QPASS: Quantitative Parallel Aptamer Selection System

核酸适体作为亲和试剂具有许多有用的特点，包括易于化学合成、可逆折叠、热稳定性和低成本，使其成为抗体和其他基于蛋白质的试剂的强大替代品。然而，在过去的二十年中，适体受到以下事实的困扰：1)传统的适体生成方法（SELEX）耗时长，劳动密集型，并且通常不能产生具有足够亲和力（< 1 nM）和特异性的适体；2)没有“标准方案”，可以普遍适用于大多数蛋白质靶点来产生适体；3)由于每个适体必须单独测量，因此测量候选适体的亲和力和特异性的表征步骤冗长且资源密集。我们认为，这些挑战是由于执行选择的传统方法的缺陷造成的，这种方法自20年前最初描述以来没有发生重大变化。我们还认为，这些问题可以通过系统地采取根本不同的方法来解决该过程的三个中心阶段-选择，分析和表征适体。我们在此建议开发这样一个系统。我们将结合三种独特的新技术-微流体选择，下一代适体测序和SPR成像-开发定量平行适体选择系统（QPASS）平台。QPASS平台将在3轮筛选中为广泛的蛋白质靶标生成具有亚纳摩尔亲和力（Kd）的特定适配体，通过下一代DNA测序和生物信息学分析确定最佳候选体，并通过平行合成和测量数千个适配体候选体的亲和力来确定最佳适配体序列。单独来看，每个组件都代表着重大的技术进步。结合这种综合方法，提供了一个机会，彻底改变适体的产生过程。

Nucleic acid aptamers possess many useful features as affinity reagents, including facile chemical synthesis, reversible folding, thermal stability and low cost, making them a powerful alternative to antibodies and other protein-based reagents. However, over the past two decades, aptamers have suffered from the fact that 1) the conventional method of aptamer generation (SELEX) is lengthy, labor intensive and often does not yield aptamers with sufficient affinity (< 1 nM) and specificity; 2) there is no "standard protocol" that can be generally applied to most protein targets to generate aptamers; and 3) the characterization steps to measure the affinity and specificity of candidate aptamers are lengthy and resource-intensive, because each aptamer must be measured individually. We believe that these challenges arise from deficiencies in the conventional methodology of performing the selection, which has not changed significantly since its initial description 20 years ago. We also believe that these problems can be solved, by systematically taking fundamentally different approaches towards the three central stages of the process - selection, analysis and characterization of the aptamers. We propose here the development of such a system. We will combine three distinctly novel technologies -microfluidic selection, next-generation aptamer sequencing, and SPR Imaging - to develop the Quantitative Parallel Aptamer Selection System (QPASS) platform. The QPASS platform will generate specific aptamers with sub-nanomolar affinities (Kd) for a wide range of protein targets within 3 rounds of selection, identify a pool of the best candidates by next generation DNA sequencing and bioinformatic analysis, and home in on the optimal aptamer sequence by the parallel synthesis and measurement of the affinities of thousands of aptamer candidates. Individually, each component represents a significant technological advance. Combined this integrated approach offers an opportunity to revolutionize the process of aptamer generation.