FRET-based Ratiometric MicroRNA Detection with G-quadruplex-stabilized Silver Nanoclusters
Received date: 2017-09-15
Online published: 2017-11-07
Supported by
Project supported by the National Natural Science Foundation of China (Nos. 21475004 and 21535006).
Fluorescent DNA-stabilized Ag nanoclusters (DNA-Ag NCs), a class of excellent luminescence probes with excellent optical properties, have been applied in assorted sensing and imaging fields. To date, most of the quantifications were based on the direct signal change of DNA-Ag NCs caused by target recognition, which inevitably jeopardizes the reproducibility and robustness of methods when experimental settings or detecting conditions are changed. In this work, using the highly fluorescent G-quadruplex-stabilized Ag NCs (GQ-Ag NCs) and Cy5 as the donor-acceptor pair, we for the first time developed a FRET based ratiometric method for miRNA detection. A rationally optimized hairpin recognition structure was attached to the G-quadruplex template of the Ag nanocluster. The introduction of target sequence opened the hairpin, led to the approximation of the donor nanocluster and the acceptor Cy5, enabled the energy transfer between the FRET pair, and thus generated the optical signal change. The Cy5 tag sequence was designed to be universal, simplifying the experimental design and reduced the cost in applications. The optical signal for quantitation was determined by the signal difference between the Ag nanocluster and the Cy5 fluorophore, with the fluorescence intensity of Cy5 used as internal reference in order to prevent signal variation. MicroRNAs (miRNA) are short RNA molecules that have emerged as a kind of key post-translational regulators of gene expression in eukaryotic organisms. In this study, microRNA let-7a was chosen as the model target of our FRET-based ratiometric detection for demonstration. The linear range and the detection limit of the method on let-7a was 12~300 nmol/L and 6.9 nmol/L, respectively. The proposed method presented reasonable selectivity amongst the members of the same let-7 family. The remarkable recovery in total RNA extracted from HepG2 cell lines demonstrated the potential in clinical applications. The highlights of our work extended the application of DNA-templated Ag NCs and facilitated more understanding of DNA-Ag NCs as the energy donor in FRET design.
Key words： G-quadruplex; silver nanoclusters; FRET; ratiometric; miRNA
Lin Ruoyun , Chen Yang , Tao Guangyu , Pei Xiaojing , Liu Feng , Li Na . FRET-based Ratiometric MicroRNA Detection with G-quadruplex-stabilized Silver Nanoclusters[J]. Acta Chimica Sinica, 2017 , 75(11) : 1103 -1108 . DOI: 10.6023/A17090407
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