Measuring Quenching Efficiencies

See Notes 3-5 and Fig. 6 for examples of assays measuring quenching efficiencies for probe evaluation. Experimental conditions, such as temperature and buffer composition, can dramatically affect quenching efficiency and dye fluorescence (see Notes 6 and 7).

3.2.1. Sample Preparation and Analysis

1. Probe concentrations in samples for fluorescence measurements should be less than 0.5 |M. This is because fluorescence measurements can become distorted owing to the re-absorption of emitted light (8).

2. All fluorescence intensities should be corrected by subtracting the fluorescence intensity of a buffer blank.

3. Quenching data is often reported as a signal/background ratio where the background is from the intact (quenched) probe and the signal is recorded after hybridization or nuclease treatment (dequenched). Percentage quenching is calculated by dividing the signal of the dequenched probe (minus buffer blank) by the signal of the quenched probe (minus buffer blank), multiplying the result by 100, and then subtracting the result from 100.

3.2.2. Hybridization Assay

1. In a hybridization assay, it is important to use a buffer that contains Mg2+, such as PCR buffer (10 mM trizma hydrochloride, 50 mM KCl, and 3.5 mM MgCl2).

Rster Resonance Energy Transfer
Fig. 3. Structures used by Marras et al. (5) to measure percentage quenching via Förster resonance energy transfer and contact quenching.

2. If a complementary sequence has extra bases on each end, the binding is stronger than if exact complement is used. (The complementary sequences used in Fig. 5 have three extra T bases on each end.)

3. A probe should be used that has a melting temperature above room temperature (see Note 8).

4. After adding a fivefold molar excess of complement, the fluorescence intensity should be monitored until it reaches a final value. This usually takes between 5 and 15 min.

3.2.3. Nuclease Digestion Assay

1. There are several nucleases that can be used for digestion assays. Snake venom phosphodiesterase with bacterial alkaline phosphatase yield the nucleoside monomers through exonuclease digestion (9). DNase I and Bal 31 are both endonu-cleases that degrade both single and double-stranded DNA (USB, cat. no. 14367 and 70011Y).

2. The probe should be dissolved in the buffer for enzyme digestion and split into two fractions, one of which is a control that does not receive the digestion enzyme.

3. The extent of oligo digestion can be monitored by anion-exchange high-performance liquid chromatography.

4. After incubation, both the control and the digested sample should be diluted with buffer (e.g., PCR buffer) and the fluorescence intensities can be measured.

3.2.4. Determination of Quenching Mechanism

In order to distinguish definitively between FRET and static quenching it is necessary to measure fluorescence lifetimes. The fluorescence lifetime is the average time before a dye emits a photon after the dye has absorbed a photon.

Dynamic quenching, which includes FRET, decreases both fluorescence intensity and fluorescence lifetime of the reporter dye by the same factor. In

Fig. 4. Selected percentage quenching data from Marras et al. (5). Dark lines show contact quenching and striped lines show Förster resonance energy transfer quenching.
Fig. 5. Percentage quenching with dual-labeled P-actin probes. The P-actin sequence is 5'-d-ATG-CCC-TCC-CCC-ATG-CCA-TCC-TGC-G-3'. Dark lines show hybridization and striped lines nuclease digestion.

contrast, static quenching involves formation of a dye-quencher dimer. This effectively decreases the concentration of the fluorescent dye by creating a new, nonfluorescent reporter-quencher dimer. Because this dimer is formed before the dye absorbs a photon, static quenching does not change the reporter dye's fluorescence lifetime.

In a fluorogenic assay with a dual-labeled probe, if both the fluorescence intensity (I) and fluorescence lifetime (t) change by the same amount going from the quenched to dequenched species, i.e., IquencheAequenched = Tquenched/Tdequenched, it can be concluded that dynamic quenching is the mechanism at work.

If T quenched = Tdequenched while Iquenched/Idequenched is less than On^ Static q^nchmg is the mechanism at work. Another indication of static quenching is a change in the absorption spectrum of the probe in the quenched vs dequenched states (Fig. 7).

Linear Probe

Molecular Probe with Beacon G. S. Complex

Quenched Forms:

Fluoresce™ Release

Fluoresce™ Release

Fig. 6. Quenched probe structures and the release of fluorescence via digestion by nuclease and hybridization.

