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Fluorogenic probes dual-labeled with reporter and quencher dyes use a change in fluorescence to monitor biochemical events (e.g., substrate binding or enzyme digestion). Such events change the reporter-quencher distance, which affects fluorescence. Recently, it is has been shown that static quenching through intramolecular dimers is an important mechanism that can sometimes be more efficient than Förster resonance energy transfer (FRET).

The group II azoreductase BTI1 utilizes NADPH to directly cleave azo bonds in water-soluble azo dyes, including quenchers of fluorescence. Unexpectedly, optimal reduction was dye specific, ranging from a pH of 8.3 for flame orange.

Fluorescent energy transfer within dual-labeled oligonucleotide probes is widely used in assays for genetic analysis. Nucleic acid detection/amplification methods, such as real-time polymerase chain reaction, use dual-labeled probes to measure the presence and copy number of specific genes or expressed messenger RNA. Fluorogenic probes are labeled with both a reporter and a quencher dye. Fluorescence from the reporter is only released when the two dyes are physically separated via hybridization or nuclease activity. Fluorescence resonance energy transfer (FRET) is the physical mechanism that is most often cited to describe how quenching occurs. We have found that many dual-labeled probes have enhanced quenching through a nonFRET mechanism called static quenching. Static quenching, which is also referred to as contact quenching, can occur even in "linear" oligonucleotide probes that have no defined secondary structure to bring the reporter and quencher pair into proximity. When static quenching accompanies FRET quenching, the background fluorescence of probes is suppressed. This chapter describes how to pair reporter and quencher dyes for dual-labeled probes to maximize both FRET and static quenching. Data comparing various reporter-quencher pairs is presented as well as protocols for evaluation and optimization of the probes.

Fluorescent energy transfer within dual-labeled oligonucleotide probes is widely used in assays for genetic analysis. Nucleic acid detection/amplification methods, such as real-time polymerase chain reaction, use dual-labeled probes to measure the presence and copy number of specific genes or expressed messenger RNA. Fluorogenic probes are labeled with both a reporter and a quencher dye. Fluorescence from the reporter is only released when the two dyes are physically separated via hybridization or nuclease activity. Fluorescence resonance energy transfer (FRET) is the physical mechanism that is most often cited to describe how quenching occurs. We have found that many dual-labeled probes have enhanced quenching through a nonFRET mechanism called static quenching. Static quenching, which is also referred to as contact quenching, can occur even in "linear" oligonucleotide probes that have no defined secondary structure to bring the reporter and quencher pair into proximity. When static quenching accompanies FRET quenching, the background fluorescence of probes is suppressed. This chapter describes how to pair reporter and quencher dyes for dual-labeled probes to maximize both FRET and static quenching. Data comparing various reporter-quencher pairs is presented as well as protocols for evaluation and optimization of the probes.

Lanthanides are attractive as biolabels because their long luminescence decay rates allow time-gated detection, which separates background scattering and fluorescence from the lanthanide emission. A stable and highly luminescent terbium complex based on a tetraisophthalamide (TIAM) chelate is paired with a polyaromatic-azo dark quencher (referred to as a Black Hole Quencher or BHQ) to prepare a series of 5'TIAM(Tb)/3'BHQ dual-labeled oligonucleotide probes with no secondary structure. Luminescence quenching efficiency within terbium/BHQ probes is very dependent on the terbium-BHQ distance. In an intact probe, the average terbium-BHQ distance is short, and Tb --BHQ energy transfer is efficient, decreasing both the terbium emission intensity and lifetime. Upon hybridization or nuclease digestion, which spatially separate the Tb and BHQ moieties, the Tb luminescence intensity and lifetime increase. As a result, time-gated detection increases the emission intensity ratio of the unquenched probe/quenched probe due to the shorter lifetime of the quenched species. A 40-mer probe that has a 3-fold increase in steady-state luminescence upon digestion has a 50-fold increase when gated detection is used. This study demonstrates that time gating with lanthanide/dark quencher probes in energy transfer assays is an effective means of improving sensitivity.

A molecular beacon oligonucleotide probe covalently labeled with Ru(II)(bpy)3 and Black Hole Quencher-2 is synthesized, and hybridization assays are performed using photoluminescence and electrochemiluminescence methods of excitation.

The cover picture shows a 96-well plate illuminated by a UV lamp and viewed through a cutoff filter. All wells contain a Cy3.5-BHQ1 (reporter quencher) dual-labeled oligonucleotide probe in buffer. Complementary sequence was added to three columns. The probe is virtually nonfluorescent due to static quenching through formation of an intramolecular dimer. Addition of complement separates the dye and quencher, causing fluorescence. Intramolecular dimers as a new design strategy for fluorescence-quenched probes is discussed by M. K. Johansson and R. M. Cook in the Concepts article on p. 3466 ff.