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

粒子显示：超高通量高通量发现的新范例

• 批准号: 8976164
• 负责人: Qiang Gong
• 金额: $ 62.53万
• 依托单位: APTITUDE MEDICAL SYSTEMS, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-01 至 2017-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8976164
• 关键词: Adoption; Affinity; Antibodies; Apolipoprotein E; Binding; Binding Sites; Bioinformatics; Biological; Biological Assay; Biological Sciences; Bovine Serum Albumin; Chemicals; Color; DNA; Data; Diagnosis; Diagnostic; Emulsions; Environment; Enzyme-Linked Immunosorbent Assay; Exhibits; Fluorescence; Generations; Goals; Health; Human; Immunoassay; Incubated; Interleukin-6; Investigation; Kinetics; Label; Libraries; Liquid substance; Mainstreaming; Measures; Membrane Proteins; Methods; Monoclonal Antibodies; Mus; Nucleic Acids; Outcome; Performance; Phase; Plasminogen Activator Inhibitor 1; Process; Production; Property; Proteins; Proteome; Rattus; Reagent; Reporting; Saliva; Sampling; Serum; Signal Transduction; Small Business Innovation Research Grant; Specificity; TNF gene; Testing; Therapeutic; Thrombin; Variant; Work; aptamer; base; cost; cytokine; diagnostic assay; improved; innovation; meetings; next generation sequencing; novel; particle; real world application; research and development; screening; small molecule; success; thrombin aptamer; tool

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.

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，适体的主要优点包括适用于非免疫原性靶标、化学和热稳定性、生产成本低以及无批次间差异。然而，由于发现过程中的瓶颈，适配体的潜力尚未完全发挥：迄今为止，用于发现适配体的方法完全是基于选择的（即SELEX）。不幸的是，由于文库合成偏差、PCR 偏差和非特异性结合等多种干扰因素，这些方法常常无法在起始文库中发现最高性能的适体。此外，之前的研究表明，这些方法只能发现约30%的人类蛋白质组的DNA适配体，极大地限制了适配体的应用范围。以往发现过程中的创新都是SELEX的变种，主要集中在提高选择效率；因此，基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”），以克服适配体发现障碍。我们将文库中的每个适体以 105 个拷贝展示在适体颗粒 (AP) 上，这使我们能够使用 FACS 以超高通量的方式定量测量每个 AP 上适体的结合特性。因此，PD 能够精确分离通过定义的性能阈值的适体。此外，我们可以使用多色FACS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。该项目的假设是PD可以克服 传统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体。 在第一阶段，我们建议首先开发用于筛选高靶点亲和力的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筛选适配体对以实现夹心测定，筛选生物样本中的即用型适配体，并验证PD在不同靶标类型中的普遍适用性。在第二阶段结束时，我们预计将建立Particle Display以高通量方式开发高性能适配体的能力，从而满足治疗、诊断和生命科学研发领域的各种未满足的需求。

项目成果

Multiparameter Particle Display (MPPD): A Quantitative Screening Method for the Discovery of Highly Specific Aptamers.

多参数粒子显示 (MPPD)：一种用于发现高度特异性适体的定量筛选方法。

• DOI: 10.1002/anie.201608880
• 发表时间: 2017-01-16
• 期刊: Angewandte Chemie
• 作者: Wang J;Yu J;Yang Q;McDermott J;Scott A;Vukovich M;Lagrois R;Gong Q;Greenleaf W;Eisenstein M;Ferguson BS;Soh HT
• 通讯作者: Soh HT