Hybridization Chain Reaction: In Situ Amplification for Biological Imaging

项目摘要 杂交链式反应：生物成像的原位扩增 多细胞有机体中的每个细胞都包含相同的基因组，但其中编码的调节电路 基因组实施的发育计划产生了显著的空间异质性和复杂性。就地 杂交方法是阐明发育和病理过程的基本工具，使 从亚细胞到组织长度尺度的形态背景下的信使核糖核酸表达成像。由于 样本之间的变异性，准确绘制不同调控基因座之间的空间关系图 基因需要多个实验，在这个实验中，多个mRNA被成像在一个生物样本中。使用 目前的原位杂交方法中，同时检测多个靶点的表达是具有挑战性的 完整脊椎动物胚胎中的mRNAs。这一缺陷严重阻碍了对交互作用的研究 与人类发展和疾病关系最密切的系统中的管理要素。 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原位AM-AM。 基于杂交链式反应(HCR)机制的分枝。使用这种方法，RNA探针COM- 补充mRNA靶标引发链式反应，荧光团标记的RNA发夹在其中自组装 变成系留的荧光放大聚合物。在第一个资助期，我们设计了正交HCR 同时在同一样品中独立工作的放大器。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个靶向mRNA时。 此外，HCR放大器具有良好的样品穿透性、高的信号背景比和尖锐的信号 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，以使目标类别和 对该技术进行验证和优化的生物，并就地设计下一代HCR 具有改进性能的放大器。我们的主要目标是： 全胚胎图像中信使核糖核酸丰度的准确和精确的相对定量。 斑马鱼全套胚胎单个mrna转录本的亚细胞成像研究。 MiRNAs和选择性剪接的mRNAs的多路映射在整个 安装斑马鱼胚胎。 推广HCR原位扩增在不同生物体中的应用。 设计新一代HCR现场放大器，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，提供一个不同的领域。 用于相互作用RNA研究的多路复用、定量、灵敏度和分辨率的先例组合 完整的脊椎动物胚胎和其他不同生物样本中的调节元件。

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.