A Pipeline for Production of Bivalent Synthetic Antibodies to the Human Proteome

在蛋白质组范围内创建蛋白质亲和试剂是人类医学的下一个重大挑战。高质量的蛋白质捕获试剂对于阐明蛋白质功能、分子诊断甚至治疗都是至关重要的。随着蛋白质发现的加速，我们将需要几乎所有蛋白质的亲和试剂。然而，使用在动物身上产生的抗体是一种繁琐的方法，既昂贵又耗时，而且对许多抗原常常失败。单链抗体和适体等替代方法可以解决这些问题，但仍然需要多轮选择才能获得高质量的试剂。我们开发了一种新的创新概念，用于创建称为“DNA合体”的二价亲和试剂。我们将两个与目标蛋白具有中等亲和力（Kd<1 pM）的肽结合成一个单分子，以低nM亲和力结合其目标蛋白。这个概念的核心是使用合成DNA作为刚性支架，将一个肽定位在义链上，另一个肽定位在反义链上。使用DNA作为支架可以简单可靠地调整两个肽之间的间距和角度。我们的初步数据表明，DNA合体可以直接产生并结合低纳米亲和力的靶标。此外，它们的序列和位置是精确已知的，因此它们很容易再生，生产和储存容易且价格低廉，甚至可以通过电子邮件发送到另一个实验室。DNA合体具有明确定义的化学结构，易于用标签或标记修改，并且与常见的功能分析兼容。在这个应用程序中，我们将开发必要的方法来创建一个完整的高通量管道来生产人类蛋白质的DNA合体。研究计划包括四个阶段：靶标生产、亲和试剂生产、亲和试剂表征与验证、亲和试剂分配。对于每个零件，我们将优化技术并制定适合大规模生产的标准操作程序。我们还将建立一个包含所有相关信息的数据库，包括DNA合体组成、结合亲和力、动力学参数、特异性——所有这些我们将与标准的商业抗体进行比较。

Creating protein affinity reagents on a proteome-wide scale is the next grand challenge in human medicine. High quality protein capture reagents are critical for the elucidation of protein function, molecular diagnostics and even therapeutics. With the acceleration in protein discovery, we will require affinity reagents to nearly all proteins. Yet the cumbersome use of antibodies produced in animals is expensive, time consuming, and often fails for many antigens. Alternative approaches like single chain antibodies and aptamers solve some of these problems, but still require many rounds of selection to get high quality reagents. We have developed a new and innovative concept for creating bivalent affinity reagents called "DNA synbodies." We combine two peptides with moderate affinity (Kd<1 pM) to a target protein into a single molecule that binds its target with the low nM affinity. Central to this concept is the use of synthetic DNA as a rigid scaffold that positions one peptide on the sense strand and the second peptide on the antisense strand. The use of DNA as a scaffold allows simple and reliable adjustment of the spacing and angle between the two peptides. Our preliminary data demonstrates that DNA synbodies are straightforward to produce and bind their targets with low nM affinity. Moreover, their sequences and positions are known precisely, so they are readily renewable, easy and inexpensive to produce and store, and they can even be E-mailed to another lab. DNA synbodies have a well-defined chemical structure that is easy to modify with labels or tags and are compatible with common functional assays. In this application we will develop the methodology necessary to create a complete high throughput pipeline to produce DNA synbodies to human proteins. The research plan consists of four phases: target production, affinity reagent production, affinity reagent characterization and validation, and affinity reagent distribution. For each part, we will optimize the technology and develop standard operating procedures amenable to large-scale production. We will also establish a database that contains all relevant information, including DNA synbody composition, binding affinities, kinetic parameters, specificity-all of which we will compare to standard commercial antibodies.