Hybridization Chain Reaction: Highly Multiplexed Quantitative RNA and Protein Imaging

项目摘要 杂交链反应：高度复合的定量RNA和蛋白质成像 编码在每个生物体的基因组中，生物回路指导着发育，在脸上保持完整性 攻击，控制对环境刺激的反应，有时还会出现故障导致疾病。RNAIn 原位杂交(RNA-ISH)和免疫组织化学(IHC)方法为生物学家、药物开发商和 病理学家对这一回路的空间组织进行了关键窗口的研究，从而能够对mRNA和 蛋白质在解剖学背景下的表达。虽然希望执行多路复用实验，其中 使用传统的rna-ish，在单个样本中以高分辨率对一组靶标进行定量成像。 和免疫组化法在包括全套脊椎动物胚胎和FFPE组织在内的高度自动fl荧光样本中的应用 部分，多路传输是繁琐或不切实际的，空间分辨率经常受到 报告分子，并且染色是非定量的。这些几十年来的技术缺陷是fi的标志- 不能阻碍生物研究以及药物开发和病理分析的进步， 防止对发育和与疾病相关的监管网络进行高维度的量化分析 一个解剖学的背景。 为了克服这些挑战，基于杂交链式反应机制的原位扩增fi (HCR)借鉴了动态核酸纳米技术这一新兴学科的概念，实现了四个 高度自动化的fl荧光样本中核糖核酸的突破，包括整装脊椎动物胚胎，厚脑 切片和FFPE组织切片：1)直接多路复用，一步定量信号放大fi阳离子 同时检测多达5个靶基因；2)亚细胞分辨率的模拟信使核糖核酸相对定量 解剖学背景；3)数字信使核糖核酸在解剖学中的单分子分辨率绝对定量 上下文；4)在整个协议中自动背景抑制，显著增强了性能和 易于使用。拟议的研究将建立在HCR的独特能力基础上，以实现下一代 RNAISH的多重水平，以扩展一步多重定量无酶hcr的fits 并开发同时用于fi-ISH/IHC第一次fi原位扩增产物， 同时对靶RNA和蛋白质进行一步hcr信号fi扩增。在第一阶段，我们 将设计分子组件以实现高度复用的信号放大fi阳离子，并建立统一的fi边缘 核糖核酸和蛋白质成像的框架。在第二阶段，我们将开发强大的技术并将其商业化 对于对学术研究、药物开发和/或至关重要的关键样本类型的高度多路复用的RNA-ISH/IHC 临床诊断学。这些新的HCR产品将使生物学家、药物开发商和病理学家能够 以前不可能对大片的mRNA和蛋白质进行全高分辨率的定量分析 解剖学背景。

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.