Suppressing Background Fluorescence by Dual Quencher Labeling

The sensor design described above is highly sensitive and selective for Pb2+. However, the detection has to be performed at 4°C. If the temperature at which the experiment is carried out is raised to room temperature, high background fluorescence is observed, with only 60% fluorescence increase upon addition of Pb2+, as compared to the approx 300% increase at 4°C (13). At elevated temperatures, a fraction of uncleaved substrate dissociates from the enzyme strands (Fig. 4A), resulting in increased background fluorescence (Fig. 4C)

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Fig. 4. Further improvement of DNAzyme fluorescent sensors (13). (A) The DNAzyme complex is stable at 4°C. At room temperature a small portion of the substrate can dissociate from the DNAzyme, which increases background fluorescence. (B) By adding a second quencher on the other end of the substrate strand, the fluorescence of free substrate is also suppressed, resulting in decreased background. (C) With the design shown in (A), only 60% fluorescence increase is observed at room temperature. (D) With the design shown in (B), 660% fluorescence increase is observed. (E) Sensor response in the presence of different metal ions with differing concentrations. d

Fig. 4. Further improvement of DNAzyme fluorescent sensors (13). (A) The DNAzyme complex is stable at 4°C. At room temperature a small portion of the substrate can dissociate from the DNAzyme, which increases background fluorescence. (B) By adding a second quencher on the other end of the substrate strand, the fluorescence of free substrate is also suppressed, resulting in decreased background. (C) With the design shown in (A), only 60% fluorescence increase is observed at room temperature. (D) With the design shown in (B), 660% fluorescence increase is observed. (E) Sensor response in the presence of different metal ions with differing concentrations. d

(13). To suppress background fluorescence at higher temperatures, methods to increase the stability of the DNAzyme complex can be employed, such as increasing NaCl concentration, increasing the length of recognition arms, or increasing the G-C basepair content of the recognition arms. All these methods increase the stability of the DNAzyme complex, and, therefore, should decrease background fluorescence at room temperature. However, these methods cannot be generally applied to a wide temperature range. To observe metal-induced fluorescence increase, the substrate strand has to first be cleaved by the enzyme, and then dissociate from the enzyme strand. Therefore, the binding of the substrate to the enzyme strand should not be too strong. With the previously mentioned methods to increase binding affinity, a detection condition that works at a higher temperature may not be used at lower temperatures, because the cleavage product release may be inhibited. Therefore, we chose to design the sensor as shown in Fig. 4B (13). A second quencher is added to the other end of the substrate strand. This intramolecular quencher partially quenches the fluorescence of free substrate. Thus, the new design contains two quenchers. The advantage of the inter-molecular quencher (the quencher on the enzyme strand) is that it quenches the fluorophore on the opposing strand with almost 100% efficiency owing to the very close fluorophore-to-quencher distance. However, not all substrate strands are annealed to enzyme strands, which causes background fluorescence. This is corrected by the intramolecular quencher (the quencher on the substrate strand). The new design takes advantage of both quenchers, and shows a 660% fluorescence increase at room temperature (Fig. 4D) (13). The response of the sensor with two quenchers in the presence of different metal ions with different concentrations is presented in Fig. 4E. High fluorescence intensity is observed only when Pb2+ is present. Described next are the detailed protocols for performing Pb2+ detection using the new design. Because most procedures are identical to the design previously described, only new procedures are presented.

1. Assay sample preparation: Dissolve FAM and dabcyl dual-labeled substrate (17DS-FD, see Table 1 for sequence), and 17E-Dy to the final concentration 50 nM each in 50 mM Tris-acetate buffer, pH 7.2, with 50 mM NaCl (see Note 5 for the choice of fluorophore-quencher pair). Anneal the sample using the protocol previously described.

2. Assay protocol: use a 96-well plate for the assay. Fill a row of 8 wells in the 96-well plate with 95 pL (in each well) of the above annealed DNA sample at room temperature. Fill another row of the plate with seven different 20X concentrated metal ion stock solutions, and fill the final well in that row with water as an internal standard for comparison. Initiate the cleavage reaction by adding 5 pL of each metal ion solution to the corresponding wells containing the sensor solution with an 8-channel pipet.

3. Fluorescence monitoring: acquire the fluorescence signal on a multi-functional three-laser fluorescence image analysis system. Set the excitation wavelength at 473 nm, and the filter at 520 nm to monitor the FAM fluorescence.

4. Notes

1. Preparation of Pb2+ solution: Lead acetate Pb(OAc)2 should be dissolved in acid instead of water. Prepare a stock solution of 100 mM lead acetate by dissolving solid lead acetate in 5% (by volume) acetic acid. Serial dilutions should be performed by diluting stock in 50 mM acetic acid to prevent hydrolysis. Because the final Pb2+ concentration used in an assay is usually less than 10 |M, and the metal ion solution added is less than 5% of the total volume, the effect on pH variation is minimal. Pb(OAc)2 hydrolyzes if it is dissolved directly in H2O, which decreases the effective Pb2+ concentration.

2. The annealing step is important for the success of DNAzyme-based metal detections. After heating to 90°C, the sample should be cooled slowly to 4°C, instead of directly transferring it to a 4°C environment as sudden cooling may lead to misfolding of DNAzymes.

3. After annealing the substrate and enzyme strands, the sample should be used within several hours because the enzyme may very slowly cleave the substrate in the presence of trace amount of divalent metal ions. The annealed samples should be freshly prepared to assure high sensitivity, although the substrate and enzyme strands can be prepared in advance and stored separately for years in a freezer.

4. For detection of Pb2+ in water samples, such as lake water, DNA should be annealed at higher concentrations in the buffer, for example 500 nM each of the substrate and the enzyme. Add a small volume (i.e., 10% of the volume of the water sample to be tested) of the high concentration DNAzyme sensor into the water sample. Annealing DNA samples at very high concentrations such as 100 |M should be avoided, because with the increase of DNA concentration, the likelihood of forming inactive DNAzyme dimer may also increase (19).

5. The choice of fluorophore-quencher pair has large effects on the success of the sensor design. There are at least two mechanisms for fluorophore-quencher interactions. The first mechanism is through fluorescence resonance energy transfer (FRET), when the fluorophore and quencher are separated by 10-100 A. The emission of the fluorophore should have large overlap with the absorption spectrum of the quencher for higher quenching efficiency. Another, static quenching mechanism acts through ground-state complex formation when the fluorophore-to-quencher distance is too short. In this case, the spectral overlap is not important. Both of these quenching mechanisms can be utilized. For example, dabcyl is not a good quencher for TAMRA, from the point of FRET-based quenching. However, in the first design, the TAMRA-dabcyl pair is still capable of efficient quenching, because of the very short distance between them. In the second design, the fluorophore is changed from TAMRA to FAM, because in this case, the intramolecular quenching is based on FRET, and FAM can be efficiently quenched by dabcyl.

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