Novel affinity reagents for epigenetics markers

描述(申请人提供)：组蛋白尾部的翻译后修饰(PTM)是转录激活和沉默的主要标志。因此，检测和捕获翻译后修饰的组蛋白是表观遗传学研究中的关键一步。这一研究领域的一个主要技术瓶颈是缺乏高质量的亲和试剂。多克隆抗体和单克隆抗体是目前唯一广泛可用的组蛋白尾部亲和试剂，但它们在重复性、可扩展性、存储和生产吞吐量以及费用等方面都有根本的局限性。该项目的长期目标是开发一个创新和强大的技术平台，为含有PTM的组蛋白尾部提供高质量的亲和试剂，并使一套标准的此类亲和试剂广泛适用于表观遗传学研究社区。这个项目建立在我们团队最近建立的一个创新的蛋白质工程概念上。这一概念被称为亲和力钳制，它利用了存在于所谓相互作用域中的固有特异性，并通过附加一个“增强子域”并随后通过组合文库的定向进化来优化其相互作用界面，从而极大地提高了它们的亲和力和特异性。由此产生的具有翻盖结构的亲和试剂，统称为“亲和钳”，从而“夹住”目标，导致更高的亲和力和特异性数量级。以这种方式建立的蛋白质文库易于与特定类型的多肽基序结合(例如，带有甲基化赖氨酸的组蛋白尾部)，它们实际上保证了针对预定义多肽基序的高性能亲和试剂的成功设计。亲和钳制代表了亲和试剂生成的范式转变。由于亲和钳是在大肠杆菌中生产的完全重组的试剂，它们可以很容易地大量生产和分发。此外，它们还可以重组为适合体外和体内应用的各种融合蛋白。我们的概念验证实验已经成功地证明了亲和力夹持概念的总体可行性，并暗示了其巨大的潜力。由于存在许多与翻译后修饰的组蛋白尾部弱结合的相互作用结构域，我们有信心将亲和夹持策略应用于各种组蛋白基序，以生产高质量的亲和试剂。拟议的项目将严格评估将亲和力钳制技术应用于表观遗传组蛋白标记的可行性和潜力。项目初期的具体目标是(I)为具有良好特性的组蛋白赖氨酸甲基化位点生产“组蛋白钳”，并将其与商业上可获得的单抗进行比较，以确定其在常用分析中的性能；以及(Ii)为没有高质量抗体的组蛋白甲基化位点生产组蛋白钳。这样的组蛋白钳将被提供给表观遗传学社区，这将对表观遗传学研究的质量、规模和类型产生重大影响。 与公共卫生相关：准确测量化学修饰形式的组蛋白的类型和数量，并捕获它们进行下游分析是表观遗传学研究中的主要技术挑战。该项目将建立一种全新的方法，以方便地产生用于这些目的的高性能试剂。这项创新和强大的技术将填补目前表观遗传学研究中的一个重大空白，该项目的产品被称为“组蛋白钳”，将使建立一套可广泛分布于社区的表观遗传学捕获试剂的标准集成为可能，并开辟表观遗传学研究的新途径。

DESCRIPTION (provided by applicant): Post-translational modifications (PTMs) of histone tails are major marks for transcription activation and silencing. Accordingly, detecting and capturing post-translationally modified histones represent a critical step in epigenetic research. A major technological bottleneck in this area of research is a paucity of high-quality affinity reagents. Polyclonal and monoclonal antibodies are the only widely available affinity reagents for histone tails, but they have fundamental limitations in reproducibility, scalability, storage and production throughput and expenses. The long-term goals of this project are to develop an innovative and powerful technology platform for facile production of high-quality affinity reagents for histone tails containing PTMs and to make a standard set of such affinity reagents broadly available to the epigenetics research community. This project is built on an innovative protein-engineering concept that our group has recently established. The concept, termed Affinity Clamping, harnesses the inherent specificity present in the so-called interaction domains and dramatically enhances their affinity and specificity by attaching an "enhancer domain" and subsequently optimizing its interaction interface by directed evolution of combinatorial libraries. The resulting affinity reagents with clamshell architecture, collectively termed "Affinity Clamps" thus "clamp" the target, leading to orders-of-magnitude higher affinity and specificity. Protein libraries made in this manner are predisposed to binding to a specific class of peptide motifs (e.g. histone tails with a methylated lysine), and they virtually guarantee successful engineering of high-performance affinity reagents for a predefined peptide motif. Affinity Clamping represents a paradigm shift in affinity reagent generation. Because Affinity Clamps are fully recombinant reagents produced in E. coli, they can be easily produced in large quantities and distributed. Also they can be reformatted into a variety of fusion proteins suitable for in vitro and in vivo applications. Our proof-of-concept experiments have successfully demonstrated the general feasibility of the Affinity Clamping concept and suggest its enormous potential. Because there exist a number of interaction domains that weakly bind to post-translationally modified histone tails, we are confident that we can apply the Affinity Clamping strategy to produce high-quality affinity reagents to a variety of histone motifs. The proposed project will critically evaluate the feasibility and potential of applying the Affinity Clamping technology to epigenetic histone marks. The specific aims of the initial project period are (i) to produce "Histone Clamps" for well-characterized histone lysine methylation sites and benchmark them against commercially available monoclonal antibodies for their performance in commonly used assays; and (ii) to produce Histone Clamps for histone methylation sites for which no high-quality antibodies exist. Such Histone Clamps will be provided to the epigenetic community, which will have a major impact on the quality, scale and types of epigenetics research. PUBLIC HEALTH RELEVANCE: Accurately measuring the type and amounts of chemically modified forms of histones and capturing them for downstream analysis are major technological challenges in epigenetic research. This project will establish a totally new approach to facile generation of high-performance reagents for these purposes. This innovative and powerful technology will fill a major void in the currently epigenetic research, and products from this project, termed "Histone Clamps", will make it feasible to establish a standard set of epigenetic capture reagents that can be distributed broadly to the community and open new avenues of epigenetics research.