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Switchable DNA tweezer and G-quadruplex nanostructures for ultrasensitive voltammetric determination of the K-ras gene fragment

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Abstract

Voltammetric detection of the K-ras gene fragment was accomplished through the combined application of (a) a switchable DNA nanostructure, (b) the use of hairpin probe and exonuclease III (Exo III)-assisted signal amplification, (c) a split G-quadruplex, and (d) by exploiting the redox activity of DNAzyme. Three assistant oligonucleotides were designed to construct a DNA tweezer on a gold electrode. It is in “open state” in the absence of K-ras DNA. Then, a hairpin probe was introduced, whose stem-loop structure can be opened through hybridization with the K-ras DNA. Exo III is added which hydrolyzes the complementary region of the hairpin sequence to release a single-stranded rest fragment. The ssDNA hybridizes with the DNA tweezer on the electrode which thereby is switched to the “closed state”. This leads to the formation of G-quadruplex due to the shortened distance of the split G-quadruplex-forming sequences in the tweezer. The voltammetric signal of the G-quadruplex-hemin complex, with a peak near −0.3 V vs. Ag/AgCl, is used as the signal output. Under the optimal conditions, the current response in differential pulse voltammetry (DPV) increases linearly with the concentration of K-ras DNA in the range of 0.01–1000 pM, and the detection limit is 2.4 fM. The assay can clearly discriminate K-ras DNA from a single-base mutation. The method has excellent selectivity and was applied to the determination of K-ras DNA in (spiked) serum samples.

Schematic representation of a method for the determination of the K-ras gene fragment through a combination of switchable DNA tweezer, split G-quadruplex, and exonuclease III (ExoIII)-assisted target recycling signal amplification.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (no. 31271860), the Six Talent Peaks Project in Jiangsu Province (JY-078) and National First-class Discipline Program of Light Industry Technology and Engineering (LITE2018-07).

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  1. The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China

    Haohan Chen, Xiaofan Sun, Rongfeng Cai, Yaping Tian & Nandi Zhou

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Correspondence to Nandi Zhou.

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Chen, H., Sun, X., Cai, R. et al. Switchable DNA tweezer and G-quadruplex nanostructures for ultrasensitive voltammetric determination of the K-ras gene fragment. Microchim Acta 186, 843 (2019). https://doi.org/10.1007/s00604-019-3993-5

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