Novel recombinant sensors to study histone ubiquitin signaling

研究组蛋白泛素信号传导的新型重组传感器

基本信息

批准号：
10600926
负责人：
Zu-Wen Sun
金额：
$ 127.68万
依托单位：
EPICYPHER, INC.
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：
2023-06-01 至 2025-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10600926
关键词：
Affinity Aging Antibodies Bar Codes Binding Biochemical Biological Assay Cell Count Cells Chromatin Collaborations Colorado DNA DNA Damage DNA Double Strand Break DNA biosynthesis Data Detection Development Disease Double Strand Break Repair Epitopes Exhibits Gene Activation Genetic Transcription Genomics High Pressure Liquid Chromatography Histones Inflammation Link Malignant Neoplasms Maps Methods Modification Monoubiquitination Noise Nucleosomes Pathogenesis Performance Phase Play Post-Translational Protein Processing Process Reaction Reader Reagent Recombinants Regulation Research Role Sampling Signal Transduction Site Specificity Structure Surface Technology Testing Ubiquitin Universities Validation biomarker discovery cell type commercial launch design drug discovery gene repression genome-wide improved in vivo in vivo imaging innovation manufacture nanomolar next generation novel nuclease rational design repaired response sensor stability testing synergism tool

项目摘要

PROJECT SUMMARY Monoubiquitination (Ub) of histones is an important post-translational modification (PTM) that regulates multiple DNA-related processes including DNA replication, transcription, and repair. Aberrant regulation of histone Ub is strongly linked to the pathogenesis of diverse diseases, including cancer, aging, and inflammation. However, the ability to map the distribution and dynamics of histone Ub signals has been challenged by a lack of high-quality tools, most notably detection reagents. EpiCypher performed a screen of commercial histone Ub antibodies using a panel of monoubiquitinated recombinant designer nucleosomes (Ub-dNucs) and found that nearly every commercial histone ubiquitin antibody is not specific for its intended target. Thus, next-generation detection reagents are greatly needed to advance our understanding of histone Ub signaling, opening the door to new avenues of high value drug and biomarker discovery. In this Direct to Phase II application, EpiCypher is developing UbSensors™, a novel class of detection reagents that leverage chromatin reader domains to specifically detect Ub on nucleosomes to enable analysis of histone Ub signaling dynamics in vivo. Recombinant UbSensors are designed using a structure-guided approach to combine binding domains that interact with a nucleosomal surface (Anchor) and ubiquitin (UBD) with a rationally designed Linker, providing high selectivity towards site-specific nucleosomal Ub targets. In Phase I equivalent studies, we collaborated with Drs. Robert Cohen and Tingting Yao (Colorado State University), who developed UbSensors to target H2AK119ub1, H2BK120ub1, H2AK15ub1, or any histone Ub moiety (pan-Ub). We validated these reagents in a biochemical binding assay using Ub-dNucs, demonstrating that the UbSensors are highly specific for their intended Ub-Nuc targets and vastly outperform current best-in- class antibodies. In addition, we successfully applied our UbSensors to interrogate the distribution of histone Ub genome-wide using CUT&RUN (Cleavage Under Targets & Release Using Nuclease), a breakthrough genomic mapping technology that generates high quality data with low cell input and sequencing requirements. In Phase II, we will expand upon our structure-guided designs and develop additional UbSensors, and apply these reagents to characterize Ub-Nuc signaling dynamics using cutting-edge genomic mapping assays. In Aim 1, we will develop additional UbSensors to target new Ub-Nuc modifications as well as explore various epitope tags to improve performance in downstream assays. In Aim 2, we will further develop the application of UbSensors for CUT&RUN by developing a DNA-barcoded Ub-dNuc spike-in panel and performing rigorous validation with a range of cell types, sample processing methods, and inputs. In Aim 3, we will prepare for commercial launch at the conclusion of Phase II by scaling UbSensor manufacturing and developing a lot release strategy, as well as performing external validation and demonstrating the utility of UbSensors for high-value genomics research by generating the first reliable maps of nucleosomal Ub signals in DNA double strand break repair.

项目概要组蛋白的单泛素化 (Ub) 是一种重要的翻译后修饰 (PTM)，可调节多种 DNA 相关过程，包括 DNA 复制、转录和修复的异常调节。组蛋白 Ub 与多种疾病的发病机制密切相关，包括癌症、衰老和炎症。然而，绘制组蛋白 Ub 信号分布和动态的能力受到了缺乏的挑战。高质量的工具，最著名的检测试剂进行了商业组蛋白 Ub 的筛选。使用一组单泛素化重组设计核小体 (Ub-dNucs) 的抗体，发现几乎所有商业组蛋白泛素抗体都不是针对其预期靶标的特异性，因此，下一代。迫切需要检测试剂来增进我们对组蛋白 Ub 信号传导的理解，打开大门高价值药物和生物标志物发现的新途径。在这个直接进入第二阶段的应用中，EpiCypher 正在开发 UbSensors™，一种新型检测方法利用染色质阅读器域特异性检测核小体上的 Ub 以便进行分析的试剂重组 UbSensor 是使用结构引导设计的。组合与核小体表面（Anchor）和泛素（UBD）相互作用的结合域的方法具有合理设计的连接器，为特定位点的核小体 Ub 靶标提供高选择性。 I 期等效研究，我们与 Robert Cohen 博士和 Tingting Yao（科罗拉多州立大学）合作 University），开发了针对 H2AK119ub1、H2BK120ub1、H2AK15ub1 或任何组蛋白 Ub 的 UbSensors 我们使用 Ub-dNucs 在生化结合测定中验证了这些试剂，证明了 UbSensors 对其预期的 Ub-Nuc 目标具有高度针对性，并且远远优于当前的最佳产品此外，我们成功地应用我们的 UbSensors 来询问组蛋白 Ub 的分布。使用 CUT&RUN（目标下切割并使用核酸酶释放）进行全基因组分析，这是一项突破性基因组技术绘图技术可生成高质量数据，且细胞输入和同相测序要求较低。 II，我们将扩展我们的结构引导设计并开发更多的 UbSensors，并应用这些在目标 1 中，我们使用尖端基因组作图分析来表征 Ub-Nuc 信号动力学。将开发额外的 UbSensors 以针对新的 Ub-Nuc 修饰，并探索各种表位标签在目标 2 中，我们将进一步开发 UbSensors 的应用。 CUT&RUN，通过开发 DNA 条形码 Ub-dNuc 掺入面板并使用在目标 3 中，我们将准备商业发布。通过扩大 UbSensor 制造规模和制定批次发布策略来结束第二阶段，以及执行外部验证并展示 UbSensors 在高价值基因组学研究中的实用性生成 DNA 双链断裂修复中核小体 Ub 信号的第一个可靠图谱。