Microfluidic Selection of Aptamers for Biological Purification Applications

用于生物纯化应用的适体的微流体选择

• 批准号: 7762499
• 负责人: Qiao Lin
• 金额: $ 19.6万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7762499
• 关键词: Address; Affinity; Affinity Chromatography; Binding; Biological; Biomedical Research; Cells; Characteristics; Complex Mixtures; DNA; Dependence; Development; Diagnostic; Disease; Engineering; Equilibrium; Evolution; Exhibits; In Vitro; Interdisciplinary Study; Kinetics; Libraries; Ligands; Mechanics; Medicine; Methods; Microfluidics; Modeling; Nucleic Acids; Oligonucleotides; Polymerase Chain Reaction; Principal Investigator; Procedures; Property; Proteins; Public Health; Research; Stimulus; System; Techniques; Technology; Temperature; Thermodynamics; Time; Universities; Validation; Work; aptamer; base; drug discovery; instrument; microchip; nucleic acid structure; prototype; public health relevance; small molecule

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.

描述（由申请人提供）：用于生物纯化应用适体的微流体选择主要研究者：哥伦比亚大学适体Qiao Lin是与小分子或蛋白质结合的寡核苷酸。通过体外选择和放大程序分离适体，称为配体的系统演变（SELEX）。该过程基于亲和力选择，然后放大来自大型随机文库的“粘合剂”的核酸。可以为具有高亲和力的非常广泛的分析物开发适体，可以具有良好的目标选择性，并且可以合成以结合具有预定义特征的目标。特别是，通常在一般中的适体结合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，并在适度不同但也预定义的温度下与目标脱离。该特性对生物医学应用（例如亲和力纯化）有吸引力，因为它可以通过热激活的释放和分析物的等值释放来实现特定的纯化。传统的SELEX仪器在适体方面取得了巨大进展，但它们的使用通常是劳动力密集的且耗时的。这些限制可以通过利用微流体技术来解决。我们建议追求概念验证证明微流体SELEX系统，该系统集成了SELEX方法的所有步骤，以允许具有预定义的温度依赖性结合特性的适体的自动化开发，以应用于分析物的亲和力纯化。我们的具体目的包括：（1）在微通道中开发基于珠的聚合酶链反应（PCR）技术，以确定其对微流体SELEX的适用性； （2）整合微芯片DNA选择和放大以创建原型微流体SELEX系统； （3）通过使用该原型系统来选择适合蛋白的适体，从而验证和验证原型系统。拟议的研究与公共卫生有关，因为它将提供一个平台，以选择适体，以纯化分析物，例如蛋白质，小分子和参与疾病生物医学研究的细胞。此外，该系统还可以用于开发适合用于靶向验证，药物发现，诊断和治疗的适当人。 公共卫生相关性（由申请人提供）：用于生物纯化应用适用应用的微流体选择主要研究者：哥伦比亚大学Qiao Lin这项研究将遵循概念概念的证明，证明了微流体系统，该系统整合了由指数富集（Electectement Emarthiphment（Seleex）的指数富集的所有步骤）。该系统将允许具有预定义温度依赖性结合特性的适体的自动开发，以用于分析物纯化应用。它与公共卫生有关，因为它将提供一个平台，用于选择适体，以纯化分析物，例如蛋白质，小分子和参与疾病生物医学研究的细胞。此外，该系统还可以用于开发适合用于靶向验证，药物发现，诊断和治疗的适当人。