Zhou Yubin, Chi Hong, Wu Yuanyuan, Marks Robert S, Steele Terry W J
School of Materials Science & Engineering, College of Engineering, Nanyang Technological University50 Nanyang AvenueSingapore639798Singapore.
School of Materials Science & Engineering, College of Engineering, Nanyang Technological University50 Nanyang AvenueSingapore639798Singapore; Department of Biotechnology Engineering, Faculty of Engineering Sciences, Ben Gurion University of the NegevP.O. Box 653Beer Sheva84105Israel.
Talanta. 2016 Nov 1;160:172-182. doi: 10.1016/j.talanta.2016.06.067. Epub 2016 Jul 1.
Aptamer-ligand binding has been utilized for biological applications due to its specific binding and synthetic nature. However, the applications will be limited if the binding or the ligand is unstable. Malachite green aptamer (MGA) and its labile ligand malachite green (MG) were found to have increasing apparent dissociation constants (Kd) as determined through the first order rate loss of emission intensity of the MGA-MG fluorescent complex. The fluorescent intensity loss was hypothesized to be from the hydrolysis of MG into malachite green carbinol base (MGOH). Random screening organic additives were found to reduce or retain the fluorescence emission and the calculated apparent Kd of MGA-MG binding. The protective effect became more apparent as the percentage of organic additives increased up to 10% v/v. The mechanism behind the organic additive protective effects was primarily from a ~5X increase in first order rate kinetics of MGOH→MG (kMGOH→MG), which significantly changed the equilibrium constant (Keq), favoring the generation of MG, versus MGOH without organic additives. A simple way has been developed to stabilize the apparent Kd of MGA-MG binding over 24h, which may be beneficial in stabilizing other triphenylmethane or carbocation ligand-aptamer interactions that are susceptible to SN1 hydrolysis.
适体-配体结合因其特异性结合和合成性质已被用于生物应用。然而，如果结合或配体不稳定，应用将会受到限制。通过孔雀石绿适体（MGA）-孔雀石绿（MG）荧光复合物发射强度的一级速率损失测定发现，孔雀石绿适体及其不稳定配体孔雀石绿具有不断增加的表观解离常数（Kd）。荧光强度损失被推测是由于MG水解成孔雀石绿醇碱（MGOH）。发现随机筛选的有机添加剂可降低或保留MGA-MG结合的荧光发射及计算得到的表观Kd。随着有机添加剂的体积百分比增加至10%，保护作用变得更加明显。有机添加剂保护作用背后的机制主要源于MGOH→MG的一级反应动力学增加了约5倍（kMGOH→MG），这显著改变了平衡常数（Keq），与没有有机添加剂时相比，有利于生成MG而非MGOH。已开发出一种简单方法来使MGA-MG结合的表观Kd在24小时内保持稳定，这可能有助于稳定其他易受SN1水解影响的三苯甲烷或碳正离子配体-适体相互作用。
