Bioconjugate Chem. 2003, 14, 1133−1139
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Inter- and Intramolecular Fluorescence Quenching of Organic Dyes by Tryptophan Nicole Marme´,† Jens-Peter Knemeyer,† Markus Sauer,*,‡ and Ju¨rgen Wolfrum† Physikalisch-Chemisches Institut, Universita¨t Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg, Germany and Fakulta¨t fu¨r Physik, Angewandte Laserphysik und -spektroskopie, Universita¨t Bielefeld, Universita¨tsstrasse 25, 33615 Bielefeld, Germany. Received July 23, 2003; Revised Manuscript Received September 23, 2003
Steady-state and time-resolved fluorescence measurements were performed to elucidate the fluorescence quenching of oxazine, rhodamine, carbocyanine, and bora-diaza-indacene dyes by amino acids. Among the natural amino acids, tryptophan exhibits the most pronounced quenching efficiency. Especially, the red-absorbing dyes ATTO 655, ATTO 680, and the oxazine derivative MR 121 are strongly quenched almost exclusively by tryptophan due to the formation of weak or nonfluorescent ground-state complexes with association constants, Kass., ranging from 96 to 206 M-1. Rhodamine, fluorescein, and bora-diaza-indacene derivatives that absorb at shorter wavelengths are also quenched substantially by tyrosine residues. The quenching of carbocyanine dyes, such as Cy5, and Alexa 647 by amino acids can be almost neglected. While quenching of ATTO 655, ATTO 680, and the oxazine derivative MR121 by tryptophan is dominated by static quenching, dynamic quenching is more efficient for the two bora-diaza-indacene dyes Bodipy-FL and Bodipy630/650. Labeling of the dyes to tryptophan, tryptophan-containing peptides, and proteins (streptavidin) demonstrates that knowledge of these fluorescence quenching processes is crucial for the development of fluorescence-based diagnostic assays. Changes in the fluorescence quantum yield of dye-labeled peptides and proteins might be used advantageously for the quantification of proteases and specific binding partners.
INTRODUCTION
To date, the use of fluorescently labeled proteins and nucleic acids in various biological and medical relevant assays represents standard technology. Usually, the presence or absence of target molecules is monitored by an increase or decrease in fluorescence intensity of one dye interacting via short-range electronic or long-range dipole-dipole interactions with a second dye (1-5). Deteriorations of the interaction geometry or efficiency are caused, for example, by specific binding or cleavage events. Especially, the use of fluorescence resonance energy transfer (FRET) between a donor, D, and an acceptor dye, A, has increased considerably in the field of biodiagnostics during the past decade (6-8). Other important methods rely on two-color cross-correlation to monitor specific binding events at the single molecule level (9-11). That is, probe and target molecule are labeled with two spectrally distinguishable fluorescent dyes. The two dyes are excited using two different lasers or two-photon excitation with a single laser line and their fluorescence is recorded on two spectrally separated detectors. Colocalization of the two dyes in the observation volume is reflected by a higher cross correlation amplitude. On the other hand, the absorption and emission spectra, fluorescence quantum yield, and fluorescence lifetime of dyes often vary with environmental conditions. Hence, instead of using interactions between two extrinsic probes, many fluorescent dyes can be used as sensors * Corresponding author. Phone: +49-521-106-5450. Fax: +49-521-106-2958. E-mail:
[email protected]. † Universita ¨ t Heidelberg. ‡ Universita ¨ t Bielefeld.
