Initial state of DNA-Dye complex sets the stage for protein induced fluorescence modulation

• Published in: Nature communications Vol. 10; no. 1; pp. 2104 - 14
• Main Authors: Rashid, Fahad, Raducanu, Vlad-Stefan, Zaher, Manal S., Tehseen, Muhammad, Habuchi, Satoshi, Hamdan, Samir M.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 08.05.2019; Nature Publishing Group; Nature Portfolio
• Subjects: 631/57; 631/57/2265; 631/57/2267; 631/57/2272; 639/766/747; Deoxyribonucleic acid; Design of experiments; DNA; DNA - chemistry; DNA - metabolism; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - isolation & purification; DNA-Binding Proteins - metabolism; Dyes; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - isolation & purification; Escherichia coli Proteins - metabolism; Experimental design; Flap Endonucleases - chemistry; Flap Endonucleases - isolation & purification; Flap Endonucleases - metabolism; Fluorescence; Fluorescence Resonance Energy Transfer - methods; Fluorescent Dyes - chemistry; Humanities and Social Sciences; Microscopy, Fluorescence - methods; multidisciplinary; Obsolescence; Oligonucleotides - chemistry; Oligonucleotides - metabolism; Protein Binding; Proteins; Quenching; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Science; Science (multidisciplinary); Single Molecule Imaging - methods; Staining and Labeling; Viral Proteins - chemistry; Viral Proteins - isolation & purification; Viral Proteins - metabolism; Viscosity
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-019-10137-9

Protein-induced fluorescence enhancement (PIFE) is a popular tool for characterizing protein-DNA interactions. PIFE has been explained by an increase in local viscosity due to the presence of the protein residues. This explanation, however, denies the opposite effect of fluorescence quenching. This work offers a perspective for understanding PIFE mechanism and reports the observation of a phenomenon that we name protein-induced fluorescence quenching (PIFQ), which exhibits an opposite effect to PIFE. A detailed characterization of these two fluorescence modulations reveals that the initial fluorescence state of the labeled mediator (DNA) determines whether this mediator-conjugated dye undergoes PIFE or PIFQ upon protein binding. This key role of the mediator DNA provides a protocol for the experimental design to obtain either PIFQ or PIFE, on-demand. This makes the arbitrary nature of the current experimental design obsolete, allowing for proper integration of both PIFE and PIFQ with existing bulk and single-molecule fluorescence techniques. Protein-induced fluorescence enhancement (PIFE) is a popular tool for characterizing protein-DNA interactions. Here, authors provide a perspective on understanding the general phenomenon of induced fluorescence modulation