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，包括500多种不同的化合物， 13个不同的结构多样化的家庭，并广泛滥用作为大麻的替代品。的影响 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诊断，包括自我报告或 大麻拟毒素组和阴性药物筛选两者的存在。因此，需要 可用于ER的血清中SCB/代谢物的准确筛选方法，以诊断和 将SCB过量与其他精神或神经系统疾病区分开来， 治疗方案和处置措施。已经针对某些SCB开发了免疫测定。这些 分析依赖于药物/代谢物和抗体之间的特异性结合，提供高特异性， 灵敏度然而，SCB的核心结构经常被引入微小的修改，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。与 目前市场上的SCBs多达数百种，新化合物不断涌现，开发SCBs已迫在眉睫 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， 原始的均相核酸酶辅助（NA）-SELEX结合并行和串行选择 提出了分离与所有SCB及其代谢物交叉反应的高亲和力DNA适体的策略 来自吲唑-3-甲酰胺家族。NA-SELEX利用结构化的DNA文库和高保真的 限制性内切酶，以有效地将靶结合的适体与文库的其余部分分离。具体地说， 靶向结合导致适体经历构象变化， 酶消化，而未结合的寡核苷酸是活的酶底物， 并被淘汰出下一轮选拔将执行计数器-SELEX以确保分离的 适体不与包括植物大麻素、结构相似的内源性 物质、处方药、违禁药物以及与ER访视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许多SCB的交叉反应性适体， 代谢物基于其共享的核心结构，使得外围化学修饰不应 有意义地影响适体的结合亲和力。基于此的电化学适体传感器 然后制造交叉反应性适体以检测所有亲本SCB的总血清浓度， 其代谢物，达到临床相关的检测限和长检测窗口。得到的传感器 可以在急诊室中使用，以帮助诊断SCB过量，大大改善公共卫生和安全。的 所提出的技术可以容易地适用于分离其他家族的高亲和力、交叉反应性适体 或用于体内治疗和诊断应用。