Fluorescence resonance energy transfer (FRET) is a quantum process that occurs between two dye molecules. From the donor, molecule excitation is shifted to an acceptor fluorophore by the dipole-dipole interaction without photon emission. Consequently, quenching of donor molecule fluorescence results in the excitation of the acceptor molecule. Afterwards, it loses energy by the emission of heat or fluorescent known as sensitized emission. In FRET the intensity of donor fluorescence along with the lifetime and quantum efficiency decline thereby increase in the fluorescence intensity of the acceptor. Numerous studies identified DNA binding proteins that act as promotor during gene transcription and ligand-receptor interactions. FRET-based hybridization assays possess advantages such as real-time observation within the solution of the hybridization reaction without the requirement of any solid support as well as washing Methods, speed and simplicity, allowing for the ease of adaptation to automated performance making possible of in-vivo hybridisation within the living cells (Didenko, 2001).
In 1996, Parkhurst and co-workers while studying the interaction of Saccharomyces cerevisiae TATA-binding protein (TBP) with the TATA-promoter element applied the FRET technique. A 14-bp 5′-GGGCTATAAAAGGG-3′ based on the adenovirus major late promoter was specially labelled with tetramethylrhodamine (TMR) and fluorescein at 5′- and 3′-terminals, correspondingly. In the absence of TBP, rigid DNA duplex nature holds the fluorophores separately resultant in a low FRET response. Still, the addition of TBP bends the DNA thus bringing 3′- and 5′ terminals of the duplex closer to each other causing almost 37% reduction in the emission of fluorescein at 520nm with a saturation ratio of 14:1 (protein: DNA) thus rhodamine which is an acceptor moiety give rise to concomitant rise in the emission (Leung, 2012).
Mostly in FRET-based studies of DNA, the distance among base pairs is of prime importance thus has been analysed. The occurrence of FRET is due to the duplex formation between two labelled oligonucleotides, taking the donor near acceptor dyes. On the other hand, the hairpin configuration of an oligonucleotide labelled with both donor and acceptor fluorophores can outcome in FRET signal generation. The detection of FRET and its disruption are both applied for analyses purpose. The detection of FRET points toward distance between the oligonucleotide that is the two fluorophore molecules are physically within a few nanometers. FRET disruption shows the alteration in the relative positioning of the molecules with the new distance between the donor and acceptor oligonucleotides prohibit the incidence of FRET (Didenko, 2001). Differences in structures and flexibilities of DNA duplexes is also a vital parameter for the recognition by DNA binding proteins. In recent times, FRET technology is employed to study complex DNA structures. One such example is the UspDBD/EcRDBD-hsp27pal complex topology in which fluorescence data signifies that UspDBD governs the architecture of the heterocomplex UspDBD/EcRDBD which is due to the substantial deformation (DNA bending) of the response element, while specific helper molecule is considered to be the Scribd. None of the protein is known to forms a complex structure within the solution in the absence of the response element (Krusiński et al, 2010).
Melting temperature is associated with the FRET signal generation. The assembly and disassembly of the DNA duplex molecule are permitted by the precise placement of FRET fluorophores pair on two constituent oligonucleotides. The change in interfluorophore distance depends upon temperature which is associated with conformational changes. Correct assembly occurs upon cooling thus FRET pair is bought into proximity thereby maximum FRET efficiency occurs at low temperatures. Whereas, upon thermal melting i.e. at the high-temperature separation of the FRET pair takes place thus induces nominal FRET efficiency (Nangreave et al, 2009).
Didenko, V. V. (2001). DNA Probes Using Fluorescence Resonance Energy Transfer (FRET): Designs and Applications. BioTechniques, 31(5), 1106–1121.
Chung-Hang Leung, Daniel Shiu-Hin Chan, Hong-Zhang He, Zhen Cheng, Hui Yang, Dik-Lung Ma. (2012). Luminescent detection of DNA-binding proteins, Nucleic Acids Research, 40(3), 941–955.
Tomasz Krusiński, Andrzej Ożyhar, Piotr Dobryszycki; Dual FRET assay for detecting receptor protein interaction with DNA, Nucleic Acids Research, Volume 38, Issue 9, 1 May 2010, Pages e108, https://doi.org/10.1093/nar/gkq049.
Nangreave, J., Yan, H., & Liu, Y. (2009). Studies of thermal stability of multivalent DNA hybridization in a nanostructured system. Biophysical Journal, 97(2), 563-571.