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

用于检测组蛋白翻译后修饰的工程超亲和力试剂

• 批准号: 10382046
• 负责人: Zu-Wen Sun
• 金额: $ 102.43万
• 依托单位: EPICYPHER, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-20 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10382046
• 关键词: Address; Affinity; Antibodies; Automobile Driving; Avidity; Bar Codes; Binding; Biochemical; Biological Assay; Cell Count; Cell Line; Cell physiology; Cells; Chromatin; Clinical; Clinical Research; DNA; DNA Damage; DNA Repair; Detection; Development; Engineering; Environment; Epigenetic Process; Epitopes; Exhibits; Gene Expression; Genomics; High Pressure Liquid Chromatography; Histones; Laboratories; Libraries; Maps; Methods; Noise; Nucleosomes; Performance; Phase; Physiological; Population; Post-Translational Protein Processing; Preparation; Reader; Reagent; Recombinants; Research; Research Personnel; Role; Running; Sampling; Services; Signal Transduction; Site-Directed Mutagenesis; Specificity; Technology; Testing; Time; Validation; Work; clinical biomarkers; commercialization; drug development; epigenomics; improved; innovation; novel; nuclease; repaired; response; stability testing; tool

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.

项目概要 组蛋白翻译后修饰（PTM）因其在控制中的不同作用而被广泛研究。 基因表达的研究很大程度上依赖于抗体；然而，我们已经证明大多数 商业组蛋白 PTM 抗体存在效率低和/或抗体质量低的问题。 特别关注超低细胞输入方法，这需要检测试剂表现出高目标 表位结合（即效率）与最小的脱靶结合（即特异性），以最大限度地提高有限的产量 EpiCypher 已开始解决这一问题。 通过开发携带多种基因的重组设计核小体 (dNuc) 底物文库来克服障碍 组蛋白 PTM 并利用该技术严格评估抗体结合特异性和目标 然而，一些 PTM 目标（例如 H4K20me2）和未修改的状态（例如 H3K4me0）仍然存在。 棘手（即不存在好的抗体），和/或由于目标低而不适合低投入研究 因此，需要新的检测试剂来获取历史上具有挑战性的目标并实现这一目标。 超低细胞输入方法，为有效研究生物学相关的 PTM 和 临床生物标志物/药物开发。 EpiCypher 正在开发 Super Readers™：超灵敏、一流的重组检测 表现出更高的特异性和亲和力（与抗体相比）的试剂，以实现 1) 检测历史 具有挑战性的 PTM 目标和 2) 超低输入 PTM 映射具有高天然染色质阅读器域。 特异性，但在分离时通常表现出低亲和力，这种方法的创新在于多聚化。 染色质阅读器域，利用多价相互作用的亲合力效应来创造高性能 这些突破性工具将使用 EpiCypher 专有的 DNA 条形码进行开发。 dNuc 技术严格验证生理相关底物的特异性，并实现交叉 CUT&RUN（目标下切割并使用核酸酶释放）中的样品比较，一种新型超灵敏 为了概念验证，我们开发了一个超级阅读器，证实了其特异性。 dNucs，并将其应用于 CUT&RUN，展示其在 PTM 映射方面的实用性和可靠性。 总共开发了六个超级阅读器，并在一系列条件下（包括超条件）在 CUT&RUN 中验证它们 此外，我们将应用 Super Reader 来分析没有可用优质抗体的 PTM。 (H4K20me2)，从而为该标记创建了第一个准确的基因组图谱，并描述了它在 DNA 中的作用 最后，我们将优化六个超级阅读器的损坏制造，组装测试套件，执行。 外部验证，并将这些工具集成到内部 CUT&RUN 服务中，这项工作将进行分析。 导致新型高性能检测试剂的开发和商业化 提供对历史上具有挑战性的 PTM 的首次访问，并实现超低单元输入 PTM 映射，从而 推动以前无法实现的新颖的表观遗传学研究和临床进展。