to probe their local environments in biological and analytical applications. For example, interactions of fluorophores with DNA bases can be used for the specific detection of DNA or RNA sequences at the single molecule level (12, 13). Nucleobase-specific interactions have been reported for coumarin (14), rhodamine (15-17), oxazine (12, 13, 18), and bora-diaza-indacene (19) dyes. Nucleobase-specific quenching relies on the differences in specific properties of naturally occurring nucleotides, in particular, the low oxidation potential of the DNA base guanosine (14, 20) and the tendency of many fluorophores to aggregate in aqueous environment to decrease their water accessible area. Thus, dependent on the reduction potential of the fluorophore used, efficient photoinduced electron transfer (PET) can occur between the first excited singlet-state of the dye and the ground-state guanosine (18, 21). With careful design of conformationally flexible probes, e.g. DNA-hairpins that carry several guanosine residues in the complementary stem sequence and the use of appropriate fluorophores (rhodamine and oxazine dyes are well suited candidates), efficient single molecule sensitive DNA-hairpins can be produced (12). If quenching interactions between the fluorophore and the guanosine residue are deteriorated upon specific binding to the target, for example, due to binding of a complementary DNA sequence, or due to cleavage by an endonuclease enzyme, fluorescence of the DNA-hairpin is restored. Although this quenching effect is not always desirable, it can be used to develop new, highly sensitive probes for the detection of specific DNA sequences or proteins (12, 13, 18). Intra- and intermolecular fluorescence quenching experiments with different dyes and amino acids in water have revealed that a similar mechanism holds true for
10.1021/bc0341324 CCC: $25.00 © 2003 American Chemical Society Published on Web 11/01/2003
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Figure 1. Molecular structures of fluorescein, rhodamine B, tetramethylrhodamine (TMR), Bodipy-FL, MR 121, ATTO 590, and Bodipy 630/650 investigated in inter-, and intramolecular quenching experiments. The molecular structures of some of the investigated dyes are available, while for other dyes the structures are proprietary (Alexa 647, ATTO 655, ATTO 680).
some amino acids. It has been shown that the fluorescence quantum yield of some fluorescein and bora-diazaindacene derivatives is quenched substantially by tryptophan, and to a smaller degree partly by tyrosine and methionine residues (22-24). The oxidation potential, Eox., of tryptophan is ∼1V (25, 26) even lower than that of dG, so the resulting quenching efficiency can be expected to be relatively strong. The strong fluorescence quenching efficiency of oxazine derivatives by tryptophan residues has been used in oxazine-labeled peptide epitopes to detect the presence of p53-autoantibodies in serum samples of patients (27). The underlying method involves the detection of reversible fluorescence quenching of dyes by tryptophan residues in labeled short peptides derived from the antibody recognition sequence of p53. In the free peptide in aqueous solution fluorescence is reduced, due to the formation of ground-state complexes between the dye and the tryptophan residue and subsequent efficient fluorescence quenching. Binding to the antibody via the tryptophan-containing recognition sequence initiates a spontaneous conformational change in the peptide accompanied by a strong increase in fluorescence intensity. Similar quenching interactions between an oxazine derivative and a tryptophan residue have been used to study intramolecular contact formation in peptides at the single molecule level (28). In contrast to electronic energy transfer-based systems where long-range dipole-dipole interactions occur, efficient electron transfer-based fluorescence quenching within these peptides requires close contact between the fluorophore and the tryptophan. Consideration of these quenching effects of fluorescent dyes in peptides or proteins by neighboring amino acids is crucial for the development of new fluorescence-based assays. Motivated by these considerations, we measured steady-state and time-resolved intermolecular fluores-
