Homogeneous Nuclease-Assisted SELEX for Rapid Isolation of Cross-Reactive, Functionalized Aptamers for Synthetic Cannabinoids

均质核酸酶辅助 SELEX 用于快速分离合成大麻素的交叉反应功能化适体

• 批准号: 10392763
• 负责人: Yi Xiao
• 金额: $ 11.36万
• 依托单位: NORTH CAROLINA STATE UNIVERSITY RALEIGH
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10392763
• 关键词: Homogeneous; Nuclease; Assisted; SELEX; Rapid

Synthetic cannabinoids (SCBs) are a class of designer drugs comprising more than 500 distinct compounds in thirteen different structurally diverse families, and are widely abused as an alternative to cannabis. The effects of consuming SCBs include adverse psychological and physiological effects and even death. SCB overdose is currently diagnosed in emergency room (ER) settings from indirect evidence, including self-reporting or the presence of both a cannabimimetic toxidrome and a negative drug screen. Therefore, there is a need for accurate screening methods for SCBs/metabolites in serum that can be employed in the ER, to diagnose and distinguish SCB overdoses from other psychiatric or neurological diseases for establishing appropriate treatment protocols and disposition measures. Immunoassays have been developed for certain SCBs. These assays rely on specific binding between drugs/metabolites and antibodies, offering high specificity and sensitivity. However, minor modifications are often introduced to the SCB core structure to evade regulation. These modifications can greatly impair binding by existing antibodies, resulting in false-negative results. With hundreds of SCBs on the market and new compounds continuously emerging, it is therefore urgent to develop new cross-reactive bioaffinity elements that can recognize structurally similar SCBs. To address this problem, an original homogeneous nuclease-assisted (NA)-SELEX in conjunction with a parallel-and-serial selection strategy is proposed to isolate a high-affinity DNA aptamer that cross reacts to all SCBs and their metabolites from the indazole-3-carboxamide family. NA-SELEX utilizes a structured DNA library and a high-fidelity restriction enzyme to efficiently separate target-bound aptamers from the remainder of the library. Specifically, target binding causes aptamers to undergo a conformational change that renders them inaccessible to enzymatic digestion, whereas unbound oligonucleotides are viable enzyme substrates and are rapidly digested and eliminated from the next round of selection. Counter-SELEX will be performed to ensure that the isolated aptamer does not bind to interferent molecules including phytocannabinoids, structurally-similar endogenous substances, prescription drugs, illicit drugs as well as structurally-dissimilar drugs associated with ER visits. This process is expected to yield the first cross-reactive aptamer capable of recognizing numerous SCBs and metabolites based on their shared core structure, such that peripheral chemical modifications should not meaningfully affect the aptamer's binding affinity. An electrochemical aptamer-based sensor based on this cross-reactive aptamer will then be fabricated to sense the total serum concentration of all parent SCBs and their metabolites, achieving a clinically relevant detection limit and long detection window. The resulting sensor can be used in the ER to aid in the diagnosis of SCB overdose, greatly improving public health and safety. The proposed technologies can easily be adapted to isolate high-affinity, cross-reactive aptamers for other families of novel psychoactive substances or for in vivo therapeutic and diagnostic applications.

合成大麻素 (SCB) 是一类设计药物，包含 500 多种不同的化合物 十三个不同的结构多样化的家庭，并被广泛滥用作为大麻的替代品。效果 食用 SCB 的后果包括不良的心理和生理影响，甚至死亡。 SCB 过量是 目前在急诊室 (ER) 环境中通过间接证据进行诊断，包括自我报告或 大麻模拟毒理学和阴性药物筛选的存在。因此，需要 血清中 SCB/代谢物的准确筛查方法可用于 ER、诊断和 将 SCB 过量与其他精神或神经系统疾病区分开来，以建立适当的治疗方法 治疗方案和处置措施。已经针对某些 SCB 开发了免疫测定法。这些 检测依赖于药物/代谢物和抗体之间的特异性结合，提供高特异性和 敏感性。然而，SCB 核心结构经常会进行细微修改以逃避监管。 这些修饰会极大地削弱现有抗体的结合，导致假阴性结果。和 市场上有数百种SCB，新化合物不断涌现，因此迫切需要开发 新的交叉反应生物亲和元件可以识别结构相似的 SCB。为了解决这个问题， 原始同质核酸酶辅助 (NA)-SELEX 与并行和串行选择相结合 提出了分离高亲和力 DNA 适体的策略，该适体与所有 SCB 及其代谢物发生交叉反应 来自吲唑-3-甲酰胺家族。 NA-SELEX 利用结构化 DNA 库和高保真度 限制性内切酶可有效地将靶标结合的适体与文库的其余部分分离。具体来说， 目标结合导致适体发生构象变化，使其无法被 酶消化，而未结合的寡核苷酸是可行的酶底物并且被快速消化 并在下一轮选拔中被淘汰。将执行 Counter-SELEX 以确保隔离 适体不与干扰分子结合，包括植物大麻素、结构相似的内源性分子 药物、处方药、非法药物以及与急诊室就诊相关的结构不同的药物。 该过程预计将产生第一个能够识别大量 SCB 的交叉反应适体 基于其共享核心结构的代谢物，因此外围化学修饰不应 有意义地影响适体的结合亲和力。基于此的电化学适配体传感器 然后将制造交叉反应适体来感测所有亲本 SCB 的总血清浓度，并 它们的代谢物，实现了临床相关的检测限和长检测窗。由此产生的传感器 可在急诊室用于辅助诊断 SCB 过量，从而大大改善公众健康和安全。这 所提出的技术可以很容易地用于分离其他家族的高亲和力、交叉反应适体 新型精神活性物质或用于体内治疗和诊断应用。