Particle display: a new paradigm in high throughput discovery of ultra-high perfo

描述(申请人提供)：核酸适配子在治疗学、诊断学和研发工具中显示出作为单抗的合成替代品的巨大前景。与抗体相比，适配子的主要优势包括对非免疫原性靶标的适用性、化学和热稳定性、低生产成本以及不同批次的变化。然而，由于发现过程中的瓶颈，适配子的潜力尚未完全实现：到目前为止，用于发现适配子的方法完全是基于选择的(即SELEX)。遗憾的是，由于文库合成偏差、聚合酶链式反应偏差和非特异性结合等多种干扰因素，这些方法往往无法在起始文库中发现最高性能的适配子。此外，以往的研究表明，这些方法可能只发现约30%的人类蛋白质组的DNA适配子，这极大地限制了适体的应用范围。以前在发现过程中的创新都是SELEX的变体，主要集中在提高选择效率；因此，根本的适体性能问题尚未得到解决。该SBIR项目的目的是开发一种基于筛选的方法(在本提案中称为“粒子显示”或“PD”)，以克服适体发现障碍。我们将文库中的每个适配子展示在105个拷贝的适配子粒子(AP)上，这使我们能够使用FAC以超高通量的方式定量测量每个AP上的适配子的结合性质。因此，PD能够精确分离通过定义的性能阈值的适配子。此外，我们可以使用多色流式细胞仪同时测量AP的多个荧光波长(每个波长代表一种结合属性)，从而唯一地能够筛选出多种有利的结合属性，如亲和力、特异性和结合动力学。这个项目的假设是帕金森病可以克服 克服了传统的基于选择的方法的局限性，并能够有效地发现最高性能的适配子。在第一阶段，我们建议首先开发PD平台，用于筛选高亲和力的靶点。然后，我们将致力于开发多颜色PD，以同时筛选多种结合属性。在第二阶段，我们将使用多颜色PD来筛选适配子对，以实现三明治分析，在生物样本中筛选可应用的适配子，并验证PD在不同靶点类型中的普遍适用性。在第二阶段结束时，我们希望建立粒子显示公司以高通量方式开发高性能适配子的能力，该适配子可以服务于治疗、诊断和生命科学研发中各种未得到满足的需求。

DESCRIPTION (provided by applicant): Nucleic acid aptamers have shown great promise as the synthetic alternative to monoclonal antibodies in therapeutics, diagnostics, and R&D tools. Key advantages of aptamers comparing to antibodies include applicability to non-immunogenic targets, chemical and thermal stability, low production cost, and no batch-to-batch variation. Nevertheless, the potential of aptamers has not been fully realized due to the bottleneck in the discovery process: to date, the methods used to discover aptamers have been exclusively selection-based (i.e. SELEX). Unfortunately, these methods often fail to discover the highest performance aptamer in the starting library, due to a multitude of interfering factors such as library synthesis bias, PCR bias and non-specific binding. Furthermore, previous investigations suggest that these methods may discover DNA aptamers for only ~30% of the human proteome, greatly limiting aptamers' application scope. Previous innovations in the discovery process are all variants of SELEX and mainly focus on improving selection efficiency; in consequence, the fundamental aptamer performance issue has not yet been solved. The purpose of this SBIR project is to develop a screening-based method (referred as "Particle Display" or "PD" in this proposal) that overcomes the aptamer discovery barrier. We display each aptamer in the library on an aptamer particle (AP) at 105 copies, which allows us to use FACS to quantitatively measure the binding property of the aptamer on each AP in an ultra-high-throughput fashion. PD thus enables precise isolation of aptamers that pass a defined performance threshold. Moreover, we can use multi-color FACS to simultaneously measure multiple fluorescence wavelengths of the APs (each wavelength representing one binding property), and thus uniquely enable screening of multiple favorable binding properties such as affinity, specificity, and binding kinetics. The hypothesis of this project is that PD can overcome the limitations of conventional selection-based methods, and enable efficient discovery of the highest performance aptamers. In Phase I, we propose to first develop the PD platform for screen for high target affinity. We will then work to develop multi-color PD to simultaneously screen for multiple binding properties. In Phase II, we will use multi-color PD to screen for aptamer pairs to enable sandwich assays, to screen for application-ready aptamers in biological samples, and validate universal applicability of PD in different target types. At the end of phase II, we expect to have established the capability of Particle Display in developing high performance aptamers in a high throughput manner, which can serve various unmet needs in therapeutics, diagnostics, and life science R&D.