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，适体的主要优势包括适用于非免疫原性靶标，化学和热稳定性，生产成本低，批次间无差异。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完全是基于选择的（即SELEX）。不幸的是，由于文库合成偏倚、PCR偏倚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性能最高的适配体。此外，以往的研究表明，这些方法只能发现约30%的人类蛋白质组的DNA适体，极大地限制了适体的应用范围。之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，基本的适应性性能问题尚未得到解决。这个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。我们将库中的每个适体显示在适体颗粒（AP）上105个拷贝，这使我们能够使用FACS以超高通量的方式定量测量适体在每个AP上的结合特性。因此，PD能够精确地隔离通过定义的性能阈值的适配体。此外，我们可以使用多色FACS同时测量APs的多个荧光波长（每个波长代表一种结合特性），从而独特地筛选多种有利的结合特性，如亲和力、特异性和结合动力学。这个项目的假设是PD可以克服

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.