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来评估细胞和组织中分子标记水平的空间分布，是包括癌症在内的人类疾病的诊断、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很大的限制，因为只有少数分子标记可以在单个标本上进行检查。考虑到临床标本的大小可能很小，而信息分子标记的数量可能很大，因此在单个样本上实际执行的分子和细胞分析的类型和数量经常受到损害。这些问题限制了目前通过分子标记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个标记物，这限制了充分表征单个稀有细胞和细胞龛的能力。该项目将通过开发一种新的多路重复免疫荧光成像方法来克服这些问题，这种方法称为dna催化分子生物标志物成像和扩增（DC-MBIA）。利用dna纳米技术领域的原理，DC-MBIA能够(1)对多个分子探针进行选择性荧光团标记（例如，指导含荧光团dna复合物核苷酸序列特异性反应的独特dna偶联抗体），(2)在分子标记的局部附近对荧光信号进行化学计量扩增，以及(3)通过非常温和的处理条件从样品中去除荧光团。通过这种方式，DC-MBIA允许在单个样品上重复利用荧光团；相同类型的荧光染料分子可以在分子标记物之间选择性地交换，因此，显微镜的不同荧光通道现在可以多次用于对几组分子标记物成像，即使感兴趣的标记物存在于低水平。在建立必要的基础设施以促进这一进展的同时，该项目将评估和优化DC-MBIA协议，以促进多路复用和重复标记分析。在这里，短期可行性目标是证明在单个标本上可以检测的分子标记的数量比当前技术（即，几十个标记成像，用视线来解决数百个标记）至少增加四倍。

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.