Automated Microfluidic Selection of Aptamers against Carbohydrates

针对碳水化合物的适配体的自动微流体选择

• 批准号: 8985077
• 负责人: Milan N Stojanovic
• 金额: $ 30.62万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-21 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8985077
• 关键词: Address; Affinity; Affinity Chromatography; Binding; Biological Assay; Biosensor; Carbohydrates; Ceramides; Complex; Conceptions; Coupled; Custom; Development; Devices; Engineering; Epitopes; Flow Cytometry; Fructose; Ganglioside GM1; Gangliosides; Head; Image; Imaging Techniques; In Vitro; Individual; Knowledge; Laboratories; Lead; Libraries; Ligands; Mannose; Microfluidic Microchips; Microfluidics; Molecular; Monosaccharides; Oligonucleotides; Oligosaccharides; Peptides; Phase; Polysaccharides; Positioning Attribute; Procedures; Process; Property; Proteins; Protocols documentation; Reagent; Reporting; Research; Solid; Solutions; Source; Specialist; Specificity; Surface; Testing; Time; Validation; Work; aptamer; base; flexibility; glucose receptor; in vivo; in vivo imaging; inhibitor/antagonist; novel; operation; programs; public health relevance; receptor; research study; scale up; sugar; tool

 DESCRIPTION (provided by applicant): Oligonucleotide-based receptors or aptamers are isolated from large libraries of oligonucleotides through an evolutionary process known as an in vitro selection and amplification (SELEX). Aptamers are readily adapted even in non-specialist laboratories for different functions, such as in biosensor and imaging applications, for affinity purifications, or as antagonists and in vivo targeting moieties. Once isolated and characterized, aptamers become true off-the-shelf synthetic reagents, that is, they are readily custom-made and custom- modified, being available within days from the conception of the experiment. There is by now a substantial number of idiosyncratic examples of aptamers binding to individual oligosaccharides. Also, we recently made important breakthroughs in optimization of coupled selection and counterselection (elimination of binding to closely related targets) protocols in order to isolate specific aptamers against challenging targets, including individual monosaccharides. These advances together argue that it is possible with the right selection conditions to isolate specific and high-affinity aptamers binding to any saccharide motif with an arbitrary number (three-, four-, five-, or more) of monomeric units. Furthermore, based on our progress in the development of microfluidic devices for rapid (within one day) SELEX we posit that identification of very specific binders to structural motifs (epitopes) in the context of largr glycans can be now performed in a fully automated, inject-and-collect manner, with ease of isolation limited only by the availability of synthetic and natural materials for selections. This proposal is focused on the implementation, optimization, and validation of microfluidic SELEX procedures for isolation of oligonucleotide-based receptors targeting arbitrary glycan motifs. Specifically, in our first aim we will take commercially available sets of related gangliosides and demonstrate that optimized selection protocols, consisting of individual affinity-capture and affinity-elimination steps ("modules"), can lead to specific binding to a particular epitope, eithe as in a whole glycan or as in a part of a larger motif. In our second aim we will then implement the optimized procedures in microfluidic devices, first on the same example glycans that were used to optimize procedures (gangliosides), and then validating our devices on new examples (e.g., on high-mannose glycans). The optimized device will be robust and reliable as well as allow for shortened procedure times. Individual selection and counterselection procedures, focusing on particular glycan motifs that should or should not bind to aptamers, will be readily implemented and programmed in these devices. As a result of this work, we will be ready to rapidly (within one day) isolate glycan-binding aptamers via integrated and automated microfluidic SELEX, as well as scale up the devices for parallelized isolation of aptamers against a large number of glycans, ultimately allowing aptamers with reproducible properties to become routinely and broadly available synthetic reagents in glycomics research.

 描述(申请人提供)：基于寡核苷酸的受体或适配子是通过一种称为体外选择和扩增(SELEX)的进化过程从大量寡核苷酸文库中分离出来的。即使在非专业实验室，适配子也很容易适应不同的功能，如在生物传感器和成像应用中，用于亲和纯化，或作为拮抗剂和体内靶向部分。一旦被分离和鉴定，适配子就成为真正的现成合成试剂，也就是说，它们很容易定制和定制修改，从实验构思开始几天内就可以买到。到目前为止，已经有相当数量的核酸适体与单个寡糖结合的特殊例子。此外，我们最近在耦合选择和反选择(消除与密切相关靶标的结合)方案的优化方面取得了重要突破，以分离针对具有挑战性的靶标的特定适配子，包括单个单糖。这些进展共同证明，在正确的选择条件下，有可能分离出与任何具有任意数量(三个、四个、五个或更多)单体单元的糖基序结合的特定和高亲和力适配子。此外，根据我们在开发用于快速(一天内)SELEX的微流控设备方面的进展，我们假设，在较大的多糖背景下识别非常特定的结构基序(表位)现在可以以全自动、注射和收集的方式进行，分离的简易性仅受可供选择的合成和天然材料的限制。这项建议侧重于微流控SELEX程序的实施、优化和验证，以分离以寡核苷酸为基础的针对任意糖链基序的受体。具体地说，在我们的第一个目标中，我们将获得商业上可用的相关神经节苷脂和 证明由单个亲和力捕获步骤和亲和力消除步骤(“模块”)组成的优化选择方案可以导致与特定表位的特定结合，即整个糖链或更大基序的一部分。在我们的第二个目标中，我们将在微流控设备中实施优化程序，首先在用于优化程序的同一个例子中的葡聚糖(神经节苷脂)上，然后在新的例子上验证我们的设备(例如，在高甘露糖的葡聚糖上)。优化的设备将是坚固和可靠的，并允许缩短手术时间。个体选择和反选择程序，侧重于应该或不应该与适配子结合的特定糖链基序，将很容易在这些设备中实施和编程。作为这项工作的结果，我们将准备好通过集成和自动化的微流控SELEX快速(在一天内)分离与糖结合的适配子，并扩大针对大量糖的适配子并行分离的设备，最终使具有可重复性的适配子成为糖组学研究中常规和广泛可用的合成试剂。