Hybridization Chain Reaction: In Situ Amplification for Biological Imaging

项目摘要 杂交链反应：用于生物成像的原位扩增技术 多细胞生物体中的每个细胞都包含相同的基因组，但其中编码的调控回路 基因组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， 从亚细胞到生物体长度尺度的形态学背景下的mRNA表达成像。由于 标本之间的变异性，不同的调控基因座之间的空间关系的准确映射 基因需要多重实验，其中在单个生物样品中对多个mRNA进行成像。与 目前的原位杂交方法，同时检测多个靶点的表达具有挑战性 完整脊椎动物胚胎内的mRNA。这一缺陷是研究相互作用的一个重大障碍 与人类发展和疾病最相关的系统中的调节元件。 在这里，我们借鉴核酸纳米技术领域的概念来设计和验证原位am- 基于杂交链反应（HCR）机制的增强剂。使用这种方法，RNA探针com- 作为mRNA靶点的补充，触发了荧光团标记的RNA发夹自组装的链式反应 到栓系的荧光放大聚合物中。在第一个资助期内，我们设计了正交HCR 放大器在同一时间在同一样本中独立工作。实现了稳健的性能 当在固定的整装和横切的斑马鱼胚胎中同时成像五种靶mRNA时。 此外，HCR放大器表现出优异的样品穿透，高信号背景，和尖锐的信号 本地化在第二个资助期内，我们将把核心的HCR原位扩增技术扩展到 在脊椎动物胚胎中追求前所未有的定量成像目标，使目标类别多样化， 生物，该技术已得到验证和优化，并在原位设计下一代HCR 具有改进性能的放大器。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。 miRNAs和选择性剪接mRNAs的多重定位，在整个基因组中具有高信号背景比， 安装斑马鱼胚胎。 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， 结合多重、定量、灵敏度和分辨率，用于研究相互作用的RNA 在完整的脊椎动物胚胎和其他不同的生物样品中的调节元件。

Project Summary Hybridization Chain Reaction: In Situ Amplification for Biological Imaging 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 am- plifiers based on the mechanism of hybridization chain reaction (HCR). Using this approach, RNA probes com- plementary 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 un- precedented combination of multiplexing, quantitation, sensitivity, and resolution for the study of interacting RNA regulatory elements within intact vertebrate embryos and other diverse biological samples.