QPASS: Quantitative Parallel Aptamer Selection System

QPASS：定量平行适体选择系统

• 批准号: 8335434
• 负责人: LLOYD M SMITH
• 金额: $ 106.28万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA BARBARA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-25 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8335434
• 关键词: Adsorption; Affinity; Algorithms; Antibodies; Architecture; Binding; Bioinformatics; Biotechnology; Carbon; Complex Mixtures; DNA; DNA Sequence; Data; Development; Devices; Generations; Growth Factor; Home environment; Image; Integrins; Measurement; Measures; Methodology; Methods; Microfluidics; Nucleic Acids; Performance; Process; Proteins; Protocols documentation; Reagent; Resources; Selection Bias; Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Staging; System; Techniques; aptamer; base; chemical synthesis; cost; new technology; next generation; novel strategies; pressure; prototype; software development

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.

作为亲和力试剂，核酸的适应体具有许多有用的特征，包括易于化学化学合成，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。但是，在过去的二十年中，适体遭受了这样的事实，即1）常规的适体产生方法（SELEX）冗长，劳动密集，并且通常不会产生具有足够亲和力（<1 nm）和特异性的适体； 2）没有通常可以应用于大多数蛋白质靶标生成适体的“标准方案”； 3）测量候选适体的亲和力和特异性的表征步骤是冗长且资源密集的，因为每个适体必须单独测量。我们认为，这些挑战是由执行选择的常规方法中的缺陷引起的，自20年前最初的描述以来，这种挑战并没有发生重大变化。我们还认为，可以通过系统地采用从根本上采取不同的方法来解决该过程的三个核心阶段 - 选择，分析和表征适中，可以解决这些问题。我们在这里提出了这样一个系统的开发。我们将结合三种新颖的技术 - 微流体选择，下一代适体测序和SPR成像 - 以开发定量的平行适体选择系统（QAPS）平台。 QPASS平台将生成具有亚纳米尔亲和力（KD）的特定适体，以在3轮选择内为广泛的蛋白质靶标生成特定的适体，通过下一代DNA测序和生物信息学分析确定最佳候选者的库，并通过平行的综合和测量量的最佳适应性序列在最佳的适应性序列中进行居住。单独地，每个组件代表着重要的技术进步。这种集成的方法相结合提供了一个彻底改变适体生成过程的机会。