cence quenching of different fluorophores (MR121, ATTO 655, ATTO 680, Cy5, ATTO 590, Alexa 647, tetramethylrhodamine, rhodamine B, Bodipy-FL, Bodipy 630/650, and fluorescein) by several amino acids. Among the fluorophores investigated, the red-absorbing oxazine derivative MR121 and the dyes ATTO 655 and ATTO 680 show the most pronounced quenching efficiency by tryptophan. Other amino acids (tyrosine, phenylalanine, methionine, and histidine) showed only minor quenching efficiencies. The fluorescence intensity and lifetime of the carbocyanine dyes Cy5 and Alexa 647 are only slightly influenced by the presence of amino acids. To investigate intramolecular fluorescence quenching, the fluorophores were covalently attached to amino acids, a tryptophancontaining peptide, and streptavidin. Each hydrophobic binding pocket of streptavidin contains four tryptophan residues that are involved in strong biotin binding (23, 29). We demonstrate that knowledge of the fluorescence quenching behavior of fluorophores by amino acids has to be considered for the design of biological or medical assays. EXPERIMENTAL PROCEDURES
Fluorescent Dyes and Conjugates. The dyes used in this study were obtained as free carboxy acids, or N-hydroxysuccinimidyl esters. ATTO 590, ATTO 655, and ATTO 680 were purchased from ATTO-TEC (Siegen, Germany), Cy5 from Amersham Pharmacia Biotech (Freiburg, Germany), fluorescein, rhodamine B (RB) and tetramethylrhodamine (TMR), Bodipy 630/650, and Bodipy-FL from Molecular Probes (Go¨ttingen, Germany). The oxazine derivative MR121 was kindly provided by K. H. Drexhage (Universita¨t-Gesamthochschule, Siegen). Molecular structures of the fluorophores investigated are shown in Figure 1. When dissolved in water, N-hydroxy-
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Bioconjugate Chem., Vol. 14, No. 6, 2003 1135
succinimidyl esters are believed to be deactivated within minutes to hours. MR121 and RB were converted into their N-hydroxysuccinimyl esters using an equimolar amount of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in acetonitrile. For coupling to the amino groups of amino acids or peptides, 20 µL of the activated dyes (0.1 mM in acetonitril) was mixed with an excess of amino acids or peptides in 50 µL of carbonate buffer (0.1 M, pH 8.3). Fluorescent dyes were coupled to the amino group of the lysine residue of the N-terminal protected peptide lysine-glycine-glycine-glycine-tryptophan (KGGGW). The reaction solution was incubated for 6 h at room temperature, and the product was purified by HLPC (HewlettPackard, Bo¨blingen, Germany) using a reversed-phase column (Knauer, Berlin, Germany) with octadecylsilanehypersil C18. Separation was performed in 0.1 M triethylammonium acetate, using a linear gradient from 0% to 75% acetonitrile in 20 min. Yields >90% were obtained. To ensure low labeling degrees, the coupling to streptavidin was carried out in 50 µL carbonate buffer (0.1 M, pH 8.3) using 20 µL of the activated dye solution (0.1 mM in acetonitrile) and a 100-fold excess of streptavidin. Purification of the product was performed with NAPcolumns (Sephadex G-25). Absorption measurements revealed an average label degree of 0.95 (Table 2). Figure 2a-c show three typical static and dynamic Stern-Volmer plots. Bodipy-FL (Figure 2a) shows a combination of dynamic and static quenching while Cy5 exhibits only weak dynamic quenching (Figure 2b). In the case of the oxazine derivative MR121, and the two dyes ATTO 655 and ATTO 680, the Stern-Volmer plots are dominated by strong static quenching (Figure 2c). In addition, Stern-Volmer plots obtained from steady-state measurements display upward curvatures for almost all dyes investigated. These deviations from linearity with increasing quencher concentration indicate the presence of at least two populations of fluorophores. The fact that (i) static quenching is much higher than expected for a diffusion-controlled bimolecular reaction and (ii) all dyes investigated show almost monoexponential fluorescence decays independent of the quencher concentration, strongly support the idea of the formation of weak or nonfluorescent ground-state complexes between the fluorophores and Trp in aqueous solutions. Dependent on the relative conformations and resulting interaction geometries, the complexes can be considered as essentially nonfluorescent. Then, the association constant, Kass., can be calculated via eq 4 by plotting (F0/F)/(τ0/τ) versus the quencher concentration (Figure 2a and 2c).
