Jeong Ji-Eun, Kim Boram, Woo Shinjae, Hwang Sungu, Bazan Guillermo C, Woo Han Young
Department of Nanofusion Engineering , Department of Cogno-Mechatronics Engineering , Pusan National University , Miryang , Gyeongsangnam-do 627-706 , Republic of Korea . Email:
Department of Nanomechatronics Engineering , Pusan National University , Miryang , Gyeongsangnam-do 627-706 , Republic of Korea.
Chem Sci. 2015 Mar 1;6(3):1887-1894. doi: 10.1039/c4sc03258f. Epub 2015 Jan 7.
A strategy to extend the detection range of weakly-binding targets is reported that takes advantage of fluorescence resonance energy transfer (FRET)-based bioassays based on molecular beacon aptamers (MBAs) and cationic conjugated polyelectrolytes (CPEs). In comparison to other aptamer-target pairs, the aptamer-based adenosine triphosphate (ATP) detection assays are limited by the relatively weak binding between the two partners. In response, a series of MBAs were designed that have different stem stabilities while keeping the constant ATP-specific aptamer sequence in the loop part. The MBAs are labeled with a fluorophore and a quencher at both termini. In the absence of ATP, the hairpin MBAs can be opened by CPEs a combination of electrostatic and hydrophobic interactions, showing a FRET-sensitized fluorophore signal. In the presence of ATP, the aptamer forms a G-quadruplex and the FRET signal decreases due to tighter contact between the fluorophore and quencher in the ATP/MBA/CPE triplex structure. The FRET-sensitized signal is inversely proportional to [ATP]. The extension of the detection range is determined by the competition between opening of the ATP/MBA G-quadruplex by CPEs and the composite influence by ATP/aptamer binding and the stem interactions. With increasing stem stability, the weak binding of ATP and its aptamer is successfully compensated to show the resistance to disruption by CPEs, resulting in a substantially broadened detection range (from millimolar up to nanomolar concentrations) and a remarkably improved limit of detection. From a general perspective, this strategy has the potential to be extended to other chemical- and biological-assays with low target binding affinity.
报道了一种扩展弱结合靶点检测范围的策略，该策略利用基于分子信标适体（MBA）和阳离子共轭聚合物（CPE）的荧光共振能量转移（FRET）生物测定法。与其他适体-靶点对相比，基于适体的三磷酸腺苷（ATP）检测测定法受到两者之间相对较弱结合的限制。作为回应，设计了一系列MBA，它们具有不同的茎稳定性，同时在环部分保持恒定的ATP特异性适体序列。MBA在两端分别标记有荧光团和猝灭剂。在没有ATP的情况下，发夹状MBA可以被CPE通过静电和疏水相互作用打开，显示出FRET敏化的荧光团信号。在有ATP的情况下，适体形成G-四链体，由于ATP/MBA/CPE三聚体结构中荧光团和猝灭剂之间更紧密的接触，FRET信号降低。FRET敏化信号与[ATP]成反比。检测范围的扩展取决于CPE对ATP/MBA G-四链体的打开与ATP/适体结合和茎相互作用的复合影响之间的竞争。随着茎稳定性的增加，ATP与其适体的弱结合得到成功补偿，以显示对CPE破坏的抗性，从而导致检测范围大幅拓宽（从毫摩尔到纳摩尔浓度），检测限显著提高。从一般角度来看，该策略有可能扩展到其他具有低靶点结合亲和力的化学和生物测定中。
