Integrated Selection of Thermally Responsive Aptamers for Specific Purification and Enrichment of Biomolecules

用于生物分子特异性纯化和富集的热响应适体的综合选择

• 批准号: 0854030
• 负责人: Qiao Lin
• 金额: $ 30万
• 依托单位: Columbia University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0854030&HistoricalAwards=false
• 关键词: Integrated; Selection; Thermally; Responsive; Aptamers

0854030LinAptamers, or oligonucleotides that bind with high affinity to chemical and biological targets, are isolated through an in vitro selection and amplification procedure called systematic evolution of ligands by exponential enrichment (SELEX). Aptamers can be developed for an extremely broad spectrum of targets, such as small molecules, proteins, cells, viruses, and bacteria, with well controlled target selectivity and predefined binding 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 affinity purification, as it can enable specific purification with thermally activated release and isocratic elution of analytes, as well as regeneration of the purification instrument. Additionally, thermally activated isocratic elution frees experimental procedures from unnecessary and potentially harmful reagents, and simplifies the purification procedure. Conventional SELEX instruments are widely used but are generally labor-intensive and time-consuming. These limitations can be addressed by leveraging microfluidic technology. We propose to develop 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 processes to create a prototype microfluidic SELEX system; and (3) validating the prototype system and demonstrating its utility with selection of aptamers targeting proteins which are involved in cancer detection and therapy, and for which established aptamers are available.The proposed research will have broader impacts in healthcare and biotechnology. Applied to therapeutics, aptamers can be tuned to target and inhibit particular proteins which are the cause of diseases. Given their selectivity, drug delivery mechanisms can be devised where aptamer molecules for a specific disease initiated protein are released into the system for location and suppression of the protein. Alternatively, aptamers can also be used to screen potential drug candidates. Aptamers specific to the prospective drug can be used to immobilize the molecule to undergo extensive characterization. Moreover, the proposed research has potential in diagnosis and biosensing for environmental and food monitoring, antiterrorism, and pathology. Aptamers can be selected for toxins, pathogens and parasites which can contaminate air, water and food supplies. Also, aptamers are of utility in cell manipulation. Cell separation with thermally sensitive aptamers offers attractive alternatives to current techniques in cell counting. In particular, cells, such as mesenchymal stem cells, can benefit from specific extraction, enrichment and isocratic elution using aptamers. Finally, a strong educational component in this research will involve undergraduate- and graduate-level teaching, interdisciplinary training of graduate and undergraduate students including those from minorities and underrepresented groups, and active educational outreach activities in New York City.

0854030LinApmer，即与化学和生物靶标具有高亲和力的寡核苷酸，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分离。适体可以用于非常广泛的靶标，如小分子、蛋白质、细胞、病毒和细菌，具有良好的靶标选择性和预定义的结合特性。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实现特定的纯化，以及纯化仪器的再生。此外，热激活等度洗脱使实验过程从不必要的和潜在的有害试剂中解放出来，并简化了纯化程序。传统的SELEX仪器被广泛使用，但通常都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。我们建议开发一种微流控SELEX系统，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选择和扩增过程，创建微流控SELEX系统的原型；以及(3)验证原型系统，并通过选择与癌症检测和治疗相关的蛋白质和已建立的适配子来验证其有效性。将适体应用于治疗学，可以调整适配子来靶向和抑制导致疾病的特定蛋白质。考虑到它们的选择性，可以设计出药物传递机制，其中特定疾病启动蛋白的适体分子被释放到系统中，以定位和抑制该蛋白。或者，适配子也可以用来筛选潜在的候选药物。特定于预期药物的适配子可用于固定化分子以进行广泛的表征。此外，拟议的研究在环境和食品监测、反恐和病理学的诊断和生物传感方面具有潜力。可以选择适配子来检测毒素、病原体和寄生虫，这些物质可以污染空气、水和食物供应。此外，适配子在细胞操作中也是有用的。用热敏适配子分离细胞提供了当前细胞计数技术的有吸引力的替代方案。特别是，细胞，如间充质干细胞，可以从特定的提取、浓缩和使用适配子的等度洗脱中受益。最后，这项研究的一个重要教育部分将涉及本科生和研究生水平的教学，对研究生和本科生，包括少数群体和代表性不足群体的跨学科培训，以及在纽约市积极的教育外联活动。