Hybridization Chain Reaction: In Situ Amplification for Biological Imaging

描述(申请人提供)：多细胞生物体中的每个细胞都含有相同的基因组，但该基因组中编码的调控电路实施了一个发育计划，产生了显著的空间异质性和复杂性。原位杂交方法是阐明发育和病理过程的重要工具，能够在从亚细胞到组织长度的形态范围内对mRNA的表达进行成像。由于样本之间的变异性，准确绘制不同基因调控基因座之间的空间关系需要多个实验，其中多个mRNAs被成像在单个生物样本中。用目前的原位杂交方法，同时检测多个靶基因在完整脊椎动物胚胎中的表达是具有挑战性的。这一缺陷严重阻碍了对与人类发展和疾病关系最密切的系统中相互作用的调控因素的研究。在这里，我们借鉴了核酸纳米技术领域的概念来设计和验证基于杂交链式反应(HCR)机制的原位扩增。使用这种方法，互补于mRNA靶标的RNA探针触发链式反应，在这些反应中，荧光团标记的RNA发夹自组装成系留的荧光放大聚合物。在第一个资助期内，我们设计出了在同一样本中同时独立工作的正交HCR放大器。在固定、整装和横切的斑马鱼胚胎中，同时成像5个靶mRNA时，获得了稳健的性能。此外，HCR放大器具有良好的样品穿透性、高的信号本底比和清晰的信号定位。在第二个资助期，我们将扩展核心HCR原位扩增技术，以在脊椎动物胚胎中追求前所未有的定量成像目标，使该技术得到验证和优化的目标和生物类别多样化，并设计性能更好的下一代HCR原位扩增技术。我们的主要目标是：准确和精确地对整个胚胎图像中的mRNA丰度进行相对定量。斑马鱼全套胚胎单个mrna转录本的亚细胞成像研究。斑马鱼整体胚胎中miRNAs和具有高信号背景比的选择性剪接mRNAs的多重定位。推广HCR原位扩增在不同生物体中的应用。设计新一代HCR原位放大器，大幅提高增益、一致性、速度和成本。这些目标的实现将对生物科学的研究产生广泛的影响，为相互作用的RNA调控研究提供前所未有的多重、定量、敏感性和分辨率的组合。 完整的脊椎动物胚胎和其他不同生物样本中的元素。 公共卫生相关性：我们建议设计分子工具，在单个完整的脊椎动物胚胎中同时定量绘制多个基因调控元件的表达模式。这项技术将为生物学家提供关键工具，以阐明生物回路在人类发育和疾病中扮演的角色。

DESCRIPTION (provided by applicant): Each cell in a multi-cellular organism contains the same genome, yet the regulatory circuits encoded within this genome implement a developmental program yielding significant spatial heterogeneity and complexity. In situ hybridization methods are an essential tool for elucidating developmental and pathological processes, enabling imaging of mRNA expression in a morphological context from sub-cellular to organismal length scales. Due to variability between specimens, accurate mapping of spatial relationships between the regulatory loci of different genes requires multiplexed experiments in which multiple mRNAs are imaged in a single biological sample. With current in situ hybridization approaches, it is challenging to simultaneously detect the expression of multiple target mRNAs within intact vertebrate embryos. This shortcoming is a significant impediment to the study of interacting regulatory elements in systems most relevant to human development and disease. Here, we draw on concepts from the field of nucleic acid nanotechnology to design and validate in situ amplifiers based on the mechanism of hybridization chain reaction (HCR). Using this approach, RNA probes complementary to mRNA targets trigger chain reactions in which fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. During the first funding period, we engineered orthogonal HCR amplifiers that operate independently in the same sample at the same time. Robust performance was achieved when imaging five target mRNAs simultaneously in fixed whole-mount and cross-sectioned zebrafish embryos. Moreover, HCR amplifiers exhibited excellent sample penetration, high signal-to-background, and sharp signal localization. During the second funding period, we will extend the core HCR in situ amplification technology to pursue unprecedented quantitative imaging goals in vertebrate embryos, to diversify the classes of targets and organisms for which the technology is validated and optimized, and to engineer next-generation HCR in situ amplifiers with improved properties. Our major goals are: Accurate and precise relative quantitation of mRNA abundance across whole-embryo images. Sub-cellular imaging of single mRNA transcripts with quantitative yield in whole-mount zebrafish embryos. Multiplexed mapping of miRNAs and alternatively spliced mRNAs with high signal-to-background in whole- mount zebrafish embryos. Generalizing HCR in situ amplification for use in diverse organisms. Engineering next-generation HCR in situ amplifiers with dramatically improved gain, uniformity, speed, and cost. Realization of these goals would have a broad impact on research in the biological sciences, providing an unprecedented combination of multiplexing, quantitation, sensitivity, and resolution for the study of interacting RNA regulatory elements within intact vertebrate embryos and other diverse biological samples. PUBLIC HEALTH RELEVANCE: We propose to engineer molecular instruments for quantitatively mapping the expression patterns of multiple genetic regulatory elements at the same time within a single intact vertebrate embryo. This technology will provide biologists with crucial tools for elucidating the roles that biological circuits play in human development and disease.