Quenching Mechanisms in Probes |
Fluorescence Quenching Mechanisms in Probes for Genetic Analysis
Fluorescent probes are used in biochemical assays to monitor specific events such as binding, cleavage or conformational changes. Dual labeled probes with a fluorophore and a quencher have many applications in genetic analysis. The efficiency of fluorescence quenching is very distance dependent – if the reporter fluorophore and quencher are far apart, there is fluorescence; if the reporter and quencher are close together in space, fluorescence is suppressed. Typically, the reporter and quencher are placed at specific sites in an oligonucleotide such that a change in their distance will produce a maximal change in fluorescence and effectively signal the event being monitored (often hybridization or nuclease activity). The oligonucleotide acts as a flexible tether linking the fluorescent reporter and quencher.
Until 2002 it was accepted or assumed that reporter-quencher pairings should be made according to Förster theory. However, many dyes are known to aggregate, self-associate, form dimers, trimers, etc. This tendency for dyes to aggregate is the basis of the static quenching mechanism.
Static Quenching (also known as contact quenching):
A reporter (such as FAM) and a quencher (such as BHQ-1 label) can bind together to form a new, nonfluorescent species called an intramolecular dimer. The biomolecule linking the reporter and quencher acts as a tether and enables intramolecular association between the reporter and quencher. Static quenching efficiency depends on the affinity of the reporter and quencher for each other. The reporter and quencher often are planar, hydrophobic molecules that stack together to avoid contact with water. Static quenching is less important at high temperature. FRET quenching and static quenching can occur together.
In 2002 two seminal papers were published:
 Title: Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes. Authors: Salvatore A. E. Marras, Fred Russell Kramer and Sanjay Tyagi Journal: Nucleic Acids Research, 2002, 30, e122. This publication compares many reporter-quencher pairs and assesses their FRET and contact quenching efficiencies. |
 Title: Intramolecular Dimers: A New Strategy to Fluorescence Quenching in Dual-Labeled Oligonucleotide Probes. Authors: Mary Katherine Johansson, Henk Fidder, Daren Dick and Ronald M. Cook. Journal: J. Am. Chem. Soc. 2002, 124, 6950. This publication carefully describes and characterizes a previously overlooked fluorescence quenching mechanism in dual-labeled oligonucleotide probes |
Other Biosearch publications describing static quenching: Title: Choosing Reporter-Quencher Pairs for Efficient Quenching Through Formation of Intramolecular Dimers. Authors: Johansson, M.K. Book: Methods in Molecular Biology, v. 335; V.V. Didenko, Ed; Humana Press: Totowa, NJ, 2006; pp 17-29. |
 Title: Intramolecular Dimers: A New Design Strategy for Fluorescence-Quenched Probes. Authors: Johansson, M.K.; Cook, R.M. Journal: Chem.-Eur. J. 2003, 9, 3466. This is the featured cover article. |
FRET (Förster Resonance Energy Transfer) Quenching:
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| Spectral Overlap for FRET |
Unlike static quenching, FRET is a through-space mechanism in which energy from the reporter dye is transferred to the quencher without absorption or emission of light.
Efficient FRET requires:
a) Proximity: the donor and acceptor molecules must be close to each other (approx. 10 – 100 Å). FRET is extremely distance dependent.
b) Spectral overlap: the absorption spectrum of the acceptor must overlap with the emission spectrum of the donor.
c) Relative donor-quencher orientation: in most assays with fluorescent probes, it is assumed that the relative orientation of the dyes is random.
Key equation for FRET: 
Where E is the efficiency of FRET energy transfer, R0 is the Förster distance which is the donor-acceptor distance at which energy transfer is 50%, and r is the distance between the donor and acceptor.
Distinctions between Static Quenching and FRET
Static and FRET quenching can occur together.
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| Physical contact between reporter & quencher | Reporter and quencher interact through space | |
| Temperature and solvent dependent | Not very temperature and solvent dependent | |
| Absorption spectrum of reporter distorted | Absorption spectrum unchanged | |
Quenching in a dual-labeled probe can occur through a combination of static and FRET quenching.
Difficulty Applying Förster Distance Data to Oligo Probes
Förster distances can be calculated from spectral data. However, this data is often of limited use for oligo probes for the following reasons:
1) The flexible tethers linking the dyes to the oligo makes distance estimates difficult - this complicates both measuring and applying Förster distances.
2) Förster quenching is not only distance dependent, but dependent on the relative dye-quencher orientation.
3) If there is good spectral overlap, a reporter dye-quencher pair will have efficient FRET quenching. In other words, good pairs can be "eyeballed". The reporter-quencher pairs that Biosearch recommends for dual-labeled BHQ probes have been tested in many PCR assays as indicated in Biosearch's Dye Selection Chart.
4) Static quenching (ground state complex formation or contact quenching) can be another important quenching mechanism that can operate simultaneously.
Illustration of Extreme Distance Dependence with FRET
In this example we assume a Förster distance of 55 Å and use the equation E = R06 / (R06 + r6) where E is the energy transfer efficiency, R0 is the Förster distance and r is the experimental distance. The graph below illustrates the dependence of quenching efficiency and signal/background with the reporter-quencher distance.
| Distance | Quenching efficiency | Signal/background (signal = 100%) |
| 55 Å | 50% | 2.0 |
| 48 Å | 69% | 3.2 |
| 34 Å | 95% | 20 |
Thus, in this FRET pair, going from a reporter-quencher distance of 34 Å to 48 Å causes a dramatic change in the
signal/background ratio: from 20 to 3.2.
Choice of Quenchers: The evolution from TAMRA to Dabcyl to BHQ labels
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FAM-TAMRA probes, oligonucleotide probes with fluorescein (FAM) as a 5' label and tetramethylrhodamine (TAMRA) as a 3' label, were commonly used before the availability of dark quenchers and are still used by some groups today. With these probes, FAM is the reporter dye and TAMRA is the acceptor dye. These probes work well due to the good spectral overlap between the FAM fluorescence and TAMRA absorption curves. In a FRET assay, the FAM dye is excited at 490 nm and transfers energy to TAMRA. The FRET mechanism returns the FAM reporter to the ground state and generates TAMRA in an electronically excited state without any light emission or absorption. Due to FRET, the amount of FAM fluorescence loss at 520 nm is proportional to the amount of TAMRA fluorescence gain at 580 nm.
Unfortunately, when a fluorescent dye, such as TAMRA, is used as a quencher, its fluorescence can contribute to background signal. In addition, such quencher fluorescence makes multiplexing, in which more than one reporter-quencher pair is used simultaneously in a reaction, more difficult. These limitations led to the use of Dabcyl which is known as a dark quencher because it is not fluorescent. Dabcyl, however, has limited utility as a FRET quencher because it has an absorption maximum at 453 nm, which is blue-shifted and far removed from the emission maxima of most reporters. Therefore, Dabcyl has poor spectral overlap and consequently minimal FRET quenching.
Black Hole Quencher® (BHQ®) labels are a family of dark quenchers with excellent spectral overlap with all common reporter dyes for efficient FRET quenching. As a result, BHQ labels have become the industry standard for quenchers in dual-labeled oligonucleotide probes. The most recent or “highly evolved” addition is the BHQplus™ probe. The BHQplus probe is a novel, compact probe for qPCR with a duplex stabilizing technology terminated with Biosearch’s Black Hole Quencher dye. The stabilizing technology permits the design of shorter oligonucleotides which enables greater specificity as well as the detection of more difficult targets such as AT-rich regions. BHQplus probes are especially well suited to detect single nucleotide polymorphyisms.
