QPASS: Quantitative Parallel Aptamer Selection System

核酸适配子作为亲和试剂具有化学合成简便、可逆折叠、热稳定性好、价格低廉等特点，是抗体和其他蛋白质类试剂的有力替代品。然而，在过去的二十年中，适配子一直受到以下事实的影响：1)传统的适配子产生方法(SELEX)冗长、劳动密集型，而且往往不能产生具有足够亲和力(1 NM)和特异性的适配子；2)没有可以普遍应用于大多数蛋白质靶标来产生适配子的“标准方案”；以及3)测量候选适配子的亲和力和特异性的表征步骤冗长且耗费资源，因为每个适配子都必须单独测量。我们认为，这些挑战源于执行选择的传统方法的缺陷，自20年前最初描述以来，这种方法没有发生重大变化。我们还认为，通过系统地对进程的三个中心阶段--适配子的选择、分析和表征--采取根本不同的方法，这些问题是可以解决的。我们在这里建议开发这样一个系统。我们将结合三项全新的技术-微流控选择、下一代适配子测序和SPR成像-开发定量平行适配子选择系统(QPASS)平台。QPASS平台将在3轮筛选内为多种蛋白质靶标产生亚纳摩尔亲和力(Kd)的特定适体，通过下一代DNA测序和生物信息学分析确定最佳候选者，并通过并行合成和测量数千个候选者的亲和力来确定最佳适配子序列。就个人而言，每个组件都代表着一项重大的技术进步。这种综合的方法结合在一起，提供了一个彻底改变适配子产生过程的机会。

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.