Fluorescence-Quenching

A fluorescent reporter is a molecule that absorbs light (excitation) at a characteristic wavelength and emits light (fluorescence) at a lower-energy, longer wavelength. A quencher is a molecule that absorbs or dissipates energy from an excited fluorophore (the reporter), returning it to the ground state without any fluorescent emission by that fluorophore. Capture and transfer of light energy in this fashion is referred to as "quenching". For a more detailed description of fluorescent dyes, click here.

Quenchers

Quenchers can be selected from a wide variety of compounds, many of which are also fluorescent dyes. Energy from a "higher energy" dye can be captured or transferred to a "lower energy" dye, one with an absorption max at a longer wavelength. Energy transfer from the reporter excites the quencher, which itself emits a fluorescent signal at the wavelength characteristic for that dye. For example, a combination of Fam and Tamra will absorb at 492 (excitation peak for fluorescein) and emit at 580 (emission peak for Tamra).

The example above invokes fluorescence resonance energy transfer (FRET) as the quenching mechanism. FRET permits interactions over relatively long distances and oligonucleotide probes can have the reporter and quencher groups separated by > 30 bases and still achieve useful quenching. Quenching is highly efficient within the Förster radius of the donor/acceptor pair (which is often in the 50 - 60 Å range). Outside this distance, quenching efficiency falls off rapidly, decreasing by the inverse sixth power of the intermolecular separation. The Förster radius is unique for each reporter/quencher pair. When employing a FRET quenching mechanism, the reporter/quencher pair must be carefully chosen to have a compatible spectral overlap. The emission spectra of the reporter must overlap with the excitation spectra of the quencher for FRET to occur. For a more detailed examination of FRET, click here.

Interestingly, Dabcyl can be used as a quencher for Fluorescein even though the absorbance maximum for Dabcyl occurs at a higher energy than the Fluorescein emission peak. Here, reporter and quencher must be in close physical proximity for quenching to be efficient. The utility of this combination is limited to probe designs where distance between the reporter and quencher is minimized, such as short end-labeled oligos, longer oligos that employ internal labeling (keeping R/Q distance < 8-10 bases), or any oligo in which secondary structure brings the reporter and quencher into contact. For example, in Molecular Beacons, the reporter group and Dabcyl are brought together by hairpin/stem formation, and the reporter is quenched. Upon hybridization with target, the stem structure is lost and the reporter and Dabcyl groups become separated by the full length of the probe (usually >20 bases); quenching no longer occurs and fluorescence of the reporter molecule is observed. For more information about the design and use of Molecular Beacons, click here.

New "dark quenchers" are available which capture energy from an excited reporter molecule without subsequent emission of light, i.e., they do not fluoresce. Dark quenchers have advantages over fluorescent quenchers in many applications.

Their benefits include:

• Probes made using dark quenchers tend to be more sensitive in quantitative detection systems (such as real time PCR), primarily due to lower background fluorescence than probes that employ fluorescent quenchers.
• Dark quenchers enable use of a wider range of reporter dyes, expanding the options available for multiplexed assays.
• Dark quenchers enable design of visual assay formats.

Click on a quencher to view its absorption spectra.

Quencher	Abmax	Availability
Dabcyl	453 nm	3'-CPG or NHS-ester
BHQTM-1	534 nm	3'-CPG
QSYTM-7	574 nm	NHS-ester
BHQTM-2	579 nm	3'-CPG
QSYTM-7 is licensed from Molecular Probes, Eugene, OR. BHQTM (Black Hole Quenchers) are licensed from Biosearch Technologies, Novato, CA.

Figure 1: Spectral Overlap of Dark Quenchers and Fluorescent Dyes

• Even a small percentage of probe molecules that are missing the quencher group can significantly increase background fluorescence and can interfere with an assay. IDT therefore strongly recommends that all oligos made with dark quenchers be purified by HPLC.
• BHQ-1^TM and BHQ-2^TM are available from IDT as 3'-end modifications which are attached during oligo synthesis using BHQ-CPG. QSY-7^TM is attached to oligos using NHS-ester chemistry after synthesis and can be positioned at the 5'-end, 3'-end, or at an internal amino-dT base. IDT recommends use of BHQs for 3'-end modification and QSY-7 when 5'-end or internal modification is needed.
• Dabcyl should only be used when the reporter and quencher will be in close physical proximity, such as Molecular Beacons.
• Black Hole Quenchers^TM produce an extra peak if examined using MALDI-TOF Mass Spectroscopy at 300 daltons below the true molecular weight that results from dye cleavage during laser desorption. This peak is an artifact and is not a concern. For more information about Mass Spectroscopy analysis of oligonucleotides, click here.

Mark Behlke, M.D., Ph.D.