Interdepartmental Toxicology Program, ‡Department of Chemistry, §Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University , Ames, Iowa 50011, United States.
Anal Chem. 2016 Aug 2;88(15):7715-23. doi: 10.1021/acs.analchem.6b01633. Epub 2016 Jul 11.
The cocaine aptamer has been seen as a good candidate for development as a probe for cocaine in many contexts. Here, we demonstrate that the aptamer binds cocaine, norcocaine, and cocaethylene with similar affinities and aminoglycosides with similar or higher affinities in a mutually exclusive manner with cocaine. Analysis of its affinities for a series of cocaine derivatives shows that the aptamer specificity is the consequence of its interaction with all faces of the cocaine molecule. Circular dichroism spectroscopy and 2-aminopurine (2AP) fluorescence studies show no evidence of large structural rearrangement of the cocaine aptamer upon ligand binding, which is contrary to the general view of this aptamer. The aptamer's affinity for cocaine and neomycin-B decreases with the inclusion of physiological NaCl. The substitution of 2AP for A in position 6 (2AP6) of the aptamer sequence eliminated the effect of NaCl on its affinities for cocaine and analogues, but not for neomycin-B, showing a selective effect of 2AP substitution on cocaine binding. The affinity for cocaine also decreased with increasing concentrations of serum or urine, with the 2AP6 substitution blunting the effect of urine. Its low affinities for cocaine and metabolites and its ability to bind irrelevant compounds limit the opportunities for application of this aptamer in its current form as a selective and reliable sensor for cocaine. However, these studies also show that a small structural adjustment to the aptamer (2AP exchanged for adenine) can increase its specificity for cocaine in physiological NaCl relative to an off-target ligand.
可卡因适体已被视为在许多情况下开发可卡因探针的良好候选物。在这里，我们证明该适体以相互排斥的方式与可卡因、去甲可卡因和可卡乙基结合具有相似的亲和力，与氨基糖苷结合具有相似或更高的亲和力。对一系列可卡因衍生物的亲和力分析表明，适体的特异性是其与可卡因分子所有面相互作用的结果。圆二色性光谱和 2-氨基嘌呤（2AP）荧光研究表明，在配体结合时，可卡因适体没有大的结构重排的证据，这与该适体的一般观点相反。可卡因适体与可卡因和新霉素-B 的亲和力随着生理 NaCl 的加入而降低。将适体序列中的位置 6 的 A 替换为 2-氨基嘌呤（2AP6）消除了 NaCl 对其与可卡因和类似物亲和力的影响，但对新霉素-B 没有影响，表明 2AP 取代对可卡因结合具有选择性影响。可卡因的亲和力也随血清或尿液浓度的增加而降低，2AP6 取代削弱了尿液的影响。其对可卡因和代谢物的低亲和力及其结合无关化合物的能力限制了该适体作为可卡因选择性和可靠传感器的应用机会。然而，这些研究还表明，适体的微小结构调整（用腺嘌呤代替 2AP）可以增加其在生理 NaCl 中对可卡因的特异性，相对于非靶标配体。
