可卡因适体的特异性和配体亲和力：结构特征和生理NaCl的影响,Analytical Chemistry

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可卡因适体的特异性和配体亲和力：结构特征和生理NaCl的影响
Analytical Chemistry ( IF 6.7 ) Pub Date : 2016-07-11 00:00:00 , DOI: 10.1021/acs.analchem.6b01633
Ashish Sachan ₁ , Muslum Ilgu ₁ , Aaron Kempema ₁ , George A. Kraus ₁ , Marit Nilsen-Hamilton ₁

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在许多情况下，可卡因适体已被视为可卡因探针的良好发展候选物。在这里，我们证明了适体以可互斥的方式与可卡因结合在一起，可卡因，去甲可卡因和可卡因具有相似的亲和力，而氨基糖苷具有相似或更高的亲和力。分析其对一系列可卡因衍生物的亲和力表明，适体特异性是其与可卡因分子所有表面相互作用的结果。圆二色光谱和2-氨基嘌呤（2AP）荧光研究表明，没有证据表明可卡因适体在配体结合后发生大的结构重排，这与该适体的一般观点相反。适体对可卡因和新霉素-B的亲和力随生理性氯化钠的加入而降低。用2AP替换适体序列第6位的A（2AP6）消除了NaCl对可卡因和类似物的亲和力的影响，但对新霉素B的亲和力没有影响，显示了2AP替换对可卡因结合的选择性作用。对可卡因的亲和力也随着血清或尿液浓度的增加而降低，而2AP6取代减弱了尿液的作用。它对可卡因和代谢产物的亲和力低，并且与无关化合物结合的能力限制了将这种适体以其当前形式用作可卡因的选择性和可靠传感器的机会。然而，这些研究还表明，适体（2AP交换为腺嘌呤）的小结构调整可以增加其相对于脱靶配体在生理NaCl中对可卡因的特异性。

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Specificity and Ligand Affinities of the Cocaine Aptamer: Impact of Structural Features and Physiological NaCl

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.

更新日期：2016-07-11

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