STTR Phase I: Cooperative Selection of Aptamer Pairs by Unlocking Parallel Enrichment Paths

这个小企业技术转让（STTR）第一阶段项目的更广泛的影响/商业潜力将是开发生产适体对的可靠技术。 适体是新兴市场的核心试剂，被视为治疗、诊断和成像中单克隆抗体的替代品。它们具有几个吸引人的品质：易于体外合成，灵活的修饰，广泛的靶向范围，可重复使用性和高热/化学稳定性。到2019年，市场价值预计将达到54亿美元，复合年增长率为73.5%。适体在实际应用中的一个突出的负面方面是缺乏适体对。单克隆抗体的配对由于其定制的靶结合位点而相当容易实现。相比之下，适体是在没有这种知识的情况下从随机池中富集的，因此不可能用目前可用的方法来分配池中单个适体的结合位点。所提出的开发适体的创新允许直接从随机池中筛选适体对。这种从未经表征的池中直接选择的类型与拟议的简单工作流程是在适体行业产生高收入潜力的市场驱动力。该STTR第一阶段项目提议进一步开发和验证从未表征的适体池中筛选适体对的方法。目前，可用的工具涉及具有多个选择后步骤的大规模筛选，这些步骤严重依赖于仪器，例如下一代测序、自动化克隆工具和基于珠的多重结合测定。这些“蛮力”策略最终可能会产生适体对，但它们耗时、昂贵且效率低下，这也许可以解释为什么今天很少有适体对存在。所提出的适体对选择方法通过在针对自由溶液靶标的选择过程中仅允许对存活来解决该行业中未满足的需求。建议的选择平台的同质性使这个工具是可扩展的和可复用的。这项技术已经在初步的原理验证实验中付诸实践。在NSF STTR第一阶段项目中，目标是通过应用/改进工具来测试创新的普遍性，以找到针对各种蛋白质靶标的更多DNA/RNA适体对。

The broader impact/commercial potential of this Small Business Technology Transfer (STTR) Phase I project will be to develop reliable technology for the production of aptamer pairs. Aptamers are core reagents in emerging markets, regarded as alternatives to monoclonal antibodies in therapeutics, diagnostics, and imaging. They possess several appealing qualities: Ease of in vitro synthesis, flexible modification, broad target ranges, reusability, and high thermal/chemical stability. The market value is expected to reach $5.4 billion by 2019 with a compound annual growth rate of 73.5%. One prominent negative aspect of aptamers in practical use is the lack of aptamer pairs. Pairing of monoclonal antibodies is fairly easily accomplished due to their tailor-made target binding sites. In contrast, aptamers are enriched from a random pool without such knowledge, so that it is impossible to assign the binding site of individual aptamers in pools with currently available methodologies. The proposed innovation in developing aptamers permits screening of pairs of aptamers directly from random pools. This type of direct selection from uncharacterized pools with the proposed simple workflow is a market driver to generate high revenue potential in the aptamers industry.This STTR Phase I project proposes to further develop and validate methodology for screening aptamer pairs from uncharacterized aptamer pools. Currently, available tools involve massive screening with multiple post-selection steps that heavily depend on instrumentation such as next generation sequencing, automated cloning tools, and bead-based multiplex binding assays. These "brute-force" strategies may eventually produce aptamer pairs, but they are time-consuming, expensive, and inefficient, perhaps explaining why few aptamer pairs exist today. The proposed aptamer pair selection methodology addresses this unmet need in the industry by allowing only pairs to survive during the selection process against free-solution targets. The homogeneous nature of the proposed selection platform enables this tool to be scalable and multiplexable. This technology has been reduced to practice in preliminary proof-of-principle experiments. During this NSF STTR Phase I project, the objective is to test the generalizability of the innovation by applying/improving the tool to find more DNA/RNA aptamer pairs against variety of protein targets.