Microfluidic Selection of Aptamers for Biological Purification Applications

主要研究者：乔林，哥伦比亚大学适体体是结合小分子或蛋白质的寡核苷酸。适体的分离是通过体外选择和扩增过程称为配体的系统进化指数富集（SELEX）。该程序基于亲和选择，然后扩增核酸，这些核酸是来自大型随机文库的“结合物”。核酸适配体可用于极宽谱的分析物，具有高亲和力，具有良好的目标选择性控制，并且可以合成结合具有预定义特征的目标。特别是适体结合一般表现出强烈的温度依赖性；因此，适体可以在预定义的温度下特异性地结合目标分析物，并在适度不同的温度下与目标物可逆地解耦，但也是预定义的温度。这一特性对于诸如亲和纯化之类的生物医学应用具有吸引力，因为它可以通过热激活释放和等温洗脱来实现特定的纯化。传统的SELEX仪器在适配体方面取得了很大的进展，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体技术来解决。我们建议进行微流体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温度依赖结合特性的适配体，用于分析物的亲和纯化。我们的具体目标包括：(1)在微通道中开发基于微球的聚合酶链反应（PCR）技术，以确定其对微流体SELEX的适用性；(2)集成微芯片DNA选择与扩增，构建微流控SELEX系统原型；(3)通过对已建立的蛋白质选择适体来表征和验证原型系统。这项拟议的研究与公共卫生有关，因为它将为选择适合体用于分析物（如蛋白质、小分子和涉及疾病生物医学研究的细胞）的亲和纯化提供一个平台。此外，该系统还可用于开发用于靶标验证、药物发现、诊断和治疗的适体。

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.