Hybridization Chain Reaction: In Situ Amplification for Biological Imaging

杂交链式反应：生物成像的原位放大

• 批准号: 8531239
• 负责人: NILES A PIERCE
• 金额: $ 37.12万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-22 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8531239
• 关键词: Amplifiers; Biological; Biological Sciences; Brain; Cells; Chick Embryo; Culicidae; DNA; Development; Developmental Process; Drosophila genus; Embryo; Engineering; Exhibits; Formalin; Funding; Genes; Genome; Goals; Heterogeneity; Human; Human Development; Image; In Situ; In Situ Hybridization; Label; Length; Maps; Messenger RNA; Methods; Molecular; Mus; Muscle Fibers; Nanotechnology; Nucleic Acid Regulatory Sequences; Nucleic Acids; Organism; Paraffin Embedding; Pathologic Processes; Pattern; Penetration; Performance; Play; Polymers; Population; Property; RNA; RNA Probes; RNA Splicing; Reaction; Regulatory Element; Relative (related person); Research; Resolution; Role; Sampling; Signal Transduction; Specimen; Speed; System; Technology; Time; Transcript; Whole Organism; Zebrafish; base; bioimaging; cellular imaging; cost; design; fluorophore; human disease; human tissue; improved; instrument; mRNA Expression; microbial; next generation; programs; research study; spatial relationship; tool

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.

项目概要 杂交链式反应：生物成像的原位放大 多细胞生物体中的每个细胞都包含相同的基因组，但其中编码的调节电路 基因组执行一个发育程序，产生显着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，使 从亚细胞到生物体长度尺度的形态学背景下 mRNA 表达的成像。由于 标本之间的变异性，准确绘制不同调控位点之间的空间关系 基因需要多重实验，其中多个 mRNA 在单个生物样品中成像。和 目前的原位杂交方法，同时检测多个靶标的表达具有挑战性 完整脊椎动物胚胎内的 mRNA。这一缺陷严重阻碍了交互作用的研究。 与人类发育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技术 基于杂交链式反应（HCR）机制的放大器。使用这种方法，RNA 探针可 与 mRNA 靶标互补，引发链式反应，其中荧光团标记的 RNA 发夹自组装 成系留荧光放大聚合物。在第一个资助期间，我们设计了正交 HCR 同时在同一样本中独立运行的放大器。实现了稳健的性能 当在固定的整体和横截面斑马鱼胚胎中同时对五个目标 mRNA 进行成像时。 此外，HCR 放大器表现出出色的样品穿透性、高信号背景比和尖锐的信号 本土化。在第二期资助期间，我们将把核心的HCR原位放大技术扩展到 追求脊椎动物胚胎中前所未有的定量成像目标，使目标类别多样化 对其技术进行验证和优化的生物体，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： 对整个胚胎图像中 mRNA 丰度进行准确且精确的相对定量。 对整装斑马鱼胚胎中单个 mRNA 转录物进行亚细胞成像，并具有定量产量。 整体上具有高信号背景的 miRNA 和选择性剪接 mRNA 的多重作图 安装斑马鱼胚胎。 推广 HCR 原位扩增以用于多种生物体。 设计下一代 HCR 原位放大器，显着提高增益、均匀性、速度和性能 成本。 这些目标的实现将对生物科学研究产生广泛的影响，为生物科学研究提供一个新的途径。 开创性地将多重分析、定量、灵敏度和分辨率相结合，用于研究相互作用的 RNA 完整脊椎动物胚胎和其他不同生物样本中的调控元件。