Tide Fluor™ and Tide Quencher™ Dyes, Optimized for Maximizing The Power of Fluorescence Resonance Energy Transfer (FRET)

Introduction

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Fluorescence resonance energy transfer (FRET) is the transfer of the excited state energy from the initially excited donor (D) to an acceptor (A). The donor molecules typically emit at shorter wavelengths that overlap with the absorption of acceptors. The process is a distance-dependent interaction between the electronic excited states of two molecules without emission of a photon. FRET is the result of long-range dipole-dipole interactions between the donor and acceptor as illustrated in Figure 1. A few excellent recent reviews are listed in the references for further information.

As shown in Figure 1, an excited donor molecule has several routes to release its captured energy returning to the ground state. The excited state energy can be dissipated to the environment (as light or heat) or transferred directly to a second acceptor molecule, sending the acceptor to an excited state. The later process is called FRET.

• Internal Energy Conversions: The rapid return of electrons from the second (or higher) excited state to the first excited state is termed internal conversion. Energy is released through rapid solvent relaxation.
• Light Emissions: Light is released by either the transition from S₁ to S₀ (fluorescence emission) or transition from T₁ to S₀ (phosphorescence emission).
• Fluorescence Resonance Energy Transfer: FRET occurs when donor and acceptor molecules are within a specified range, usually within 10 to 100 Å. In the process of FRET, the excited-state energy of a donor is transferred to an acceptor molecule. Photons of light aren't involved in this transfer. In Figure 1, the pathway leading from the S₁ level of the donor to the S₁ level of the acceptor represents FRET. Once excited, the acceptor can return to the ground state by the same pathways as described for the donor. If the acceptor molecule is also fluorescent, it can emit light at its characteristic wavelength, which is always longer than the emission wavelength of the donor.
• Collisional Quenchings: Collisions between an excited-state fluorophore and other molecules will sometimes quench the fluorophore, returning it to the ground state without emission of a photon. Collisional quenchers include molecular oxygen and electron scavengers such as Cu²⁺, Mn²⁺, halides, isothiocyanate, nitrite and nitrate ions. Collisional quenching primarily occurs when these ions are present in the millimolar range or higher. Therefore, under most experimental conditions, collisional quenching is usually negligible.

Optimal FRET Conditions

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The efficiency (E%) and rate (k_T) of FRET are respectively expressed as follows: E% = k_T/(τ_D^-1 + k_T) [1]

k_T = R_o⁶ γ^-6 τ_D^-1 [2]

Where τ_D is the decay time of the donor in the absence of acceptor; γ is the donor-acceptor (D-A) distance; R_o is the Förster distance where FRET has 50% efficiency (typically in the range of 20-60Å). R_o is determined by the following equation:

R_o⁶ = 8.79x10²³[k²n^-4Φ_DJ(λ)] [3]

Where k² is dipole-dipole orientation factor (ranging from 0 to 4, k² = 2/3 for randomly oriented donors and acceptors); n is refractive index [The refractive index is generally known from solvent composition or estimated for macromolecules such as proteins and nucleic acids. n is often assumed to be that of water (n=1.33) for aqueous solutions, or to be that of small organic molecules (n=1.39) for organic solutions]. Φ_D is the fluorescence quantum yield of donor in the absence of acceptor. J(λ) is FRET spectral overlap integral as illustrated in Figure 2, and is determined by the following equation:

J(λ) = ∫F_D(λ)ε_A(λ)λ⁴d(λ) [4]

Where F_D(λ) is the corrected fluorescence intensity of the donor in the wavelength range λ to λ+∆λ with the total intensity (area under the curve) normalized unity; ε_A is extinction coefficient of the acceptor at λ.

From the above equations, it is easily concluded that an efficient FRET should have the following conditions:

• Distance between Donor and Acceptor: Donor and acceptor molecules must be in close proximity (typically 10–100 Å).
• Spectral Overlap: The absorption spectrum of the acceptor must overlap fluorescence emission spectrum of the donor (see Figure 2).
• Dipole Orientation: Donor and acceptor transition dipole orientations must be approximately parallel.