F0 ) (1 + Kdyn[Q])(1 + Kass.[Q]) F
(4)
Table 3. Relative Fluorescence Quantum Yields, Φf,rel, of Fluorescein, Bodipy-FL, and ATTO 655 Amino Acid Conjugates Φf,rel
Trp-dye
Tyr-dye
Phe-dye
Met-dye
His-dye
fluorescein Bodipy-FL ATTO 655
0.25 0.02 0.03
0.55 0.15 0.63
0.95 0.91 1.00
0.77 0.30 0.99
0.79 0.70 1.00
As can be seen in Table 2, bimolecular dynamic quenching constants, kq,dyn, vary between 2.0 and 5.0 × 109 M-1 s-1 for all dyes investigated. On the other hand, the bimolecular static quenching efficiencies calculated at low quencher concentrations (0-10 mM) differ significantly. Here, the red-absorbing dyes MR121, ATTO 655, and ATTO 680 exhibit the highest association constants, Kass., of 96-206 M-1 with Trp. Fluorescence quenching is dominated by the formation of nonfluorescent groundstate complexes, i.e., static quenching. Quenching of ATTO 590, fluorescein, TMR, and RB by Trp comprises static and dynamic quenching with comparable efficiency while for the two bora-diaza-indacene dyes Bodipy-FL and Bodipy630/650, dynamic quenching is more efficient than static quenching (compare Kdyn and Kass. in Table 2). Carbocyanine derivatives, such as Cy5 and Alexa 647, show solely dynamic quenching (Table 2). Intramolecular Fluorescence Quenching of Dyes by Tryptophan in Peptides and Proteins. Table 3 shows the relative fluorescence quantum yields, Φf,rel, measured for fluorescein, Bodipy-FL, and ATTO 655 coupled to the aliphatic amino group of the amino acids Trp, Tyr, Phe, Met, and His. As expected from intermolecular quenching experiments (Table 1), ATTO 655 is almost exclusively quenched when directly coupled to Trp. Besides the Trp-conjugate, only the Tyr-conjugate exhibits measurable fluorescence quenching. Comparison of the fluorescence quantum yield and fluorescence decay of the ATTO 655-labeled Trp conjugate (Figure 3a) demonstrates mainly static quenching. This supports the idea that fluorescence quenching is dominated by the formation of weak or nonfluorescent ground-state complexes that exhibit fluorescence lifetimes shorter than the time-resolution of our apparatus used to measure the time-resolved fluorescence decays (∼40 ps). The short fluorescence lifetime of 230 ps with an amplitude of 0.16 measured for the ATTO 655-Trp conjugate (Table 4) cannot account for a relative fluorescence quantum yield, Φf,rel of 0.03. The main part of the fluorescence decay is dominated by the nearly unquenched fluorescence lifetime of the free dye of ∼1.8 ns with an amplitude of 0.84. Similar fluorescence characteristics were measured for the MR121 and ATTO 680 Trp-conjugate (Table 4). The fluorescence quantum yields of the Bodipy-FL amino acid conjugates show the following sequence: Trp , Tyr < Met < His < Phe (Table 3). Φf,rel for the Trpconjugate is similar to the value measured for the ATTO 655 Trp-conjugate indicating strong quenching interactions. Furthermore, both Bodipy-FL and ATTO 655
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Bioconjugate Chem., Vol. 14, No. 6, 2003 1137
