State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, P. R. China.
Anal Chem. 2010 Mar 15;82(6):2221-7. doi: 10.1021/ac901794w.
A universal approach is proposed in this study for the development of an aptameric assay system for proteins based on aptamer structure-switching-triggered ligation-rolling circle amplification (L-RCA) upon target binding. The strategy chiefly depends on the competition for binding the aptamer probe between target protein and a complementary single-stranded DNA (CDNA) that can induce the circularization of the padlock probe. Introduction of target protein into the assay system inhibits the hybridization of the CDNA with the aptamer probe because of the formation of the target/aptamer duplex. The free CDNA can only hybridize with the padlock probe. With the assistance of DNA ligase, the padlock probe is circularized, and the subsequent RCA process can be accomplished by Phi 29 DNA polymerase. Each RCA product containing thousands of repeated sequences might hybridize with a large number of molecular beacons (detection probes), resulting in an enhanced fluorescence signal. In contrast, in the absence of target protein, no obvious change in the fluorescence intensity of the detection probe is observed. This signaling mode for target recognition and transduction events is based on the combination of aptamer recognition elements and L-RCA technology with high specificity and sensitivity. The proposed assay system not only exhibits excellent analytical characteristics (e.g., the detection limit on attomolar scale and a linear dynamic range of more than 3 orders of magnitude) but also possesses significant advantages over existing aptameric assays. The proposed strategy is universal since the sequences of aptamer probe, CDNA, and padlock probe could be easily designed to be compatible with the L-RCA based detection of other proteins without other conditions.
本研究提出了一种通用的方法，用于开发基于适配体结构切换触发连接-滚环扩增（L-RCA）的适体分析系统，用于在结合目标后对蛋白质进行分析。该策略主要依赖于目标蛋白与互补单链 DNA（cDNA）之间的竞争结合，该 cDNA 可以诱导发夹探针的环化。由于目标/适配体双链体的形成，目标蛋白的引入会抑制 cDNA 与适配体探针的杂交。游离的 cDNA 只能与发夹探针杂交。在 DNA 连接酶的辅助下，发夹探针被环化，随后的 RCA 过程可以由 Phi 29 DNA 聚合酶完成。每个包含数千个重复序列的 RCA 产物可能与大量分子信标（检测探针）杂交，从而导致荧光信号增强。相比之下，在没有目标蛋白的情况下，检测探针的荧光强度没有明显变化。这种基于适配体识别元件和 L-RCA 技术的信号转导事件的信号模式具有高度特异性和敏感性。该分析系统不仅具有优异的分析特性（例如，检测限可达飞摩尔级，线性动态范围超过 3 个数量级），而且相对于现有的适体分析方法具有显著优势。该策略具有通用性，因为适配体探针、cDNA 和发夹探针的序列可以轻松设计为与其他蛋白质的 L-RCA 检测兼容，而无需其他条件。
