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

通过信号放大对基因组相互作用进行高特异性多重成像的新策略

• 批准号: 10314777
• 负责人: Jiyoun Jeong
• 金额: $ 6.95万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10314777
• 关键词: 3-Dimensional; Address; Adopted; Biology; Cell Nucleus; Cell physiology; Cells; Cellular Assay; Chromosomes; Clinical; Color; Communication; Communities; Complement; DNA; DNA Sequence; Detection; Diagnostic; Disease; Distal; Engineering; Ensure; Environment; Exhibits; Face; Fluorescent in Situ Hybridization; Frequencies; Gene Expression; Genes; Genome; Genomic DNA; Genomics; Goals; Heterogeneity; Image; In Situ; In Situ Hybridization; Individual; Label; Maps; Measurement; Measures; Mentorship; Methodology; Methods; Microscopy; Molecular; Nature; Nuclear; Organelles; Output; Pattern; Phenotype; Positioning Attribute; Process; Proteins; RNA; Reaction; Regulatory Element; Research; Resolution; Resources; Sampling; Signal Transduction; Structure; Translations; Universities; Validation; Variant; Visualization; Yin; cell behavior; chromosome conformation capture; density; detection assay; experience; experimental study; genetic element; genome-wide; genomic locus; imaging detection; imaging probe; interest; multiplexed imaging; nanoscale; new technology; novel; novel diagnostics; novel strategies; spatiotemporal; technology development; tool

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.

概括 带有相同DNA序列的细胞可以表现出异质基因表达，从而导致表型和 健康和患病状态的功能多样性。这种异质性从根本上产生在细胞核处 通常来自基因组组织的不同时空模式的水平，这可能会差异影响 与基因表达相关的遗传元素之间物理相互作用的频率和强度。 这些相互作用模式中的破坏通常与疾病有关，因此，有一个增长 对识别基因组的异常空间模式和接触曲线的兴趣。脱氧核糖核酸 荧光原位杂交（FISH）本质上是单细胞测定法，适合探测细胞到细胞 变异以及靶向检测染色体相互作用。但是，将DNA鱼用于高 由于缺乏实现的策略，目前吞吐量和高分辨率接近性检测受到限制 具有低背景信号的多路复用和高特异性标记。该项目将设计两条单独的鱼 通过对实验室的新颖使用，可以解决DNA鱼的多路复用和标记挑战的方法 最近通过交换反应（SABER）方法（自然方法，2019）开发了信号扩增， 可以同时增加成像吞吐量和多路复用水平。具体而言，第一个目标将引入 Saber方法的一种变体，仅允许在一对物理接触时进行信号放大 鱼探针。该方法将针对DNA鱼进行优化，其广泛的多功能性将在 第二个目标，在两个单独的应用中：1）用于短DNA的高特异性和低背景标签 目标和2）无校正（即无通道比对）一步共定位测定，用于检测远端 DNA序列近距离3D接近。 Wyss研究所的杰出研究环境 哈佛大学的生物学启发工程将为成功提供许多至关重要的资源 鉴于研究所的核心，完成提议的目标并确保研究的最大影响 专注于新颖的技术开发和翻译。该项目的赞助商Peng Yin博士和他的 在开发基于DNA的分子设计，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指导。成功 因此，目标的完成将为不断增长的科学界带来新的方法论 在染色体构象捕获（3C）和用于接触曲线的交叉验证的鱼之间，将进一步 在潜在的诊断应用中扩展鱼的效用。