Novel strategies for high-specific multiplexed imaging of genomic interactions by signal amplification

摘要 携带相同DNA序列的细胞可以表现出不同的基因表达，导致表型和 健康和疾病状态下的功能多样性。这种异质性从根本上出现在原子核上。 水平通常来自不同的基因组组织时空模式，这可能会不同地影响 与基因表达有关的遗传因素之间物理相互作用的频率和强度。 这些互动模式的中断通常与疾病有关，因此，有越来越多的 对用于识别基因组的异常空间模式和接触特征的先进工具的兴趣。脱氧核糖核酸 荧光原位杂交(FISH)本质上是一种单细胞分析，适用于细胞间的探测 以及对染色体相互作用的有针对性的检测。然而，使用DNA FISH进行高- 由于缺乏能够实现的策略，吞吐量和高分辨率邻近检测目前受到限制 背景信号低的多路复用和高特异性标记。这个项目将设计出两种不同的鱼 解决DNA FISH的多重和标记挑战的方法，通过创新地使用我们实验室的 最近发展起来的交换反应信号放大(SABER)方法(自然方法，2019)，它 可以同时提高成像吞吐量和多路复用级别。具体地说，第一个目标将引入 一种SABER方法的变种，仅允许在一对 鱼探头。该方法将针对DNA FISH进行优化，其广泛的通用性将在 第二个目标，在两个不同的应用中：1)用于短DNA的高特异性和低背景标记 靶点和2)无校正(即无通道对齐)一步共定位检测远端 接近3D的DNA序列。怀斯研究所卓越的研究环境 哈佛大学的生物启发工程学将提供对成功的 完成拟议的目标，并确保研究所的核心研究产生最大影响 重点是新技术的开发和翻译。该项目的发起人彭茵博士和他的 在开发基于DNA的分子装置、成像和经验方面具有专业知识的团队 染色体研究将提供详细的技术支持和个性化的指导。成功者 因此，这些目标的完成将为不断增长的科学界带来新的方法学 在染色体构象捕获(3C)和FISH之间进行交叉验证，并将进一步 扩大FISH在潜在诊断应用中的效用。

Summary Cells carrying the same DNA sequence can exhibit heterogeneous gene expression, leading to phenotypic and functional diversity in both healthy and diseased states. This heterogeneity fundamentally arises at the nucleus level often from different spatio-temporal patterns of genomic organization, which could differentially influence the frequency and strength of the physical interactions among genetic elements related to gene expression. Disruptions in these interaction patterns are often associated with disease, and accordingly, there is a growing interest in advancing tools for identifying abnormal spatial patterns and contact profiles of the genome. DNA fluorescence in situ hybridization (FISH) is intrinsically a single-cell assay and suitable for probing cell-to-cell variation as well as targeted detection of chromosomal interactions. However, the use of DNA FISH for high- throughput and high-resolution proximity detection is presently limited due to the lack of strategies enabling multiplexing and high-specific labeling with low background signal. This project will devise two separate FISH approaches that address the multiplexing and labeling challenges of DNA FISH by making novel use of our lab's recently developed Signal Amplification By Exchange Reaction (SABER) method (Nature Methods, 2019), which can simultaneously increase imaging throughput and multiplexing levels. Specifically, the first aim will introduce a variant of the SABER method that only allows signal amplification upon physical contact between a pair of FISH probes. This method will be optimized for DNA FISH and its wide versatility will be demonstrated in the second aim, in the two separate applications: 1) for high-specific and low-background labeling of short DNA targets and 2) a correction-free (i.e. no channel alignment) one-step colocalization assay for detection of distal DNA sequences in close 3D proximity. The outstanding research environment of the Wyss Institute for Biologically Inspired Engineering at Harvard University will offer numerous resources critical for the successful completion of the proposed goals and ensure the maximum impact of the research given the institute's core focus is on novel technology development and translation. The sponsor of the project, Dr. Peng Yin, and his team who have expertise in developing DNA-based molecular devises, imaging, and experience with chromosomal studies will provide detailed technical support and personalized mentorship. The successful completion of the aims will thus bring new methodologies to the growing scientific community at the interface between chromosome conformation capture (3C) and FISH for cross-validation of contact profiles and will further expand the utility of FISH in potential diagnostic applications.