Hybridization Chain Reaction: In Situ Amplification for Biological Imaging

描述（由申请人提供）：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回路执行产生显著空间异质性和复杂性的发育程序。原位杂交方法是阐明发育和病理过程的重要工具，能够在从亚细胞到生物体长度尺度的形态学背景下成像mRNA表达。由于标本之间的变异性，不同基因的调控位点之间的空间关系的准确映射需要多重实验，其中多个mRNA在单个生物样品中成像。利用目前的原位杂交方法，同时检测完整脊椎动物胚胎中多个靶mRNA的表达是具有挑战性的。这一缺陷是一个重大的障碍，在系统中的相互作用的调节元件的研究最相关的人类发展和疾病。 在这里，我们借鉴核酸纳米技术领域的概念，设计和验证基于杂交链反应（HCR）机制的原位放大器。使用这种方法，与mRNA靶互补的RNA探针触发链式反应，其中荧光团标记的RNA发夹自组装成系留的荧光扩增聚合物。在第一个融资期内，我们设计了正交HCR放大器，可同时在同一样品中独立工作。当在固定的整装和横截面斑马鱼胚胎中同时成像五种靶mRNA时，实现了稳健的性能。此外，HCR放大器表现出优异的样品穿透性、高信号背景比和尖锐的信号定位。在第二个资助期内，我们将扩展核心HCR原位扩增技术，以实现脊椎动物胚胎中前所未有的定量成像目标，使该技术经过验证和优化的目标和生物体类别多样化，并设计具有改进性能的下一代HCR原位扩增器。我们的主要目标是：在整个胚胎图像中准确和精确地相对定量mRNA丰度。 斑马鱼胚胎中单个mRNA转录物的亚细胞成像和定量产率。 在整个安装的斑马鱼胚胎中具有高信号背景的miRNAs和选择性剪接mRNAs的多重作图。 推广HCR原位扩增用于不同的生物体。 设计下一代HCR现场放大器，大幅改善增益、均匀性、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，灵敏度和分辨率的组合。 完整的脊椎动物胚胎和其他不同的生物样品中的元素。 公共卫生相关性：我们建议设计分子仪器，用于在单个完整的脊椎动物胚胎内同时定量绘制多个遗传调控元件的表达模式。这项技术将为生物学家提供重要的工具，以阐明生物回路在人类发育和疾病中所起的作用。

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.