Microfluidic Selection of Aptamers for Biological Purification Applications

描述(申请人提供)：用于生物纯化应用的微流控选择适配子首席研究员：乔琳，哥伦比亚大学的适配子是与小分子或蛋白质结合的寡核苷酸。适配子的分离是通过一种称为指数富集型配体系统进化(SELEX)的体外选择和扩增过程来实现的。这个过程是基于亲和力选择，然后从大型随机文库中扩增作为“粘合剂”的核酸。适配子可以为极广泛的分析物开发，具有高亲和力，可以具有良好的靶标选择性，并且可以合成来结合具有预定特征的靶标。特别是，适配子结合通常表现出强烈的温度依赖性；因此，适配子可以在预定义的温度下特异性地结合目标分析物，并在略有不同但也是预定义的温度下可逆地与靶解偶。这一特性对亲和纯化等生物医学应用很有吸引力，因为它可以通过热激活释放和分析物的等度洗脱来实现特定的纯化。传统的SELEX仪器在适体方面取得了很大的进步，但它们的使用通常是劳动密集型和耗时的。这些限制可以通过利用微流控技术来解决。我们建议进行微流控SELEX系统的概念验证演示，该系统集成了SELEX方法的所有步骤，以允许自动开发具有预定义的温度依赖结合特性的适配子，用于亲和纯化分析物。我们的具体目标包括：(1)在微通道中开发基于微珠的聚合酶链式反应(PCR)技术，以确定其对微流控SELEX的适用性；(2)整合微芯片DNA选择和扩增，创建微流控SELEX系统的原型；以及(3)通过使用它来选择针对成熟蛋白质的适配子，对原型系统进行表征和验证。这项拟议的研究与公共卫生有关，因为它将为亲和纯化蛋白质、小分子和参与疾病生物医学研究的细胞等分析物提供一个选择适配子的平台。此外，该系统还可用于开发适体，应用于靶向验证、药物发现、诊断和治疗。 公共卫生相关性(由申请人提供)：用于生物净化应用的微流体适体选择首席研究员：乔琳，哥伦比亚大学这项研究将对微流体系统进行概念验证演示，该系统集成了通过指数富集法系统进化配体(SELEX)的所有步骤。该系统将允许自动开发具有预定义的温度依赖结合特性的适配子，用于分析物纯化应用。它与公众健康相关，因为它将为亲和纯化蛋白质、小分子和参与疾病生物医学研究的细胞等分析物提供一个选择适配子的平台。此外，该系统还可用于开发适体，应用于靶向验证、药物发现、诊断和治疗。

DESCRIPTION (provided by applicant): Microfluidic Selection of Aptamers for Biological Purification Applications Principal Investigator: Qiao Lin, Columbia University Aptamers are oligonucleotides that bind to small molecules or proteins. Aptamers are isolated through an in vitro selection and amplification procedure called systematic evolution of ligands by exponential enrichment (SELEX). The procedure is based on an affinity selection, followed by amplification of nucleic acids that are "binders" from large random libraries. Aptamers can be developed for an extremely broad spectrum of analytes with high affinity, can possess well controlled target selectivity, and can be synthesized to bind targets with predefined characteristics. In particular, aptamer binding in general exhibits strong temperature dependence; thus, aptamers may specifically bind target analytes at a predefined temperature and reversibly decouple from the targets at a modestly different, yet also predefined, temperature. This property is attractive to biomedical applications such as affinity purification, as it can enable specific purification with thermally activated release and isocratic elution of analytes. Conventional SELEX instruments have enabled great progress in aptamers, but their use is generally labor-intensive and time-consuming. These limitations can be addressed by leveraging microfluidic technology. We propose to pursue proof-of-concept demonstration of a microfluidic SELEX system that integrates all steps of the SELEX method to allow automated development of aptamers with predefined temperature-dependent binding characteristics for applications to affinity purification of analytes. Our specific aims include: (1) developing a bead-based polymerase chain reaction (PCR) technique in a microchannel to establish its applicability to microfluidic SELEX; (2) integrating microchip DNA selection and amplification to create a prototype microfluidic SELEX system; and (3) characterize and validate the prototype system by using it to select aptamers against well established proteins. The proposed research is relevant to public health because it will provide a platform for selecting aptamers for affinity purification of analytes such as proteins, small molecules and cells involved in biomedical research on diseases. In addition, this system could also be used to develop aptamers for applications to target validation, drug discovery, diagnostics, and therapy. PUBLIC HEALTH RELEVANCE (provided by applicant): Microfluidic Selection of Aptamers for Biological Purification Applications Principal Investigator: Qiao Lin, Columbia University This research will pursue proof-of-concept demonstration of a microfluidic system that integrates all steps of the method of systematic evolution of ligands by exponential enrichment (SELEX). The system will allow automated development of aptamers with predefined temperature-dependent binding characteristics for analyte purification applications. It is relevant to public health because it will provide a platform for selecting aptamers for affinity purification of analytes such as proteins, small molecules and cells involved in biomedical research on diseases. In addition, this system could also be used to develop aptamers for applications to target validation, drug discovery, diagnostics, and therapy.