Engineered super-affinity reagents for detection of histone post-translational modifications

项目摘要 组蛋白翻译后修饰（PTMs）由于其在调控细胞凋亡中的不同作用而被广泛研究。 基因表达。PTM的研究在很大程度上依赖于抗体;然而，我们已经表明，大多数 商业化的组蛋白PTM抗体受到低效率和/或低特异性的困扰。抗体质量为 特别关注超低细胞输入方法，其要求检测试剂表现出高靶向 表位结合（即效率）与最小的脱靶结合（即特异性），以最大限度地从有限的 输入（例如稀有细胞群）并提高测定灵敏度。EpiCypher已经开始解决这一问题 通过开发携带多种基因的重组设计核小体（dNuc）底物库， 组蛋白PTM并利用该技术严格评估抗体结合特异性和靶向 丰富。然而，一些PTM靶标（例如H4 K20 me 2）和未修饰的状态（例如H3 K4 me 0）保留 难处理的（即不存在好的抗体），和/或由于低靶点而不适合于低输入研究 丰富。因此，需要新的检测试剂来接近历史上具有挑战性的靶标，并使 超低细胞输入方法，为有效研究生物学相关PTM开辟了新途径， 推进临床生物标志物/药物开发。 在这里，EpiCypher正在开发Super Readers™：超灵敏、一流的重组检测 显示出增加的特异性和亲和力（相对于抗体）的试剂，以使得能够1）检测历史上的 挑战性PTM目标和2）超低输入PTM映射。天然染色质阅读器结构域具有高的 特异性，但在分离时通常显示低亲和力。这种方法的创新之处在于多聚化 染色质阅读器结构域，利用多价相互作用的亲合力效应， 检测试剂这些突破性的工具将使用EpiCypher专有的DNA条形码开发， dNuc技术，以严格验证生理相关底物的特异性，并使交叉 CUT&RUN（使用核酸酶进行靶下切割和释放）中的样品比较， 染色质谱分析方法。为了验证概念，我们开发了一个超级阅读器，证实了它的特异性， dNucs，并将其应用于CUT&RUN中，证明了其在PTM映射中的实用性和可靠性。在第二阶段，我们将 开发总共六个超级阅读器，并在一系列条件下在CUT&RUN中验证它们，包括超 低电池输入。此外，我们将应用超级阅读器来分析没有可用优质抗体的PTM （H4 K20 me 2），从而为该标记创建了第一个准确的基因组图谱，并表征了其在DNA中的作用。 损伤修复最后，我们将优化六个超级阅读器的制造，组装测试套件， 外部验证，并将这些工具集成到内部CUT&RUN检测服务中。这项工作将 导致一类新的高性能检测试剂的开发和商业化， 首次访问历史上具有挑战性的PTM，并实现超低单元输入PTM映射，因此 推动了新的表观遗传学研究和临床进展，这是以前无法实现的。

PROJECT SUMMARY Histone post-translational modifications (PTMs) are widely studied due to their diverse roles in controlling gene expression. The study of PTMs largely relies on antibodies; however, we have shown that the majority of commercial histone PTM antibodies are plagued by low efficiency and/or low specificity. Antibody quality is of special concern for ultra-low cell input approaches, which require detection reagents to exhibit high on-target epitope binding (i.e. efficiency) with minimal off-target binding (i.e. specificity) to maximize yields from limited inputs (e.g. rare cell populations) and improve assay sensitivity. EpiCypher has begun to address this current roadblock by developing libraries of recombinant designer nucleosome (dNuc) substrates carrying diverse histone PTMs and leveraging this technology to rigorously assess antibody binding specificity and target enrichment. However, some PTM targets (e.g. H4K20me2) and unmodified states (e.g. H3K4me0) remain intractable (i.e. no good antibodies exist), and/or are not suitable for low input studies due to low target enrichment. Thus, new detection reagents are needed to access historically challenging targets and to enable ultra-low cell input approaches, opening new avenues to effectively study biologically relevant PTMs and advance clinical biomarker / drug development. Here, EpiCypher is developing Super Readers™: ultra-sensitive, first-in-class recombinant detection reagents that exhibit increased specificity and affinity (vs. antibodies) to enable 1) detection of historically challenging PTM targets and 2) ultra-low input PTM mapping. Natural chromatin reader domains have high specificity but often display low affinity when isolated. The innovation of this approach is the multimerization of chromatin reader domains, leveraging the avidity effects of multivalent interactions to create high-performance detection reagents. These breakthrough tools will be developed using EpiCypher’s proprietary DNA-barcoded dNuc technology to rigorously validate specificity on physiologically relevant substrates, and to enable cross- sample comparisons in CUT&RUN (Cleavage Under Targets & Release Using Nuclease), a novel ultrasensitive chromatin profiling approach. For proof-of-concept, we developed a Super Reader, confirmed its specificity on dNucs, and applied it in CUT&RUN, demonstrating its utility and reliability for PTM mapping. In Phase II, we will develop a total of six Super Readers and validate them in CUT&RUN under a range of conditions, including ultra- low cell inputs. Further, we will apply a Super Reader to profile a PTM with no quality antibodies available (H4K20me2), thus creating the first ever accurate genomic map for this mark and characterizing its role in DNA damage repair. Finally, we will optimize manufacturing of the six Super Readers, assemble beta-kits, perform external validation, and integrate these tools for in-house CUT&RUN assay services. Together, this work will lead to the development and commercialization of a new class of high-performance detection reagents that will provide first-time access to historically challenging PTMs and enable ultra-low cell input PTM mapping, thus driving novel epigenetics research and clinical advances that were previously unachievable.