<i>In-situ</i> hydrophobic environment triggering reactive fluorescence probe to real-time monitor mitochondrial DNA damage

doi:10.1007/s11705-021-2063-9

Front. Chem. Sci. Eng.

2022, Vol. 16

Issue (1) : 92-102 https://doi.org/10.1007/s11705-021-2063-9

RESEARCH ARTICLE

In-situ hydrophobic environment triggering reactive fluorescence probe to real-time monitor mitochondrial DNA damage

Beidou Feng, Huiyu Niu, Hongchen Zhai, Congcong Shen, Hua Zhang(

)

School of Chemistry and Chemical Engineering, Henan Normal University, Xinxiang 453007, China

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Abstract

Mitochondrial DNA has a special structure that is prone to damage resulting in many serious diseases, such as genetic diseases and cancers. Therefore, the rapid and specific monitoring of mitochondrial DNA damage is urgently needed for biological recognition. Herein, we constructed an in situ hydrophobic environment-triggering reactive fluorescence probe named MBI-CN. The fluorophore was 2-styrene-1H-benzo[d]imidazole, and malononitrile was introduced as a core into a molecule to initiate the hydrolysis reaction in the specific environment containing damaged mitochondrial DNA. In this design, MBI-CN conjugates to mitochondrial DNA without causing additional damages. Thus, MBI-CN can be hydrolyzed to generate MBI-CHO in an in situ hydrophobic environment with mitochondrial DNA damage. Meanwhile, MBI-CHO immediately emitted a significative fluorescence signal changes at 437 and 553 nm within 25 s for the damaged mitochondria DNA. Give that the specific and rapid response of MBI-CN does not cause additional damages to mitochondrial DNA, it is a potentially effective detection tool for the real-time monitoring of mitochondrial DNA damage during cell apoptosis and initial assessment of cell apoptosis.

Keywords hydrolysis reaction mitochondrial DNA damage in situ hydrophobic environment trigger fluorescence probe apoptosis

Corresponding Author(s): Hua Zhang

Online First Date: 27 July 2021 Issue Date: 27 December 2021

Cite this article:

Beidou Feng,Huiyu Niu,Hongchen Zhai, et al. In-situ hydrophobic environment triggering reactive fluorescence probe to real-time monitor mitochondrial DNA damage[J]. Front. Chem. Sci. Eng., 2022, 16(1): 92-102.

URL:

https://academic.hep.com.cn/fcse/EN/10.1007/s11705-021-2063-9
https://academic.hep.com.cn/fcse/EN/Y2022/V16/I1/92

Fig.1 Scheme 1&chsp;The synthetic route of compound MBI-CHO and MBI-CN.

Fig.2 (a) Fluorescence changes of MBI-CN in different hydrophilic and hydrophobic environments; (b) fluorescence spectrum of MBI-CHO in water with increasing mtDNA concentration (0–100 μg?mL⁻¹); (c) linear relationship diagram between MBI-CHO and mtDNA concentration; (d) MBI-CHO and mtDNA dynamic response.

Fig.3 (a) Fluorescence response of MBI-CN (2 μmol·L⁻¹) and its intracellular microenvironment triggering product MBI-CHO (2 μmol·L⁻¹) to biological enzymes (2 mg·mL⁻¹) (F₀: the initial fluorescence intensity of probes MBI-CN or MBI-CHO; F: the fluorescence intensity after adding interference); (b) Spectra before and after the action of MBI-CHO and MBI-CHO with mtDNA in water.

Fig.4 Results of molecular docking: (a) molecular docking binding mode and (b) binding site of MBI-CN and mtDNA; (c) molecular docking binding mode and (d) binding site of MBI-CHO and mtDNA.

Fig.5 Circular dichroism analysis of the interaction between molecules and mtDNA: (a) in the Tris-HCl, the concentration of fixed mtDNA (0.25 mg·mL⁻¹) changes with the addition of MBI-CHO to the mtDNA; (b) in the Tris-HCl, the concentration of fixed mtDNA (0.25 mg·mL⁻¹), with the addition of MBI-CN, the optical rotation of mtDNA changes. Cuvette: 1 mm.

Fig.6 Cytotoxicity of (a) MBI-CN and (b) MBI-CHO in HepG 2 and 3T3 cells.

Fig.7 Cell imaging and cell colocalization experiment: (a) stained with Mito Tracker Green; (b) stained with MBI-CN; (c) merged image of Mito Tracker Green and MBI-CN; (d) intensity correlation plot of stain MBI-CN and Mito Tracker Green; (e) intracellular coregionalization of MBI-CN and Mito Tracker Green. The excitation wavelength of MBI-CN is 405 nm and the fluorescence collection range is 580–620 nm; the excitation wavelength of Mito Tracker Green is 488 nm and the fluorescence collection range is 495–535 nm. The scale bar represents 10 μm.

Fig.8 DNA digestion experiments: after fixing cells, MBI-CN cell imaging in (a) green and (b) red channel; after digestion with DNA digestion enzyme for 2.0 h, added MBI-CN and incubated for 30 min, MBI-CN cell imaging in (c) green and (d) red channel. The excitation wavelength is 405 nm, and the fluorescence collection ranges are 415–465 nm (Green channel) and 530–570 nm (Red channel). Scale bar: 10 μm.

Fig.9 Apoptosis experiment: cell apoptosis was induced by different concentrations of catechol, and then MBI-CN (7.5 μmol·L⁻¹) was added for 30 min. (a,d) Concentrations of catechol: 0 mmol·L⁻¹; (b,e) concentrations of catechol: 0.2 mmol·L⁻¹; (c,f) concentrations of catechol: 1.0 mmol·L⁻¹; (g) apoptosis imaging fluorescence intensity extraction image. The excitation wavelength is 405 nm, and the fluorescence collection ranges are 415–465 nm (Green channel) and 530–570 nm (Red channel). Scale bar: 10 μm.

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