Hybridization Chain Reaction: In Situ Amplification for Biological Imaging

描述（由申请人提供）：多细胞生物中的每个细胞都包含相同的基因组，然而在这个基因组中编码的调控回路实现了一个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，可以在亚细胞到生物体长度尺度的形态学背景下成像mRNA表达。由于标本之间的差异，准确绘制不同基因调控位点之间的空间关系需要多重实验，即在单个生物样本中成像多个mrna。利用现有的原位杂交方法，在完整的脊椎动物胚胎中同时检测多个目标mrna的表达是具有挑战性的。这一缺陷是研究与人类发育和疾病最相关的系统中相互作用的调节元件的一个重大障碍。在这里，我们借鉴了核酸纳米技术领域的概念，设计和验证了基于杂交链反应（HCR）机制的原位放大器。利用这种方法，与mRNA靶点互补的RNA探针触发连锁反应，其中荧光团标记的RNA发夹自组装成拴在一起的荧光扩增聚合物。在第一个资助期，我们设计了正交HCR放大器，在同一时间在同一样品中独立工作。当在固定的全载和横切的斑马鱼胚胎中同时成像五个目标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-背景和清晰的信号定位。在第二个资助期内，我们将扩展核心HCR原位扩增技术，以追求脊椎动物胚胎中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，并设计具有改进性能的下一代HCR原位扩增技术。我们的主要目标是：准确和精确的mRNA丰度在全胚胎图像的相对定量。全载斑马鱼胚胎中单个mRNA转录物的亚细胞成像及定量产量。全载斑马鱼胚胎中mirna和高信号背景选择性剪接的多路定位。推广HCR原位扩增用于不同生物。工程下一代HCR原位放大器显著提高增益，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RNA调控的研究提供前所未有的多路复用、定量、灵敏度和分辨率的组合

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.