FRET is a physical phenomenon used more and more in biomedical research and drug discovery today. FRET is the radiationless transmission of energy from a donor molecule to an acceptor molecule. The donor molecule is the dye or chromophore that initially absorbs the energy, and the acceptor is the chromophore to which the energy is subsequently transferred. This resonance interaction occurs over greater than interatomic distances, without conversion to thermal energy and any molecular collision. The transfer of energy leads to a reduction in the donor's fluorescence intensity and excited-state lifetime and an increase in the acceptor's emission intensity. A pair of molecules that interact so that FRET occurs is often referred to as a donor/acceptor pair. Due to its sensitivity to distance, FRET has been used to investigate molecular interactions.

Proteases are involved in several physiological processes. The detection of proteases and the screening of specific protease inhibitors are essential in discovering potential drugs for the treatment and management of protease-related diseases. A variety of donor and acceptor molecules (either fluorescent or non-fluorescent) are attached to the peptide/protein (forming the internally quenched conjugates) and are used to detect a protease depending on the sequences of the amino acids. The donor and acceptor molecules are carefully chosen so that the absorption of the acceptor overlaps with the fluorescence of the donor, thus ensuring that the fluorescence is quenched through resonance energy transfer. Enzyme hydrolysis of the substrate results in spatial separation of the donor and the acceptor molecules, thereby restoring the donor's fluorescence.

DABCYL dyes are quite useful for preparing FRET peptides. However, its low quenching efficiency for longer-wavelength dyes such as fluoresceins, rhodamines, and cyanines has limited its use in developing sensitive fluorogenic FRET probes. Additionally, the absorption spectrum of DABCYL is environment-sensitive.

To overcome the caveats associated with DABYCL dyes and other classic quenchers, AAT Bioquest has developed a series of robust non-fluorescent Tide Quencher™ chromophores covering the entire visible spectrum with unusually high FRET efficiency. For example, TQ2 has an absorption maximum perfectly matching the emission of FAM while TQ3, TQ5, and TQ7 are proven to be the best quenchers for Cy3^®, Cy5^®, and Cy7^®. Tide Quencher™ dyes are excellent dark quenchers that are individually optimized to pair with all the popular fluorescent dyes such as fluoresceins, rhodamines, and cyanines. These Tide Quencher™ dark FRET acceptors (such as TQ1, TQ2, TQ3, TQ4, TQ5, TQ6, and TQ7) are perfect to pair with our Tide Fluor™ dyes and the classic fluorophores (such as AMCA, EDANS, FAM, TAMRA, HEX, JOE, TET, ROX, Cy3^®, Cy5^®, and Cy7^®). Like our Tide Fluor™ donor dyes, our Tide Quencher™ acceptor dyes are much more cost-effective with comparable or even better performance for your desired biological applications than other similar products on the market.

Together Tide Quencher™ and Tide Fluor™ dyes can be used to produce highly sensitive FRET probes for a variety of applications, including enzyme studies, pharmacological drug screening, and qPCR analysis. Since the absorbances of Tide Quencher™ dyes are well-tuned to quench their Tide Fluor™ equivalent, substrates are completely non-fluorescent, and the potential for any background interference resulting from nonsensitized acceptor excitation is eliminated. Separating the two dyes either by proteolysis (i.e., FRET peptides) or nucleic acid hybridization (i.e., FRET nucleotides) restores fluorescence characteristic of the Tide Fluor™ label.