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

项目总结 组蛋白翻译后修饰(PTM)因其在调控中的不同作用而被广泛研究 基因表达。PTMS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抗体存在效率低和/或特异性低的问题。抗体质量为 特别关注超低细胞输入方法，这种方法要求检测试剂表现出高准确率 以最小的非靶标结合(即，特异性)最大限度地提高有限的产量 输入(例如稀有细胞群)，并提高分析灵敏度。EpiCypher已经开始解决这一问题 开发携带不同载体的重组设计核小体(DNuc)底物文库的障碍 组蛋白PTMS并利用该技术严格评估抗体结合的特异性和靶点 浓缩物。然而，一些PTM目标(例如H4K20me2)和未修改状态(例如H3K4me0)仍然存在 难治(即不存在好的抗体)，和/或由于目标低而不适合低投入研究 浓缩物。因此，需要新的检测试剂来访问历史上具有挑战性的目标并使 超低细胞输入方法，开辟了有效研究生物相关的PTM和 推进临床生物标记物/药物开发。 在这里，EpiCypher正在开发超级阅读器™：超灵敏、一流的重组检测 表现出更高的特异性和亲和力的试剂(与抗体相比)，以实现1)历史上的 具有挑战性的PTM目标和2)超低输入的PTM映射。天然染色质阅读器结构域具有高 特异性，但分离时亲和力往往较低。这种方法的创新之处在于它的多聚化 染色质阅读器结构域，利用多价相互作用的亲和力效应创建高性能 检测试剂。这些突破性的工具将使用EpiCypher的专有DNA条形码开发 DNuc技术，严格验证生理相关底物上的特异性，并使交叉 新型超灵敏试剂Cut&Run(靶下切割和核酸酶释放)中的样品比较 染色质分析方法。对于概念验证，我们开发了一款超级阅读器，确认了其在 DNucs，并将其应用于测绘，验证了其在PTM作图中的实用性和可靠性。在第二阶段，我们会 开发总共六个超级阅读器，并在一系列条件下对它们进行切割和运行验证，包括超 手机输入低。此外，我们将应用超级阅读器来分析没有高质量抗体的PTM (H4K20me2)，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，我们将优化六款超级阅读器的制造，组装测试版套件，执行 外部验证，并集成这些工具以提供内部切割和运行分析服务。共同努力，这项工作将 导致新型高效检测试剂的开发和商业化，这种试剂将 提供对历史上具有挑战性的PTM的首次访问，并启用超低单元输入PTM映射，从而 推动新的表观遗传学研究和临床进步，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.