Jiangsu Collaborative Innovation Center of Biomedical Functional Materials and Jiangsu Key Laboratory of Biofunctional Materials, School of Chemistry and Materials Science, Nanjing Normal University, Nanjing, 210023, P. R. China.
Analyst. 2021 Dec 6;146(24):7379-7385. doi: 10.1039/d1an01603b.
G-quadruplex-based complexes have been widely used in various analytical methods due to their outstanding capabilities of generating colorimetric, fluorescent or electrochemical signals. However, since loop sequences in traditional G-quadruplex structures are quite short, it is difficult to establish biosensors solely using G-quadruplex-based complexes. Herein, we attempted to lengthen the loop sequences of G-quadruplex structures and found that G-quadruplex-hemin DNAzymes (G-DNAzymes) with long loops (even 30 nucleotides) maintain high peroxidase activity. In addition, the peroxidase activity is not affected by the hybridization of the long loop with its complementary counterpart. Consequently, G-DNAzyme can be endowed with an additional function by taking the long loop as a recognition element, which may facilitate the construction of diverse colorimetric biosensors. Furthermore, by designing an apurinic/apyrimidinic site or a complementary sequence of microRNA-21 (miRNA-21) in long loops, bifunctional G-DNAzymes can be split in the presence of apurinic/apyrimidinic endonuclease 1 (APE1) or miRNA-21, decreasing their peroxidase activities. Accordingly, APE1 and miRNA-21 are quantified using 3,3',5,5'-tetramethylbenzidine as a chromophore. Using the G-DNAzyme, APE1 can be detected in a linear range from 2.5 to 22.5 U mL with a LOD of 1.8 U mL. It is to be noted that benefitting from duplex-specific nuclease-induced signal amplification, the linear range of the miRNA-21 biosensor is broadened to 5 orders of magnitude, while the limit of detection is as low as 73 fM. This work demonstrates that G-DNAzymes with long loops can both generate signals and recognize targets, providing an alternative strategy to design G-quadruplex-based analytical methods.
基于 G-四链体的复合物由于其出色的产生比色、荧光或电化学信号的能力，已被广泛应用于各种分析方法中。然而，由于传统 G-四链体结构中的环序列非常短，因此仅使用基于 G-四链体的复合物很难建立生物传感器。在此，我们试图延长 G-四链体结构的环序列，发现具有长环（甚至 30 个核苷酸）的 G-四链体-血红素 DNA 酶（G-DNAzyme）保持高过氧化物酶活性。此外，长环与互补链杂交不会影响过氧化物酶活性。因此，G-DNAzyme 可以通过将长环作为识别元件赋予其额外的功能，这可能有助于构建各种比色生物传感器。此外，通过在长环中设计无嘌呤/无嘧啶位点或 microRNA-21（miRNA-21）的互补序列，双功能 G-DNAzyme 可以在核酸内切酶 1（APE1）或 miRNA-21 的存在下分裂，降低其过氧化物酶活性。因此，使用 3,3',5,5'-四甲基联苯胺作为显色团来定量 APE1 和 miRNA-21。使用 G-DNAzyme，可以在 2.5 至 22.5 U mL 的线性范围内检测 APE1，检测限为 1.8 U mL。值得注意的是，得益于双链特异性核酸酶诱导的信号放大，miRNA-21 生物传感器的线性范围扩展到 5 个数量级，检测限低至 73 fM。这项工作表明，具有长环的 G-DNAzyme 既能产生信号又能识别靶标，为设计基于 G-四链体的分析方法提供了一种替代策略。
