Engineering m6A demethylation-activated DNAzyme for visually and sensitively sensing fat mass and obesity-associated protein

Liu Shi; Xuemei Ma; Haojie Xie; Yujia Qin; Yue Huang; Yuanyuan Zhang; Lizhou Sun; Jie Yang; Genxi Li

doi:10.1016/j.bios.2022.115007

Engineering m⁶A demethylation-activated DNAzyme for visually and sensitively sensing fat mass and obesity-associated protein

Biosens Bioelectron. 2023 Feb 15:222:115007. doi: 10.1016/j.bios.2022.115007. Epub 2022 Dec 13.

Authors

Liu Shi¹, Xuemei Ma², Haojie Xie², Yujia Qin¹, Yue Huang³, Yuanyuan Zhang⁴, Lizhou Sun⁵, Jie Yang², Genxi Li⁶

Affiliations

¹ Center for Molecular Recognition and Biosensing, School of Life Sciences, Shanghai University, Shanghai, 200444, PR China.
² State Key Laboratory of Analytical Chemistry for Life Science, School of Life Sciences, Nanjing University, Nanjing, 210023, PR China.
³ Department of Food Science and Engineering, College of Light Industry and Food Engineering, Nanjing Forestry University, Nanjing, 210037, PR China.
⁴ Department of Obstetrics and Gynecology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, PR China. Electronic address: yuanyuanzhang@njmu.edu.cn.
⁵ Department of Obstetrics and Gynecology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, PR China.
⁶ Center for Molecular Recognition and Biosensing, School of Life Sciences, Shanghai University, Shanghai, 200444, PR China; State Key Laboratory of Analytical Chemistry for Life Science, School of Life Sciences, Nanjing University, Nanjing, 210023, PR China. Electronic address: genxili@nju.edu.cn.

PMID: 36527832
DOI: 10.1016/j.bios.2022.115007

Abstract

Fat mass and obesity-associated protein (FTO) regulating the N6-methyladenine (m⁶A, the most pervasive epigenetic modification) levels within the nucleus has been identified as a potential biomarker for cancer diagnosis and prognosis. However, current methods for FTO detection are complicated or/and not sensitive enough for practical application. Herein, we propose a colorimetric biosensor for detecting FTO based on a delicate design of m⁶A demethylation-activated DNAzyme. Specifically, an m⁶A-blocked DNAzyme is constructed as a switch of the biosensor that can be turned on by target FTO. The decreased thermal stability resulting from substrate cleavage leads to a DNAzyme recycling to produce multiple primers. Then the rolling circle amplification (RCA) reactions can be initiated to generate G-quadruplex-DNAzymes catalyzing 2,2-azino-bis-(3-ethylben-zthiazoline-6-sulfonic acid (ABTS) oxidation which can be readily observed by the naked eye. Quantitative detection can also be achieved with a limit of detection (LOD) down to 69.9 fM, exhibiting higher sensitivity than previous reports. Therefore, this biosensor opens a simple and sensitive way to achieve visual assay of FTO via triple signal amplification. In addition, our biosensor has been successfully applied to FTO detection in clinical samples, which shows great potential in clinical molecular diagnostics.

Keywords: Colorimetric biosensor; DNAzyme; Demethylation; Fat mass and obesity-associated protein; N6-methyladenosine.

MeSH terms

Adipose Tissue
Alpha-Ketoglutarate-Dependent Dioxygenase FTO / genetics
Alpha-Ketoglutarate-Dependent Dioxygenase FTO / metabolism
Biosensing Techniques* / methods
Colorimetry / methods
DNA, Catalytic* / chemistry
Demethylation
G-Quadruplexes*
Humans
Nucleic Acid Amplification Techniques / methods
Obesity

Substances

Alpha-Ketoglutarate-Dependent Dioxygenase FTO
DNA, Catalytic
FTO protein, human