 

Typical Biological Applications of FRET Probes

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As discussed above, the rate and efficiency of FRET are dependent on the inverse sixth power of the intermolecular separation. FRET is highly efficient if the donor and acceptor are positioned within the Förster radius, which is typically 30-60 Å. The distance dependence of FRET has resulted in its widespread biological applications since the distances are comparable with the dimensions of biological macromolecules. For instance, one helical turn in B-DNA spans 33.2 Å. Further, FRET efficiency falls dramatically as the distance between the donor and acceptor exceeds the Förster radius, making FRET an important technique for investigating a variety of biological phenomena in which tracking physical proximity is important. If the donor molecule's fluorescence is quenched, it indicates that the donor and acceptor molecules are within the Förster radius, whereas if the donor fluoresces at its characteristic wavelength, it denotes that the distance between the donor and acceptor molecules has increased beyond the Förster radius. In recent years, FRET-based assays have found broad applications in detecting proteases, real-time assays of hybridization, PCR monitoring and DNA interactions in living cells.

Nucleic Acid Detection

Molecular Beacons, which are 'hairpin' oligonucleotides containing both a fluorescent reporter dye and a quencher as shown in Figure 3, are widely used to detect DNA hybridization. In the absence of target, the fluorescent reporter and quencher molecules are brought close together in the probe's selfcomplementary stem structure, and the fluorescent signal is suppressed. When the Molecular Beacon hybridizes to its target, the fluorescent reporter and the quencher are separated, and the reporter dye emits at its characteristic wavelength. Molecular Beacons have enhanced specificity for their targets, making them superior in hybridizationbased investigations of single nucleotide polymorphisms (SNPs). Molecular Beacons have been used in hybridization-based assays to identify rifampin-resistant Mycobacterium and to detect virus replication in HIV type 1-infected individuals. Molecular Beacons have also been widely used to detect nucleases. Dual-labeled FRET probes are commonly used to detect the 5' exonuclease activity of Taq polymerase. The two labels on the FRET oligonucleotide can be separated by as many as 25-30 bases. If the oligonucleotide is intact, the donor molecule's signal is quenched. During the course of the assay, the cleavage event separates the donor molecule from its acceptor, restoring the donor's characteristic emission. If a dark quencher is used as the acceptor molecule, multiple FRET probes can be used, each labeled with a unique fluorophore, making these probes amenable to multiplex assays.

AAT Bioquest offers a complete set of dye phosphoramidites and dye CPG supports for preparing FRET oligonucleotides including the classic dyes (such as FAM, HEX, TET and JOE) and superior oligo-labeling dyes such as our Tide Fluor™ and Tide Quencher™ dyes. Our Tide Fluor™ dyes (such as TF1, TF2, TF3, TF4, TF5, TF6, TF7 and TF8) have stronger fluorescence and higher photostability than the typical fluorophores such as fluoresceins, rhodamines and cyanines as described below. Our TF2 has the essentially same excitation and emission wavelengths as those of carboxyfluoresceins (FAM), making them readily used for the biological applications that were done with fluoresceins, but have enhanced performance with TF2. Compared to FAM probes, TF2 has much stronger fluorescence at physiological conditions, and it is much more photostable. Compared to other fluorescent dyes alternative to fluoresceins and Cy dyes (such as Alexa Fluor^® and Cy3, Cy5 and Cy7), Tide Fluor™ dyes are much more cost-effective with comparable or even better performance for your desired biological applications.

Besides our outstanding Tide Fluor™ donor dyes, AAT Bioquest has also developed the robust Tide Quencher™ acceptor dyes. These Tide Quencher™ dark FRET acceptors (such as TQ1, TQ2, TQ3, TQ4, TQ5, TQ6 and TQ7) are optimized 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.

To facility your research, we offer a variety of reactive forms for both our Tide Fluor™ donors and Tide Quencher™ acceptors. For in-synthesis labeling of oligonucleotides, we offer both phosphoramidites of our Tide Fluor™ and Tide Quencher™ dyes and their CPG supports. For post labeling of oligonucleotides, we offer both amino-reactive and thiol-reactive Tide Fluor™ and Tide Quencher™ dyes that are water-soluble.

