Novel recombinant sensors to study histone ubiquitin signaling

项目摘要 组蛋白的单泛素化（Ub）是一种重要的翻译后修饰（PTM）， 多个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信号的分布和动力学的能力受到缺乏的挑战。 高质量的工具，尤其是检测试剂。EpiCypher对商业组蛋白Ub进行了筛选， 使用一组单泛素化的重组设计者核小体（Ub-dNucs）的抗体，发现 几乎每一种市售的组蛋白遍在蛋白抗体对其预期的靶标都不是特异性的。因此，下一代 我们非常需要检测试剂来推进我们对组蛋白Ub信号传导的理解， 发现高价值药物和生物标志物的新途径。 在这个直接进入第二阶段的应用中，EpiCypher正在开发UbSensors™，这是一种新型的检测方法， 利用染色质阅读器结构域特异性检测核小体上的Ub以进行分析的试剂 组蛋白Ub信号动力学的研究。重组Ub传感器是使用结构引导的 将与核小体表面（锚）和泛素（UBD）相互作用的结合结构域联合收割机组合的方法 具有合理设计的接头，对位点特异性核小体Ub靶点具有高选择性。在 I期等效研究中，我们与Robert Cohen和Tingting Yao博士（科罗拉多州 他开发了UbSensors来靶向H2 AK 119 ub 1、H2 BK 120 ub 1、H2 AK 15 ub 1或任何组蛋白Ub 部分（pan-Ub）。我们在使用Ub-dNucs的生化结合测定中验证了这些试剂，证明了 UbSensors对它们的Ub-Nuc目标具有高度的特异性，并且远远优于目前最好的 类抗体。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（使用核酸酶进行靶下切割和释放）进行全基因组切割，这是一种突破性的基因组切割技术。 映射技术，以低细胞输入和测序要求生成高质量数据。同相 第二，我们将扩展我们的结构引导设计，开发更多的UbSensors，并应用这些 使用尖端的基因组图谱分析来表征Ub-Nuc信号传导动力学的试剂。目标1： 将开发更多的UbSensors来靶向新的Ub-Nuc修饰，并探索各种表位标签， 提高下游分析性能。在目标2中，我们将进一步开发UbSensors的应用， 通过开发DNA条形码Ub-dNuc加标面板并使用 一系列细胞类型、样品处理方法和输入。在目标3中，我们将准备在 通过扩大UbSensor制造规模和制定批次放行策略完成第二阶段，以及 进行外部验证，并展示UbSensors在高价值基因组学研究中的实用性， 在DNA双链断裂修复中产生核小体Ub信号的第一个可靠图谱。

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.