Renewable synthetic antibodies for epigenomics

描述(由申请人提供)：广泛的挑战领域：(06)使能技术特定的挑战主题：06-OD-106表观基因组研究的可再生亲和试剂。这项提议的目标是(I)建立一个技术平台，以产生高质量、经过充分验证的合成抗体，以及(Ii)产生对表观遗传学研究有用的高价值抗原的首批合成抗体。合成抗体是使用最先进的体外技术产生的，而不需要动物免疫。根据定义，它们是单克隆性的，它们的表达克隆及其免疫化学性质是永久存档的，很容易分布。总而言之，这些属性使合成抗体成为单克隆和多克隆抗体的更好替代品。表观遗传标记的亲和试剂，如含有特定翻译后修饰(PTM)的组蛋白尾部，以及参与表观遗传调控的其他蛋白质的亲和试剂，是表观基因组学研究中至关重要的工具。它们是染色质免疫沉淀(CHIP)的核心组成部分，染色质免疫沉淀是标准的、强大的工具，也是参与调控和响应表观基因组变化的蛋白质复合体的免疫组织化学和生化分析的关键。现在非常清楚的是，传统抗体存在严重的局限性。单抗生产缓慢且价格昂贵，而且对其亲和力或选择性几乎没有控制。多克隆抗体在重复性和特异性方面有一个根本性的问题，这相当于实验变异的不可接受水平。这种情况代表了我们以高度质量控制的方式执行表观基因组图谱的能力的严重差距。我们的团队包括合成抗体工程领域的三位领导者和组蛋白修饰蛋白质组学分析领域的一位领导者。我们共同开发了一套完整的完全重组抗体技术，其性能超过了天然抗体。在这个项目中，我们将结合我们互补的专业知识，进一步加强和应用这些强大的技术，以产生对组蛋白尾部PTM和其他参与表观遗传调控的蛋白质具有极高特异性和亲和力的合成抗体。该项目的具体目标是(1)开发一个综合技术平台，用于生产和表征具有精致的抗原结合特性的合成抗体；(2)产生抗组蛋白尾肽和多梳组分的合成抗体；(3)使用多种分析工具严格验证所产生的抗体用于芯片应用；以及(4)开发用于抗体分发的网络数据库和后勤基础设施。在这个项目中产生的可再生的合成抗体将对表观基因组学研究产生直接和强烈的影响，因为它们将得到高度验证，其性质(亲和力和特异性)不会改变。该项目将在迅速增长的重组抗体领域雇用和培训更多的人员。总而言之，该项目与ARRA挑战赠款的总体目标以及为表观基因组研究生成可再生亲和试剂的具体挑战完美地结合在一起。组蛋白化学成分的微妙修饰(翻译后修饰)是表观基因组转录调控中至关重要的生物标志物，这一过程对于理解细胞发育和疾病状态的发生至关重要。由于缺乏能够选择性地检测和捕获组蛋白修饰和相关蛋白的高质量“亲和试剂”，这一领域的研究受到了严重阻碍。该项目将建立一个最先进的平台，并为表观基因组靶标产生高质量、可再生的亲和试剂，从而消除表观基因组研究的一个主要瓶颈。

DESCRIPTION (provided by applicant): Broad challenge area: (06) Enabling technologies Specific challenge topic: 06-OD-106 Renewable affinity reagents for epigenomic research. The goals of this proposal are (i) to establish a technology platform for generating high-quality, well- validated synthetic antibodies and (ii) to produce the initial set of synthetic antibodies to high-value antigens useful for epigenetic research. Synthetic antibodies are generated using state-of-the-art in vitro techniques without animal immunization. They are monoclonal by definition and their expression clones, together with their immunochemical properties, are permanently archived and easily distributed. Together, these attributes make synthetic antibodies much superior alternatives to monoclonal and polyclonal antibodies. Affinity reagents to epigenetic markers such as histone tails containing specific post-translational modifications (PTMs) and to other proteins involved in epigenetic regulation are critically important tools in epigenomics research. They are the central component of the chromatin immunoprecipitation (ChIP), the standard, powerful tool and also essential for immunohistochemical and biochemical analyses of protein complexes involved in regulating and responding to epigenomic changes. It is now painfully clear that conventional antibodies present severe limitations. Monoclonal antibody production is slow and expensive, and there is little control on their affinity or selectivity. Polyclonal antibodies have a fundamental problem in reproducibility and specificity, which amount to unacceptable levels of experimental variations. This situation represents a critical gap in our ability to perform epigenomic profiling in a highly quality-controlled manner. Our team includes three leaders in the field of synthetic antibody engineering and a leader in proteomic analysis of histone modifications. We have collectively developed a complete spectrum of fully recombinant antibody technologies whose performance exceeds that of natural antibodies. In this project, we will combine our complementary expertise to further enhance and apply these powerful technologies for generating synthetic antibodies of exquisite specificity and affinity to histone tail PTMs and other proteins involved in epigenetic regulation. The specific aims of this project are (1) to develop an integrated technological platform for the production and characterization of synthetic antibodies with exquisite antigen binding properties; (2) to generate synthetic antibodies to histone tail peptides and to polycomb components; (3) to rigorously validate generated antibodies for ChIP applications using multitude of analytical tools; and (4) to develop a Web- database and logistical infrastructure for antibody distribution. Renewable, synthetic antibodies generated in this project will make an immediate and strong impact on epigenomics research, because they will be highly validated and their properties (affinity and specificity) will not change. This project will employ and train additional personnel in the rapidly growing field of recombinant antibodies. Together, this project perfectly aligns with the general goal of the ARRA challenge grants and with the specific challenge of generating renewable affinity reagents for epigenomic research. Subtle modifications (post-translational modifications) in the chemical composition of histones are critically important biomarkers in the epigenomic regulation of transcription, a process that is critical for the understanding of cell development and the onset of disease states. Research in this field has been severely hindered by a lack of high- quality "affinity reagents" that can selectively detect and capture histone modifications and proteins associated with them. This project will establish a state-of-the-art platform and generate high-quality, renewable affinity reagents for epigenomic targets, thereby eliminating a major bottleneck in epigenomic research.