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

基本信息

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
项目类别：
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

项目摘要

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.

概括携带相同 DNA 序列的细胞可以表现出异质基因表达，导致表型和健康和患病状态下的功能多样性。这种异质性从根本上出现在细胞核上水平通常来自基因组组织的不同时空模式，这可能会产生不同的影响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，越来越多的人对改进用于识别基因组异常空间模式和接触概况的工具感兴趣。脱氧核糖核酸荧光原位杂交 (FISH) 本质上是一种单细胞检测，适合探测细胞间的情况变异以及染色体相互作用的靶向检测。然而，使用 DNA FISH 进行高由于缺乏启用策略，吞吐量和高分辨率接近检测目前受到限制具有低背景信号的多重和高特异性标记。该项目将设计两个独立的 FISH 通过新颖地利用我们实验室的方法来解决 DNA FISH 的多重和标记挑战最近开发了交换反应信号放大（SABER）方法（NatureMethods，2019），该方法可以同时提高成像吞吐量和复用水平。具体来说，第一个目标将介绍 SABRE 方法的一种变体，仅允许在一对之间的物理接触时进行信号放大 FISH 探针。该方法将针对 DNA FISH 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记目标和 2) 一种无需校正（即无通道对齐）的一步共定位测定，用于检测远端 3D 接近的 DNA 序列。维斯研究所优越的研究环境哈佛大学的生物启发工程将提供大量资源，对于成功实现这一目标至关重要鉴于研究所的核心，完成拟议的目标并确保研究产生最大的影响重点是新技术开发和转化。该项目的发起人殷鹏博士及其团队团队拥有开发基于 DNA 的分子设备、成像方面的专业知识和经验染色体研究将提供详细的技术支持和个性化指导。成功者因此，目标的完成将为不断发展的科学界带来新的方法论染色体构象捕获 (3C) 和 FISH 之间的接触图交叉验证，并将进一步扩大 FISH 在潜在诊断应用中的效用。