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

描述（由申请人提供）：核酸适体作为单克隆抗体的合成替代品在治疗、诊断和研发工具中显示出巨大的前景。与抗体相比，适体的关键优势包括对非免疫原性靶标的适用性、化学和热稳定性、低生产成本和无批次间差异。然而，由于发现过程中的瓶颈，适体的潜力尚未完全实现：迄今为止，用于发现适体的方法仅基于选择（即SELEX）。不幸的是，这些方法通常不能在起始文库中发现最高性能的适体，这是由于许多干扰因素如文库合成偏倚、PCR偏倚和非特异性结合。此外，以前的研究表明，这些方法只能发现约30%的人类蛋白质组的DNA适体，大大限制了适体的应用范围。发现过程中的先前创新都是SELEX的变体，主要集中在提高选择效率;因此，基本的适体性能问题尚未解决。 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。我们在适体颗粒（AP）上以105个拷贝展示文库中的每个适体，这允许我们使用FACS以超高通量方式定量测量每个AP上适体的结合特性。因此，PD能够精确分离通过规定性能阈值的适体。此外，我们可以使用多色FACS来同时测量AP的多个荧光波长（每个波长代表一种结合特性），从而独特地能够筛选多种有利的结合特性，例如亲和力、特异性和结合动力学。本项目的假设是，PD可以克服 传统的基于选择的方法的局限性，并且能够有效地发现最高性能的适体。 在第一阶段，我们建议首先开发用于筛选高靶亲和力的PD平台。然后，我们将致力于开发多色PD，以同时筛选多种结合特性。在第二阶段，我们将使用多色PD筛选适体对，以实现夹心测定，筛选生物样品中的应用就绪适体，并验证PD在不同靶类型中的普遍适用性。在第二阶段结束时，我们预计已经建立了以高通量方式开发高性能适体的能力，可以满足治疗，诊断和生命科学研发中各种未满足的需求。

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.