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

基本信息

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
项目类别：
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

项目摘要

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）是一类设计师药物，包括500多种不同的化合物 13个不同的结构多样化的家庭，被广泛滥用作为大麻的替代品。效果消费SCB的精神和生理影响甚至死亡。 SCB过量是目前在急诊室（ER）设置中的间接证据中被诊断出，包括自我报告或大麻毒品和阴性药物筛查的存在。因此，需要可以在ER中使用的血清中SCB/代谢物的精确筛查方法，以诊断和将SCB过量与其他精神病或神经系统疾病区分开来建立适当的疾病治疗方案和处置措施。已经为某些SCB开发了免疫测定。这些测定依赖于药物/代谢产物和抗体之间的特定结合，提供高特异性和灵敏度。但是，经常将较小的修改引入SCB核心结构以逃避调节。这些修饰会严重损害现有抗体的结合，从而产生错误的阴性结果。和因此，数百种SCB在市场上，新化合物不断出现，因此迫切需要开发可以识别结构相似的SCB的新的交叉反应性生物亲和力元素。为了解决这个问题，原始均质核酸酶辅助（Na）-Slex与并行选择结合提出了策略来隔离与所有SCB及其代谢物反应的高亲和力DNA适体来自Idazole-3-羧酰胺家族。 NA-SELEX利用结构化的DNA库和高保真性限制酶有效地将目标结合的适应性与库的其余部分分开。具体来说，目标结合会导致适体经历构象变化，使它们无法访问酶消化，而未结合的寡核苷酸是可行的酶底物，并且被迅速消化并从下一轮选择中淘汰。将执行反塞氏杆菌，以确保隔离适体不与包括植物大麻素（结构相似的内源性）在内的干扰分子结合物质，处方药，非法药物以及与ER访问相关的结构性降低药物。预计该过程将产生第一个交叉反应的适体，能够识别大量的SCB和代谢物基于它们的共享核心结构，因此外周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，将制造交叉反应的适体，以感知所有母体SCB的总血清浓度和他们的代谢产物达到了临床相关的检测极限和长期检测窗口。产生的传感器可以在ER中用于帮助诊断SCB过量的诊断，从而大大改善了公共卫生和安全性。这拟议的技术很容易适应其他家庭的高亲和力，交叉反应的适体新型的精神活性物质或体内治疗和诊断应用。