Decoding chromosome structure with multiplexed super-resolution microscopy

用多重超分辨率显微镜解码染色体结构

• 批准号: 10226100
• 负责人: Peng Yin
• 金额: $ 54.67万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10226100
• 关键词: 3-Dimensional; Address; Adopted; Architecture; Binding; Biochemical; Bioinformatics; Biological Assay; Biology; Caliber; Cell Nucleus; Cells; Chromatin; Chromatin Fiber; Chromatin Loop; Chromosome Structures; Chromosome Territory; Chromosomes; Color; Crowding; DNA; DNA Damage; DNA Probes; DNA Repair; DNA biosynthesis; DNA-Protein Interaction; Defect; Detection; Development; Disease; Environment; Epigenetic Process; Event; Family; Fluorescent in Situ Hybridization; Gene Expression Profile; Genome; Genomic DNA; Genomics; Human; Image; Immunofluorescence Immunologic; In Situ; Individual; Label; Lead; Length; Malignant Neoplasms; Maps; Medical; Methods; Microscopy; Modeling; Nuclear; Oligonucleotides; Play; Population; Positioning Attribute; Process; Property; Proteins; RNA; Research; Research Personnel; Resolution; Role; Sampling; Site; Specificity; Structure; Techniques; Technology; Thick; Time; Transcriptional Regulation; Variant; Visual; Visualization; Work; X Inactivation; base; cell type; chromosome conformation capture; design; developmental disease; genomic locus; imaging modality; improved; insight; meter; molecular scale; multiplex assay; nanoscale; programs; single cell technology; single molecule; three dimensional structure; tool

Summary Decades of study have revealed that genome organization is non-random and critically impacts many nuclear processes including the regulation of transcription, DNA replication, and DNA repair, and increasing evidence suggests that the three-dimensional structures adopted by chromosomes are critical for development and are often perturbed in disease. Much of our current understanding comes from biochemical techniques performed on large populations of cells, leading to many gaps in our understanding of the mechanisms that establish and maintain organizational states, particularly in the context of individual cells. We propose to introduce a new set of single-cell technologies based on the single-molecule super-resolution imaging method DNA-PAINT to bridge this gap with a suite of tools possessing both high multiplexibility and spatial resolution. Specifically, in Aim 1 we will develop a multiplexed (>20 color) super-resolution chromosomal imaging strategy to image genomic targets ranging from kilobases to multiple megabases in Iength, which will enable us to investigate the folding properties of the chromatin fiber in single cells over a range of length-scales. In Aim 2, we will develop multiplexed assays to co-localize proteins, RNA molecules, and specific genomic sites in individual cells at the nanoscale. We will then investigate organization of architectural proteins at a model hub of large chromatin loops on the human inactive X-chromosome. In Aim 3, we will develop a proximity-dependent super-resolution method to probe specific interactions between protein and DNA targets that will allow for the sensitive detection of molecular interactions in crowded environments. We will deploy this technology to query the composition and epigenetic states of the aforementioned chromatin looping hub in individual cells. Collectively, our methods will make many questions about the positioning, composition, and epigenetic states of specific genomic loci in individual cells accessible to researchers for the first time, and promise to impact diverse fields beyond chromosome biology.

概括 数十年的研究表明，基因组组织是非随机的，对许多核的影响很大 过程包括调节转录，DNA复制和DNA修复以及越来越多的证据 表明染色体采用的三维结构对于发育至关重要，并且是 经常受到疾病的干扰。我们目前的大部分理解来自执行的生化技术 在大量细胞中，导致我们对建立和 维护组织国家，特别是在单个细胞的背景下。我们建议介绍一个新集合 基于单分子超分辨率成像方法DNA-Paint的单细胞技术 用一套具有高多重性和空间分辨率的工具桥接这一差距。具体来说，在 AIM 1我们将开发一个多路复用（> 20颜色）超分辨率的染色体成像策略 基因组靶标范围从千层酶到iTempt的多个兆座，这将使我们能够调查 在一系列长度尺度上，染色质纤维的折叠特性。在AIM 2中，我们将发展 多路复用测定以共定位的蛋白质，RNA分子和特定基因组位点的单个细胞中的特定基因组位点 纳米级。然后，我们将在大型染色质的模型中心研究建筑蛋白的组织 人类无活性X染色体的循环。在AIM 3中，我们将开发一个依赖性的超分辨率 探测蛋白质和DNA靶标之间特定相互作用的方法，该靶标将允许敏感 在拥挤的环境中检测分子相互作用。我们将部署这项技术来查询 上述染色质循环枢纽的组成和表观遗传态。共同 我们的方法将对特定的定位，组成和表观态提出许多疑问 研究人员可以首次访问的个体细胞中的基因组基因局，并承诺会影响各种领域 超越染色体生物学。

项目成果

Light-Seq: light-directed in situ barcoding of biomolecules in fixed cells and tissues for spatially indexed sequencing.

• DOI: 10.1038/s41592-022-01604-1
• 发表时间: 2022-11
• 期刊: NATURE METHODS
• 影响因子: 48
• 作者: Kishi, Jocelyn Y.;Liu, Ninning;West, Emma R.;Sheng, Kuanwei;Jordanides, Jack J.;Serrata, Matthew;Cepko, Constance L.;Saka, Sinem K.;Yin, Peng
• 通讯作者: Yin, Peng

Thermal-plex: fluidic-free, rapid sequential multiplexed imaging with DNA-encoded thermal channels.

Thermal-plex：采用 DNA 编码热通道的无流体、快速顺序多重成像。

• DOI: 10.1038/s41592-023-02115-3
• 发表时间: 2024
• 期刊: Nature methods
• 影响因子: 48
• 作者: Hong,Fan;Kishi,JocelynY;Delgado,RyanN;Jeong,Jiyoun;Saka,SinemK;Su,Hanquan;Cepko,ConstanceL;Yin,Peng
• 通讯作者: Yin,Peng