Automated Microfluidic Selection of Aptamers against Carbohydrates

 描述（由申请人提供）：通过称为体外选择和扩增（SELEX）的进化过程，从大的寡核苷酸文库中分离出基于寡核苷酸的受体或适体。适体即使在非专业实验室中也容易适应不同的功能，例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。一旦被分离和表征，适体就成为真正的现成合成试剂，也就是说，它们容易定制和定制修饰，在实验构思的几天内可用。到目前为止，有大量的特异质的例子，适体结合到个别寡糖。此外，我们最近在优化偶联选择和反选择（消除与密切相关的靶标的结合）方案方面取得了重要突破，以分离针对具有挑战性的靶标（包括单个单糖）的特异性适体。这些进展共同表明，在正确的选择条件下，可以分离出与任意数量（三个、四个、五个或更多个）单体单元的任何糖基序结合的特异性和高亲和力适体。此外，基于我们在开发用于快速（一天内）SELEX的微流体装置方面的进展，我们认为，在大聚糖的背景下，对结构基序（表位）的非常特异性的结合剂的鉴定现在可以以全自动的注射和收集方式进行，易于分离，仅受合成和天然材料的可用性限制。该提案的重点是实施，优化和验证的微流控SELEX程序，用于分离基于阿托西肽的受体靶向任意聚糖基序。具体来说，在我们的第一个目标中，我们将采用市售的相关神经节苷脂组， 证明了由单独的亲和捕获和亲和消除步骤（“模块”）组成的优化选择方案可以导致与特定表位的特异性结合，无论是在整个聚糖中还是在较大基序的一部分中。在我们的第二个目标中，我们然后将在微流体装置中实施优化的程序，首先在用于优化程序的相同示例聚糖（神经节苷脂）上，然后在新的示例（例如，高甘露糖聚糖）。优化后的器械将具有耐用性和可靠性，并可缩短手术时间。个体选择和反选择程序，侧重于应该或不应该结合适体的特定聚糖基序，将很容易在这些设备中实施和编程。作为这项工作的结果，我们将准备通过集成和自动化的微流体SELEX快速（在一天内）分离聚糖结合适体，以及扩大针对大量聚糖的适体并行分离装置，最终使具有可重现特性的适体成为糖组学研究中常规和广泛可用的合成试剂。

 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.