title: G-quadruplex fluorescent probe-mediated real-time rolling circle amplification strategy for highly sensitive microRNA detection.
abstract: Real-time PCR has revolutionized PCR from qualitative to quantitative. As an isothermal DNA amplification technique, rolling circular amplification (RCA) has been demonstrated to be a versatile tool in many fields. Development of a simple, highly sensitive, and specific strategy for real-time monitoring of RCA will increase its usefulness in many fields. The strategy reported here utilized the specific fluorescence response of thioflavin T (ThT) to G-quadruplexes formed by RCA products. Such a real-time monitoring strategy works well in both traditional RCA with linear amplification efficiency and modified RCA proceeded in an exponential manner, and can be readily performed in commercially available real-time PCR instruments, thereby achieving high-throughput detection and making the proposed technique more suitable for biosensing applications. As examples, real-time RCA-based sensing platforms were designed and successfully used for quantitation of microRNA over broad linear ranges (8 orders of magnitude) with a detection limit of 4 aM (or 0.12 zmol). The feasibility of microRNA analysis in human lung cancer cells was also demonstrated. This work provides a new method for real-time monitoring of RCA by using unique nucleic acid secondary structures and their specific fluorescent probes. It has the potential to be extended to other isothermal single-stranded DNA amplification techniques.
authors: State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, 300071, PR China; Collaborative Innovation Centre of Chemical Science and Engineering (Tianjin), Tianjin, 300071, PR China; Tianjin Key Laboratory of Biosensing and Molecular Recognition, Research Centre for Analytical Sciences, Nankai University, Tianjin, 300071, PR China.
authors: Hong-Xin
authors: HX
authors: Jiang
authors: State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, 300071, PR China; College of Life Science, Nankai University, Tianjin, 300071, PR China.
authors: Zhen-Zhen
authors: ZZ
authors: Liang
authors: State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, 300071, PR China; College of Life Science, Nankai University, Tianjin, 300071, PR China.
authors: Yan-Hong
authors: YH
authors: Ma
authors: State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, 300071, PR China; Collaborative Innovation Centre of Chemical Science and Engineering (Tianjin), Tianjin, 300071, PR China; Tianjin Key Laboratory of Biosensing and Molecular Recognition, Research Centre for Analytical Sciences, Nankai University, Tianjin, 300071, PR China. Electronic address: kongdem@nankai.edu.cn.
authors: De-Ming
authors: DM
authors: Kong
authors: State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, 300071, PR China; College of Life Science, Nankai University, Tianjin, 300071, PR China. Electronic address: hongzy@nankai.edu.cn.
authors: Zhang-Yong
authors: ZY
authors: Hong
mesh_terms: D005456:Fluorescent Dyes / Q000737:chemistry*; D054856:G-Quadruplexes; D006801:Humans; D057230:Limit of Detection; D035683:MicroRNAs / Q000032:analysis*
chemical_list: D005456:Fluorescent Dyes; D035683:MicroRNAs
keywords: G-quadruplex; MicroRNA; Real-time; Rolling circular amplification; Thioflavin T