This is because the reporter-quencher dimer has its own unique absorption spectrum. FRET quenching does not effect the probe's absorption spectrum (2).

1. There are several commercial sources for fluorogenic oligonucleotide probes and some companies offer proprietary dyes. The BHQs, Quasar, and Cal Dyes were developed at Biosearch Technologies.

2. Most real-time PCR instruments use filters to selectively monitor reporter fluorescence. The ABI PRISM® 7700 and 7900 perform spectral deconvolution. The instrument user manual may suggest combinations of reporters for multiplexing experiments.

3. There are several ways to screen combinations of reporter/quencher pairs. A series of quencher-reporter pairings were recently tested by Marras, Kramer, and Tyagi. They used complementary oligos with 5'-reporters and 3'-quenchers to bring the dyes directly together or at staggered distances to measure, respectively, contact-mediated and FRET quenching efficiencies (5) (Fig. 3). This method of bringing the dyes together in order to measure contact (static) quenching holds the reporter and quencher in a fairly constrained and fixed relative orientation. Furthermore, in this model, the reporter-quencher interaction may strongly depend on the length and rigidity of the oligo-dye linkers. Figure 4 shows the Marras et al. (5) quenching efficiencies for a series of reporter/quencher pairs with emission maxima spanning from 441 to 702 nm.

4. Notes

Fig. 7. Absorption spectrum of a Cy5/BHQ-1 probe alone (solid line) and hybridized to a complementary oligonucleotide (dashed line)

420 470 520 570 620 670 X (nm)

Fig. 7. Absorption spectrum of a Cy5/BHQ-1 probe alone (solid line) and hybridized to a complementary oligonucleotide (dashed line)

4. The data in Fig. 5 show percentage quenching that has been measured in a series of "linear" 5'-reporter-3'-quencher probes. The efficiency of static (also known as contact-mediated) quenching depends on the affinity of the reporter and quencher for each other (i.e., association constant). In a linear probe, the strength of this affinity is more critical than in a probe in which the reporter and quencher are held together through hybridization (10). Also, a linear probe can be thought of as a flexible linker that will allow the dyes to associate in a wider variety of conformations. Therefore, reporter-quencher pairs held in a fixed configuration, such as a molecular beacon or a hybrid, might have different quenching efficiencies than the same pair in a "linear" dual-labeled probe. These different structures are illustrated in Fig. 6. The quenching efficiency values measured by nuclease digestion differ from those measured via hybridization because the hybrid structure has an effect on the fluorescence intensities of many reporters (11). The 25mer linear probes with BHQ-2 as the quencher and Quasar-670, Cal Red, Quasar-570, and TAMRA as reporters all have quenching efficiencies greater than 90%. Such high quenching efficiencies in "linear" probes are indicative of static quenching.

5. The BHQ are aromatic and quite hydrophobic (in fact, many dyes are only water-soluble after conjugation to oligos). One might expect that a hydrophobic reporter dye should be used to increase reporter-BHQ association. Some reporter dyes have phosphonate or sulfonates appended in order to increase water solubility. However, there is not very much data on intramolecular heterodimers with water-soluble dyes. Surprisingly, a sulfonated Cy 3 (Amersham Biosciences, cat. no. PA13101)/BHQ-2 P-actin probe has a quenching efficiency of 93% on both hybridization and nuclease digestion. Changes in the absorption spectra suggest formation of a reporter-quencher intramolecular dimer.

6. Quenching efficiency within dual labeled probes can be temperature dependent. Association between the reporter and quencher that controls formation of the intramolecular dimer decreases with increasing temperature. Thus, efficient static (or contact) quenching at room temperature may significantly decrease at higher temperatures.

7. The fluorescence quantum yields of some dyes, such as the cyanines, decrease significantly with increasing temperature. Fluorescence intensity can also depend on pH and the local environment of the dye (12).

8. If a reporter-quencher pair that form a strong ground state complex are used in a molecular beacon (or other type of self-hybridizing probe), the additional stabilization owing to the reporter-quencher association can inhibit the molecular beacon from hybridizing to the complementary sequence at room temperature.

0 0

Post a comment