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: 9893841
• 负责人: Yi Xiao
• 金额: $ 5.99万
• 依托单位: FLORIDA INTERNATIONAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-15 至 2021-02-12
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9893841
• 关键词: Accident and Emergency department; Address; Adopted; Affect; Affinity; Antibodies; Attenuated; Binding; Biological Assay; Blood Volume; Cannabis; Cessation of life; Chemicals; Consumption; Custom; DNA; DNA Library; DNA Structure; Designer Drugs; Detection; Development; Diagnosis; Diagnostic; Digestion; Drug Prescriptions; Drug Screening; Elements; Emergency department visit; Engineering; Ensure; Environmental Monitoring; Event; Evolution; Exhibits; Family; Generations; Goals; Hospitals; Illicit Drugs; Immobilization; Immunoassay; Impairment; In Vitro; Indazoles; Law Enforcement; Libraries; Ligands; Measurement; Measures; Medical; Mental disorders; Methods; Minor; Modification; Molecular Conformation; National Institute of Drug Abuse; Nature; Nucleic Acids; Oligonucleotides; Overdose; Parents; Patient Self-Report; Performance; Peripheral; Pharmaceutical Preparations; Phase; Physiological; Process; Public Health; Reagent; Regulation; Safety; Sensitivity and Specificity; Serum; Specificity; Structure; Techniques; Technology; Testing; Therapeutic; Toxic effect; Treatment Protocols; Urine; Visit; accurate diagnosis; aptamer; base; cannabimimetics; clinically relevant; cost; cross reactivity; drug of abuse; enzyme substrate; improved; in vivo; innovation; interest; milliliter; nanomolar; nervous system disorder; novel; nuclease; phytocannabinoid; point of care; psychologic; restriction enzyme; screening; sensor; small molecule; success; synthetic cannabinoid

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)是一类特殊的药物，由500多种不同的化合物组成 13个结构多样的不同家庭，被广泛滥用，作为大麻的替代品。其影响 食用短链苯系物的风险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)环境中诊断，来自间接证据，包括自我报告或 同时存在类大麻毒素和阴性药物筛查。因此，有必要 血清中SCBS/代谢物的准确筛选方法可用于ER、诊断和 将SCB过量与其他精神或神经疾病区分开来，以建立适当的 处理方案和处置措施。已开发出针对某些SCB的免疫分析方法。这些 检测依赖于药物/代谢物和抗体之间的特异性结合，提供了高度的特异性和 敏感度。然而，渣打银行的核心结构经常被引入微小的修改，以逃避监管。 这些修饰会极大地损害现有抗体的结合，导致假阴性结果。使用 目前市场上的超临界胶体化合物数以百计，新的化合物不断涌现，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， 一种原始的均相核酸酶辅助(NA)-SELEX与并行和串联选择相结合 提出了分离与所有SCB及其代谢物交叉反应的高亲和力dna适配子的策略。 来自吲唑-3-甲酰胺家族。NA-SELEX利用结构化DNA文库和高保真 限制酶有效地将靶结合适配子从文库的其余部分分离出来。具体来说， 靶结合会导致适配子发生构象变化，使其无法与 酶消化，而未结合的寡核苷酸是活的酶底物，可以快速消化 并在下一轮选拔中被淘汰。将执行反SELEX以确保隔离的 适配子不与干扰分子结合，包括结构相似的内源性植物大麻素 与急诊室就诊有关的物质、处方药、非法药物以及结构不同的药物。 这一过程有望产生第一个能够识别大量SCB的交叉反应适配子和 代谢物基于其共享的核心结构，因此外围化学修饰不应 有意义地影响适配子的结合亲和力。一种基于此的电化学适配子传感器 然后将制造交叉反应适配子来检测所有亲本SCB的总血清浓度和 它们的代谢物，实现了临床相关的检测下限和长的检测窗口。由此产生的传感器 可用于急诊室辅助诊断SCB过量，极大地提高了公众健康和安全。这个 建议的技术可以很容易地用于分离其他家族的高亲和力、交叉反应的适配子 新的精神活性物质或用于体内治疗和诊断应用。