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

linaptamer，或与化学和生物靶标高亲和力结合的寡核苷酸，通过体外选择和扩增过程被称为配体的系统进化指数富集（SELEX）分离出来。核酸适配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，具有良好控制的靶标选择性和预定义的结合特性。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。这种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温洗脱来实现特定的纯化，以及纯化仪器的再生。此外，热活化等压洗脱使实验程序免于不必要和潜在有害的试剂，并简化了纯化程序。传统的SELEX仪器被广泛使用，但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。我们建议开发一种微流体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)整合微芯片DNA选择和扩增过程，构建微流控SELEX系统原型；(3)验证原型系统，并展示其在选择针对癌症检测和治疗中涉及的蛋白质的适体体方面的实用性，并且已建立的适体体可用。拟议中的研究将对医疗保健和生物技术产生更广泛的影响。应用于治疗学，适体可以调整目标和抑制特定的蛋白质是疾病的原因。鉴于其选择性，可以设计药物递送机制，将特定疾病引发蛋白的适体分子释放到系统中以定位和抑制蛋白质。另外，适体也可用于筛选潜在的候选药物。特定于预期药物的适体可用于固定分子以进行广泛的表征。此外，该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。核酸适配体可用于污染空气、水和食物供应的毒素、病原体和寄生虫。此外，适体在细胞操作中也很有用。细胞分离与热敏适配体提供了有吸引力的替代目前的技术在细胞计数。特别是，细胞，如间充质干细胞，可以受益于特定的提取，富集和等温洗脱使用适配体。最后，本研究的一个强大的教育组成部分将涉及本科和研究生水平的教学，研究生和本科生的跨学科培训，包括来自少数民族和代表性不足群体的学生，以及纽约市积极的教育推广活动。

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.