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: 10226792
• 负责人: NILES A PIERCE
• 金额: $ 66.5万
• 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 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10226792
• 关键词: Amplifiers; Anatomy; Biological; Biological Sciences; Cells; Complex; DNA; Diffusion; Discipline; Embryo; Engineering; Enzymes; Flow Cytometry; Genetic study; Goals; Human Development; Image; Immunohistochemistry; In Situ; In Situ Hybridization; Length; Libraries; Life; Messenger RNA; Methods; MicroRNAs; Molecular; Nanotechnology; Nucleic Acids; Organism; Performance; Proteins; RNA; RNA Sequences; RNA Splicing; Reaction; Reporter; Research; Resolution; Sampling; Signal Transduction; Small RNA; Specimen; Stains; System; Technology; Thick; Tissues; base; experimental study; genomic locus; human disease; in vivo; mRNA Expression; molecular imaging; multiplexed imaging; next generation; programs; protein expression; single molecule; spatial relationship; tool; vertebrate embryos

Project Summary Hybridization Chain Reaction: In Situ Amplification for Biological Imaging Life is orchestrated by programmable biomolecules – DNA, RNA, and proteins – interacting within complex biolog- ical circuits. RNA in situ hybridization (RNA-ISH) methods provide biologists with a crucial window into the spatial organization of this circuitry, enabling imaging of mRNA expression in an anatomical context from subcellular to organismal length scales. Due to variability between specimens, examination of detailed spatial relationships requires multiplexed experiments in which multiple target mRNAs are imaged with high resolution within a single biological sample. Using traditional RNA-ISH methods in thick autofluorescent samples including whole-mount vertebrate embryos, multiplexing is cumbersome or impractical, spatial resolution is frequently compromised by diffusion of reporter molecules, and staining is non-quantitative. The same drawbacks apply using traditional immunohistochemistry (IHC) methods to image protein expression in these challenging samples, while with tradi- tional DNA in situ hybridization (DNA-ISH) methods, it is not currently routine to image single-copy small genomic loci in any sample, much less in vertebrate embryos. These longstanding shortcomings of traditional ISH and IHC methods are a significant impediment to the study of genetic regulatory networks in systems most relevant to human development and disease. In situ amplification based on the mechanism of hybridization chain reaction (HCR) draws on concepts from the emerging discipline of dynamic nucleic acid nanotechnology to achieve three mRNA imaging breakthroughs in whole-mount vertebrate embryos and thick tissue sections: straightforward 5-channel multiplexing, subcellular relative quantitation, and single-molecule resolution and sensitivity. The proposed research will build on these unique capabilities to dramatically advance the robustness, multiplexing, and quantitation capabilities of HCR for RNA-ISH and to extend the benefits of multiplexed, quantitative, enzyme-free HCR signal amplification to IHC and DNA-ISH in thick autofluorescent samples. Major goals are: In situ HCR v3.0: automatic background suppression using cooperative probes for next-generation robust- ness and signal-to-background imaging mRNAs and short RNA targets (miRNAs, mRNA splice junctions, and closely related RNA sequences) in diverse organisms. Next-generation multiplexing (15-plex with simultaneous HCR signal amplification for all targets) and quan- titation (high-fidelity mRNA absolute quantitation with subcellular resolution and whole-embryo scale). Next-generation versatility: extend the benefits of HCR imaging to protein targets, single-copy small ge- nomic loci, and molecular complexes, enabling compatible multiplexed imaging of all target classes. Realization of these goals would have a broad impact on research in the biological sciences, providing an un- precedented combination of multiplexing, quantitation, resolution, sensitivity, and versatility for the study of genetic regulatory networks in an anatomical context.

项目摘要 杂交链反应：用于生物成像的原位扩增 生命是由可编程的生物分子-- DNA、RNA和蛋白质--在复杂的生物学中相互作用而编排的， 逻辑电路RNA原位杂交（RNA-ISH）方法为生物学家提供了一个重要的空间窗口， 组织这一电路，使成像的mRNA表达在解剖背景下，从亚细胞到 生物体长度尺度由于标本之间的变异性，检查详细的空间关系 需要多路复用实验，其中多个靶mRNA以高分辨率在单个成像系统内成像。 生物样本使用传统的RNA-ISH方法在厚的自动荧光分析样品中，包括整体封片， 在脊椎动物胚胎中，多路复用是麻烦的或不切实际的，空间分辨率经常受到以下因素的影响： 报告分子的扩散，并且染色是非定量的。同样的缺点也适用于使用传统的 免疫组织化学（IHC）方法对这些具有挑战性的样本中的蛋白表达进行成像，而传统的 常规的DNA原位杂交（DNA-ISH）方法，目前对单拷贝小基因组DNA的成像不是常规的， 基因座，更不用说脊椎动物胚胎了。传统ISH的这些长期存在的缺点， IHC方法是研究最相关系统中遗传调控网络的一个重大障碍 对人类发展和疾病的影响。 基于杂交链反应（HCR）机制的原位扩增借鉴了以下概念： 新兴学科动态核酸纳米技术实现三大mRNA成像突破 在整体安装的脊椎动物胚胎和厚组织切片中：简单的5通道复用，亚细胞 相对定量以及单分子分辨率和灵敏度。拟议的研究将建立在这些 独特的能力，大大提高了HCR的稳健性，多路复用和定量能力， RNA-ISH并将多重、定量、无酶HCR信号扩增的贝内扩展至IHC 和DNA-ISH在厚的自动荧光分析样品中的应用。主要目标是： 原位HCR v3.0：使用合作探针进行下一代鲁棒的自动背景抑制 ness和信号-背景成像mRNA和短RNA靶（miRNA，mRNA剪接点， 和密切相关的RNA序列）。 下一代多路复用（15路复用，同时对所有靶点进行HCR信号放大）和定量 滴定（亚细胞分辨率和全胚胎规模的高分辨率mRNA绝对定量）。 下一代多功能性：将HCR成像的贝内扩展到蛋白质靶点，单拷贝小基因组， 基因座和分子复合物，使得能够对所有目标类别进行兼容的多重成像。 这些目标的实现将对生物科学的研究产生广泛的影响， 多重、定量、分辨率、灵敏度和多功能性的先例组合，用于遗传学研究。 在解剖学背景下的调节网络。