Protease Detection

Proteases are involved in a number of physiological processes. The detection of proteases and the screening of specific protease inhibitors are essential in the discovery of 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. For example, EDANS and DABCYL are used to label a peptide that contains the HIV protease cleavage site. Proteolytic cleavage releases a fragment containing only the EDANS fluorophore, thus liberating it from the quenching effect of the nearby DABCYL chromophore. AAT Bioquest offers a variety of reagents for developing FRET-based protease probes and assays. Please visit our websites at www.aatbio.com for protease assays or www.bioconjugation.com for FRET probe building blocks.

HIV protease has been identified as one of the key targets for the development of anti-AIDS drugs. The aspartic HIV protease, a 10-12 kD of human immunodeficiency virus-1 (HIV-1), is required for the posttranslational cleavage of the precursor polyproteins, Prgag and Prgag-pol. These cleavages are essential for the maturation of HIV infectious particles. A Tide Fluor™ 2/Tide Quencher™ 2-derived FRET peptide has been conveniently used in our Amplite Flurorimetric HIV Protease Assay Kit for high throughput screening of HIV-1 protease inhibitors and continuous quantification of HIV-1 protease activity. The sequence of this FRET peptide is derived from the native p17/p24 cleavage site on Pr_gag for HIV-1 protease. In the FRET peptide, the fluorescence of Tide Fluor™ 2 is quenched by Tide Quencher™ 2 until this peptide is cleaved into two separate fragments by HIV1 protease.

Tide Fluor™ Donor Dyes, Optimized to Maximize FRET Performance through Enhancing Donor Fluorescence Intensity

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Although EDANS, FAM, TAMRA, ROX, Cy 3 and Cy5 have been widely used to develop a variety of FRET probes, there are still some limitations in the use of these dyes. For example, the weak absorption and environment-sensitive fluorescence of EDANS has severely limited its sensitivity for developing protease assays and nucleic acid detection probes. Compared to EDANS, fluorescein-based probes (such as FAM, HEX, JOE and TET) have stronger absorption and fluorescence. However the fluorescence of fluorescein-based probes is strongly dependent on pH. They only exhibit the strongest fluorescence at higher pH. This pH dependence makes the fluorescein-based fluorescent probes inconvenient for the assays that require low pH. In addition, most of fluorescein-based probes have quite low photostability, which limits their applications in fluorescence imaging. Among cyanine dyes, non-sulfonated Cy3 and Cy5 are widely used for developing a variety of nucleic acid probes, but they have quite low fluorescence quantum yield in aqueous media. The sulfonated Cy3 and Cy5 have improved fluorescence quantum yield than those of non-sulfonate cyanines. However, the sulfonated Cy3 and Cy5 are difficult to use in the synthesis of fluorescent oligonucleotides, and are quite cost-prohibitive.

Key Features of Tide Fluor™ Donors

• Optimized to pair with Tide Quencher™ dark acceptors to maximize the FRET potentials
• Stronger fluorescence intensity to enhance assay sensitivity
• pH-insensitive and environment-insensitive fluorescence to simplify assays
• Higher photostability to improve the quality of fluorescence imaging
• A variety of reactive forms available for conjugations

1. *Texas Red^® is the trademark of Molecular Probes, Inc.

To address these limitations, we have developed Tide Fluor™ donor dyes that are optimized as building blocks for developing FRET oligonucleotides and peptides for a variety of biological applications. Our Tide Fluor™ dyes (such as TF1, TF2, TF3, TF4, TF5, TF6, TF7 and TF8) have stronger fluorescence and higher photostability than the typical fluorophores such as fluoresceins, rhodamines and cyanines as described below. Our TF2 has the essentially same excitation and emission wavelengths to those of carboxyfluoresceins (FAM), making them readily used for the biological applications that were done with fluoresceins, but have enhanced performance with our TF2 probes. Compared to FAM probes, TF2 has much stronger fluorescence at physiological conditions, and it is much more photostable. Compared to other fluorescent dyes alternative to fluoresceins and Cy dyes (such as Alexa Fluor™ and Cy3, Cy5 and Cy7), Tide Fluor™ dyes are much more cost-effective with comparable or even better performance for your desired biological applications.

