Hybridization Chain Reaction: Highly Multiplexed Quantitative RNA and Protein Imaging

杂交链式反应：高度多重定量 RNA 和蛋白质成像

• 批准号: 10158198
• 负责人: Harry Ming Tak Choi
• 金额: $ 15万
• 依托单位: MOLECULAR INSTRUMENTS, INC.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2021-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10158198
• 关键词: Amplifiers; Anatomy; Antibodies; Biological; Biological Assay; Brain; Clinical Pathology; Development; Diffusion; Discipline; Disease; Engineering; Enzymes; Funding; Genome; Human Biology; Image; Immunohistochemistry; In Situ; In Situ Hybridization; Label; Libraries; Messenger RNA; Methods; Molecular; Nanotechnology; National Institute of Biomedical Imaging and Bioengineering; Nucleic Acids; Organism; Pathologist; Pathology; Pattern; Performance; Pharmaceutical Preparations; Phase; Proteins; Protocols documentation; Publishing; RNA; Reaction; Reporter; Research; Resolution; Sampling; Scanning; Signal Transduction; Slice; Slide; Specimen; Stains; Stimulus; Technology; Thick; Tissues; United States National Institutes of Health; analog; antibody libraries; base; biological research; clinical Diagnosis; clinical diagnostics; developmental disease; digital; disease diagnosis; drug development; experimental study; high dimensionality; human disease; mRNA Expression; next generation; novel therapeutics; prevent; protein expression; quantitative imaging; research and development; response; single molecule; technology development; vertebrate embryos

Project Summary Hybridization Chain Reaction: Highly Multiplexed Quantitative RNA and Protein Imaging Encoded in the genome of each organism, biological circuits direct development, maintain integrity in the face of attacks, control responses to environmental stimuli, and sometimes malfunction to cause disease. RNA in situ hybridization (RNA-ISH) and immunohistochemistry (IHC) methods provide biologists, drug developers, and pathologists with critical windows into the spatial organization of this circuitry, enabling imaging of mRNA and protein expression in an anatomical context. While it is desirable to perform multiplexed experiments in which a panel of targets is imaged quantitatively at high resolution in a single specimen, using traditional RNA-ISH and IHC methods in highly autofluorescent samples including whole-mount vertebrate embryos and FFPE tissue sections, multiplexing is cumbersome or impractical, spatial resolution is frequently compromised by diffusion of reporter molecules, and staining is non-quantitative. These multi-decade technological shortcomings are signifi- cant impediments to biological research as well as to advancement in drug development and pathology assays, preventing high-dimensional quantitative analyses of developmental and disease-relevant regulatory networks in an anatomical context. To overcome these challenges, 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 four RNA-ISH breakthroughs in highly autofluorescent samples including whole-mount vertebrate embryos, thick brain slices, and FFPE tissue sections: 1) straightforward multiplexing with 1-step quantitative signal amplification for up to 5 target mRNAs simultaneously; 2) analog mRNA relative quantitation with subcellular resolution in an anatomical context; 3) digital mRNA absolute quantitation with single-molecule resolution in an anatomical context; 4) automatic background suppression throughout the protocol, dramatically enhancing performance and ease-of-use. The proposed research will build on the unique capabilities of HCR to enable next-generation levels of multiplexing for RNA-ISH, to extend the benefits of 1-step multiplexed quantitative enzyme-free HCR signal amplification to IHC, and to develop the first in situ amplification product for simultaneous RNA-ISH/IHC, performing 1-step HCR signal amplification for targets RNAs and proteins simultaneously. During Phase I, we will engineer molecular components to enable highly multiplexed signal amplification, and establish a unified framework for RNA and protein imaging. During Phase II, we will develop and commercialize robust technologies for highly multiplexed RNA-ISH/IHC in key sample types critical for academic research, drug development, and/or clinical diagnostics. These new HCR products will enable biologists, drug developers, and pathologists to perform previously impossible quantitative analyses on large panels of mRNAs and proteins at high resolution with full anatomical context.

项目摘要 杂交链反应：高度多重定量RNA和蛋白质成像 编码在每个生物体的基因组中，生物电路直接发展，保持完整性， 攻击，控制对环境刺激的反应，有时功能失常导致疾病。中RNA 原位杂交（RNA-ISH）和免疫组织化学（IHC）方法为生物学家、药物开发人员和 病理学家对这种电路的空间组织有关键的了解，能够对mRNA进行成像， 蛋白质在解剖学上的表达。虽然期望进行多重实验，其中 使用传统的RNA-ISH，在单个样本中以高分辨率对一组靶进行定量成像 和IHC方法在高度自体免疫样品中的应用，包括整片脊椎动物胚胎和FFPE组织 部分，多路复用是麻烦的或不切实际的，空间分辨率经常受到扩散的影响。 报道分子，并且染色是非定量的。这些几十年来的技术缺陷是很重要的- 不能阻碍生物学研究以及药物开发和病理学测定的进展， 防止对发育和疾病相关调控网络进行高维定量分析， 解剖学背景。 为了克服这些挑战，基于杂交链反应机制的原位扩增技术 (HCR)从动态核酸纳米技术的新兴学科的概念，以实现四个 RNA-ISH在高度自动化的样品中取得突破，包括整片脊椎动物胚胎，厚脑 切片和FFPE组织切片：1）直接多路复用，1步定量信号放大 同时检测多达5种靶mRNA; 2）在细胞内亚细胞分辨率的类似物mRNA相对定量， 解剖学背景; 3）在解剖学背景中具有单分子分辨率的数字mRNA绝对定量 上下文; 4）整个协议中的自动背景抑制，极大地提高了性能， 易于使用。拟议的研究将建立在HCR的独特能力，使下一代 RNA-ISH的多路复用水平，以扩展1步多路复用定量无酶HCR的贝内 将信号放大到IHC，并开发用于同时RNA-ISH/IHC的第一个原位扩增产物， 同时对靶RNA和蛋白质进行一步HCR信号扩增。在第一阶段，我们 将设计分子组件，以实现高度多路复用的信号放大，并建立一个统一的艾德 RNA和蛋白质成像的框架。在第二阶段，我们将开发和商业化强大的技术， 用于对学术研究、药物开发和/或临床研究至关重要的关键样本类型的高度多重RNA-ISH/IHC 临床诊断学这些新的HCR产品将使生物学家，药物开发人员和病理学家能够进行 以前不可能在高分辨率下对大组mRNA和蛋白质进行定量分析， 解剖学背景