Hybridization Chain Reaction: In Situ Amplification for Biological Imaging

项目摘要 杂交链式反应：用于生物成像的原位fi扩增 生命是由可编程的生物分子-DNA、RNA和蛋白质-在复杂的Biolog中相互作用而编排的 电路。RNA原位杂交(RNA-ISH)方法为生物学家提供了一个了解空间分布的重要窗口 这一回路的组织，使得能够在解剖背景下从亚细胞到 生物体长度刻度。由于标本之间的差异，对详细的空间关系进行了检查 需要多路复用实验，在该实验中，多个靶RNA在单个 生物样本。用传统的RNA-ISH方法对包括整装在内的厚fl荧光样品进行检测 脊椎动物胚胎，多路传输是繁琐或不切实际的，空间分辨率经常受到 报告分子的扩散，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质表达进行成像，同时与传统的 传统的DNA原位杂交(DNA-ISH)方法，目前还不是单拷贝小基因组成像的常规方法 在任何样本中都有基因座，在脊椎动物胚胎中就更少了。这些长期存在的传统ISH和 IHC方法是研究最相关系统中遗传调控网络的一个重要障碍 对人类发展和疾病的影响。 基于杂交链式反应机制的原位扩增fi扩增借鉴了 新兴学科动态核酸纳米技术实现三大mRNA成像突破 在整装脊椎动物胚胎和厚组织切片中：直接的5通道多路传输，亚细胞 相对定量、单分子分辨率和灵敏度。拟议的研究将建立在这些基础上 独特的功能，可显著提升HCR的稳健性、多路复用和量化能力 Rna-ish，并将多重的、定量的、无酶的hcr信号扩增fi阳离子的好处扩展到免疫球蛋白。 和厚厚的汽车fl荧光样本中的dna-ish。主要目标是： 原位HCR v3.0：使用合作探头自动抑制背景，用于下一代Robust- Ness和信号到背景成像mRNAs和短RNA靶(miRNAs，mRNAs剪接连接， 和密切相关的RNA序列)。 下一代多路复用器(15路复用器，所有目标均可同时放大Hcr信号fi)和全数字 滴度(HIGH-fiDelity信使核糖核酸绝对定量，亚细胞分辨率和全胚胎标度)。 下一代多功能性：将HCR成像的优势扩展到蛋白质靶点，单拷贝小GE-fi 名词性基因座和分子复合体，使所有目标类别的兼容多路成像成为可能。 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。 用于遗传学研究的多重、定量、分辨率、敏感性和多功能性的先例组合 解剖学背景下的调控网络。

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.