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Fluorometric determination of RNase H via a DNAzyme conjugated to reduced graphene oxide, and its application to screening for inhibitors and activators

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Abstract

A new fluorometric method is delineated for the detection of RNase H activity by combining DNAzyme with reduced graphene oxide (rGO). In the absence of RNase H, the fluorescence of FAM-labeled probe is quenched due to the strong adsorption on the rGO. The presence of RNase H can release the active DNAzyme from the DNA-RNA chimeric strand. This triggers the cleavage of the signal probe at the rA site with the help of the cofactor Mg2+. The recycle cleavage can directly result in the amplified signal emitted by the FAM-labeled short fragment. The method allows the activity of RNase H to be detected in a linear range of 0.01 to 5 U·mL−1. The detection limit of 0.018 U·mL−1 is calculated by the principle of three-time standard deviation over the blank signal. Then, RNase H-targeting natural compounds were screened for their inhibitory action. Among the investigated compounds, five were screened as RNase H inhibitors in a concentration-dependent manner, and 4 compounds were identified as activators. Finally, the method was reliably used for discriminating the difference of RNase H activity in human serum. It is found that RNase H activity was upregulated in patients with hepatitis C virus infection.

The schematic presentation of rGO-DNAzyme-based RNase H detection. RNase H triggers the active DNAzyme releasing from the DNA-RNA chimeric strand, which can cleavage probes to FAM-labeled short fragments and make the fluorescence signal cycle amplified.

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Acknowledgements

This work was partially supported by the Natural Science Foundation of Hunan Province (h14JJ2049), the Natural Science Foundation of China (81501218, 81673579, 81874369 and 31672457).

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Authors and Affiliations

  1. College of Biology, Hunan University, Changsha, 410082, China

    Chunyi Tong, Ting Zhou, Chuan Zhao, Bin Liu, Jialong Fan & Dan Li

  2. People’s Hospital of Hunan Province, Changsha, 410082, China

    Liqun Yuan

  3. Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha, 410008, People’s Republic of China

    Ying Xu & Aiguo Zhu

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  1. Chunyi Tong
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  2. Ting Zhou
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  3. Chuan Zhao
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  6. Bin Liu
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  7. Jialong Fan
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  9. Aiguo Zhu
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Correspondence to Bin Liu or Aiguo Zhu.

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Human serum samples used in this study were collected from healthy volunteers and patients. This work was performed with the written informed consent of health and patients. The studies were approved by the medical Ethics Committee of People’s Hospital of Hunan Province and performed in accordance with the ethical standards.

The author(s) declare that they have no competing interests.

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Tong, C., Zhou, T., Zhao, C. et al. Fluorometric determination of RNase H via a DNAzyme conjugated to reduced graphene oxide, and its application to screening for inhibitors and activators. Microchim Acta 186, 335 (2019). https://doi.org/10.1007/s00604-019-3425-6

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