Tide Quencher™ Acceptor Dyes, Optimized to Maximize FRET Performance through Enhancing Quenching Efficiency

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Although DABCYL has been used to develop a variety of FRET applications, its low quenching efficiency of longer wavelength dyes (such as fluoresceins, rhodamines and cyanines) has limited its use in the development of sensitive fluorogenic FRET probes. Additionally, the absorption spectrum of DABCYL is environment-sensitive. AAT Bioquest has developed the robust Tide Quencher™ acceptor dyes for the development of longer wavelength FRET probes. These Tide Quencher™ dark FRET acceptors (such as TQ1, TQ2, TQ3, TQ4, TQ5, TQ6 and TQ7) are optimized 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™ donors 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.

Besides their broad applications in the development of Molecular Beacon probes, our Tide Quencher™ dyes have also been used to develop various protease substrates such as HIV protease (see above), MMPs and secretases. In some cases, it has demonstrated greatly improved enzyme performance. This may be partly due to the red-shifted absorption spectrum that overlaps better with the emission spectrum of fluoresceins, rhodamines and cyanines. Tide Quencher™ dyes are great choice for you to eliminate the limitations of classic quenchers. Tide Quencher ™ dyes are excellent dark quenchers that are individually optimized to pair with all of the popular fluorescent dyes such as fluoresceins and rhodamines. Our Tide Quencher™ series of nonfluorescent dyes cover the full visible spectrum with unusually high efficiency. TQ2 has absorption maximum perfectly matching the emission of FAM (See Figure 6) while TQ3 is proven to be the best quencher for TAMRA and Cy3. In summary, our Tide Quencher™ dyes have the following advantages:

• Most Powerful: enable you to explore the FRET potentials that might be impossible with other quenchers.
• Versatile Reactive Forms: convenient for self-constructing your desired FRET biomolecules.
• A Complete Set of Dyes: perfectly match your desired fluorescent donors.
• Enhanced Value: competitive price with the best performance.

1. *Texas Red^® is the trademark of Molecular Probes, Inc.

Selection of FRET Donors and Acceptors

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Probes incorporating fluorescent donor/non-fluorescent acceptor (e.g. Dabcyl) combinations have been developed primarily for the detection of proteolysis and nucleic acid hybridization. This principle has also been used to develop other FRET-based assays such as receptor/ligand interactions, distribution and transport of lipids, membrane potential sensing and cyclic AMP detection.

The donor and acceptor molecules can be the same or different. In most applications, they are different dyes. FRET can be detected either by the appearance of sensitized fluorescence of the acceptor or by the intensity ratio change of donor/acceptor (if the acceptor is fluorescent), or the fluorescence decrease of the donor. In the later case, acceptor can be either fluorescent or non-fluorescent. A number of Ro values for various D/A pairs have been published. Larger Ro values of FRET pairs give higher FRET efficiency. Fluorescein/tetramethylrhodamine pairs have been most widely used to develop FRET-based assays. EDANS/DABCYL pair has been intensively used to develop FRET-based nucleic acid probes and protease substrates. MCA (7-methoxycoumarin-4-acetic acid) and DNP (2, 4-dinitroaniline) are another pair of donor/acceptor for developing FRET-based fluorescent probes. Compared with the pair of EDANS/DABCYL, MCA/DNP pair usually has shorter and weaker wavelength of fluorescence signal. However, the pair often demonstrates better affinity or turnover rate due to their smaller sizes. DNP is also a good FRET acceptor paired with tryptophan (Trp), 2-aminobenzoic acid (Abz) or Abz derivatives such as Abz(N-Me).

1. *The value may change under different conditions; **IAEDANS = 5-((Iodoacetyl)amino)naphthalene-sulfonic acid.

Table 4 summarizes our Tide Quencher™ FRET building blocks designed for you to develop FRET probes for the demanding applications. Based on our in-house research and experience, it is recommended to use the FRET pairs marked in green. The pairs marked in cyan color are OK to use, but less efficient than the ones marked in green. We have proved that these recommended FRET pairs have demonstrated high sensitivity and low background in our protease and nucleic acid detection assays. There are also other factors that you need to consider besides the FRET efficiency, such as pH, multiplexing and buffer interference, etc.

Ordering Information

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