits fluorophore reutilization on a single specimen; the same types of fluorescent dye molecules can be selectively exchanged between molecular markers, and hence, distinct fluorescent channels of a microscope can now be used multiple times to image several sets of molecular markers, even if the markers of interest are present at low levels. While building the necessary infrastructure to facilitate this advance, this project will evaluate and optimize protocols for DC-MBIA to facilitate multiplexed and reiterative marker analyses. Here, the short-term feasibility goal is to demonstrate a minimum four-fold enhancement in the number of molecular markers that can be examined on a single specimen over that of current technologies (i.e., several tens of markers imaged, with line of sight to resolve hundreds). PUBLIC HEALTH RELEVANCE: The proposed project will create a new molecular probe technology that allows large numbers of molecular biomarkers to be examined on a single clinical biopsy, and hence, will improve the utility of biospecimens for the early detection and clinical management of cancer. Furthermore, the proposed technology will surmount current technological barriers that prohibit characterization of rare cells and low abundance markers within a single specimen, which in turn will lead to a better understanding of the molecular and cellular-level changes that occur within tumors, and assist in the future discovery of new targetable cancer markers.