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.

总结 携带相同DNA序列的细胞可以表现出异质性基因表达，导致表型和细胞周期的改变。 在健康和疾病状态下的功能多样性。这种异质性从根本上产生于核心 水平往往来自不同的时空模式的基因组组织，这可能会影响差异 与基因表达相关的遗传元件之间物理相互作用的频率和强度。 这些相互作用模式的破坏通常与疾病有关，因此， 感兴趣的先进工具，以确定异常的空间模式和接触的基因组配置文件。DNA 荧光原位杂交（FISH）本质上是一种单细胞测定法，适合于探测细胞间的 变异以及染色体相互作用的靶向检测。然而，DNA FISH用于高- 吞吐量和高分辨率邻近检测目前由于缺乏策略而受到限制 多路复用和高特异性标记，具有低背景信号。该项目将设计两个单独的FISH 解决DNA FISH的多重和标记挑战的方法， 最近开发了交换反应信号放大（SABER）方法（Nature Methods，2019）， 可以同时增加成像吞吐量和多路复用水平。具体来说，第一个目标将介绍 SABER方法的一种变体，仅允许在一对 FISH探针。该方法将被优化用于DNA FISH，其广泛的通用性将在 第二个目的，在两个单独的应用中：1）用于短DNA的高特异性和低背景标记 和2）无校正（即，无通道对齐）的一步共定位测定，用于检测远端靶向的 DNA序列在3D空间中的接近度。Wyndham研究所出色的研究环境， 生物启发工程在哈佛大学将提供许多资源的成功至关重要 完成拟议的目标，并确保研究的最大影响，因为研究所的核心 重点是新技术的开发和翻译。该项目的发起人Peng Yin博士及其 拥有开发基于DNA分子设计、成像和 染色体研究将提供详细的技术支持和个性化的指导。成功 因此，目标的完成将为接口处日益壮大的科学界带来新的方法。 染色体构象捕获（3C）和FISH之间的交叉验证的接触概况，并将进一步 扩大FISH在潜在诊断应用中的实用性。