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Highly selective detection of deoxyribonucleic acid in living cells using RecA-green fluorescent protein-single-stranded deoxyribonucleic acid filament fluorescence resonance energy transfer probe |
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| Authors: | Chunlei Wang Yuanfu Zhang Wenyue Gong Shuhao Wang |
| Institution: | 1. Key Laboratory of Food Safety and Life Analysis in Universities of Shandong, Shandong Provincial Key Laboratory of Chemical Energy Storage and Novel Cell Technology, School of Chemistry and Chemical Engineering, Liaocheng University, Liaocheng, China
Contribution: Data curation, Methodology;2. Key Laboratory of Food Safety and Life Analysis in Universities of Shandong, Shandong Provincial Key Laboratory of Chemical Energy Storage and Novel Cell Technology, School of Chemistry and Chemical Engineering, Liaocheng University, Liaocheng, China;3. Key Laboratory of Food Safety and Life Analysis in Universities of Shandong, Shandong Provincial Key Laboratory of Chemical Energy Storage and Novel Cell Technology, School of Chemistry and Chemical Engineering, Liaocheng University, Liaocheng, China Contribution: Formal analysis |
| Abstract: | A fluorescence resonance energy transfer (FRET) method was developed for double-stranded deoxyribonucleic acid (dsDNA) detection in living cells using the RecA-GFP (green fluorescent protein) fusion protein filament. In brief, the thiol-modified single-stranded DNA (ssDNA) was attached to gold nanoparticles (AuNPs); on the contrary, the prepared RecA-GFP fusion protein interacted with ssDNA. Due to the FRET between AuNPs and RecA-GFP, fluorescence of RecA-GFP fusion protein was quenched. In the presence of homologous dsDNA, homologous recombination occurred to release RecA-GFP fusion protein. Thus, the fluorescence of RecA-GFP was recovered. The dsDNA concentration was detected using fluorescence intensity of RecA-GFP. Under optimal conditions, this method could detect dsDNA activity as low as 0.015 optical density (OD) Escherichia coli cells, with a wide linear range from 0.05 to 0.9 OD cells, and the regression equation was ΔF = 342.7c + 78.9, with a linear relationship coefficient of 0.9920. Therefore, it provided a promising approach for the selective detection of dsDNA in living cells for early clinical diagnosis of genetic diseases. |
| Keywords: | deoxyribonucleic acid fluorescence resonance energy transfer gold nanoparticles RecA-GFP-ssDNA filament |