Figure 3. Fluorescence decays of the free dyes (gray) and Trp conjugates (black) (A) ATTO 655, and (B) Bodipy-FL in PBS buffer, pH 7.4. In addition, the instrument response functions (IRFs) are given. Excitation was performed using a pulsed LED (490 nm) or a laser diode emitting (635 nm). Fluorescence was detected at the emission maxima (12.5 ps/channel). Table 4. Relative Fluorescence Quantum Yields, Φf,rel, Fluorescence Lifetimes, τi, and Corresponding Amplitudes, ai, of Trp, Peptide, and Streptavidin Conjugates of Different Dyes in the Absence and Presence of an Excess of Biotin Φf,rel MR121-Trp MR121-Lys-(Gly)3-Trp MR121-streptavidin/+biotin ATTO 655-Trp ATTO 655-Lys-(Gly)3-Trp ATTO 655-streptavidin/+biotin ATTO 680-Trp ATTO 680-Lys-(Gly)3-Trp ATTO 680-streptavidin/+biotin Bodipy-FL-Trp Bodipy-FL-Lys-(Gly)3-Trp Bodipy-FL-streptavidin/+biotin fluorescein-Trp fluorescein-Lys-(Gly)3-Trp fluorescein-streptavidin/+biotin Cy5-Trp Cy5-Lys-(Gly)3-Trp Cy5-streptavidin/+biotin
0.03 0.10 0.11/0.72 0.03 0.08 0.05/0.58 0.01 0.09 0.11/0.82 0.02 0.27 0.09/0.65 0.25 0.57 0.06/0.17 0.59 0.80 0.90/0.90
τ1 (ns)/a1 τ2 (ns)/a2 0.47/0.37 0.50/0.07 0.28/0.31 0.23/0.16 1.45/1.00 0.28/0.34 0.21/0.30 1.35/1.00 0.21/0.39 0.44/0.95 2.52/1.00 1.41/0.22 1.10/0.20 2.87/1.00 0.75/0.64 0.79/1.00 0.41/0.15 0.71/0.20
1.81/0.63 1.60/0.93 1.99/0.69 1.77/0.84 1.49/0.66 1.30/0.70 1.78/0.61 4.95/0.05 5.43/0.78 1.85/0.80 2.85/0.36 0.97/0.85 1.71/0.80
exhibit similar bimolecular dynamic quenching constants, kq,dyn, of ∼4 × 109 M-1 s-1 (Table 2). However, as ATTO 655 shows a high association constant (206 M-1) promoting the formation of weak or nonfluorescent ground-state complexes, the tendency of Bodipy-FL to associate with Trp is less pronounced. That is, most of the ATTO 655 molecules are associated with the Trp residue in groundstate complexes whereas Bodipy-FL molecules are either still accessible for dynamic quenching or Bodipy-FL/Trp complexes exhibit a less optimal interaction geometry for efficient quenching. This is reflected in the fluorescence decay of the Bodipy-FL conjugate (Figure 3). The fluorescence decay can be fitted almost monoexponentially revealing a short fluorescence lifetime of 440 ps with an amplitude of 0.95 (Table 4). As can be seen in Table 2, static and dynamic quenching efficiencies measured from intermolecular fluorescence quenching experiments with fluorescein and amino acids are similar. The corresponding fluorescein conjugates show only moderate fluorescence quenching with relative fluorescence quantum yields, Φf,rel, ranging from 0.25 for the Trp-conjugate to 0.95 for the Phe-conjugate (Table 3). In the case of the Trp-conjugate, the measured fluorescence lifetimes of 1.10 ns and 1.85 ns with amplitudes of 0.20 and 0.80, respectively (Table 4), account almost for the observed decrease in fluorescence intensity. To further evaluate the quenching interactions between organic dyes and Trp residues and, in particular, the distance dependence of quenching, the dyes were
Figure 4. Model for the formation of weak or nonfluorescent ground-state complexes between the oxazine derivative MR121 and the Trp residue in the peptide MR121-Lys-(Gly)3-Trp. Due to the flexibility of the glycine residues, the oxazine derivative MR121 (red) can adopt a nearly coplanar stacking conformation with respect to the Trp residue (blue).
coupled to the terminal lysine residue of the peptide: LysGly-Gly-Gly-Trp. Because of the relatively flexible glycine residues intramolecular contact and complex formation between the dyes and the Trp residue is possible. Table 4 shows the relative fluorescence quantum yields and fluorescence lifetimes of the Trp and peptide conjugates of MR121, ATTO 655, ATTO 680, Bodipy-FL, fluorescein, and Cy5. For all dyes investigated, fluorescence quenching is diminished in the peptide conjugates. The low fluorescence quantum yields measured for MR121, ATTO 655, and ATTO 680 demonstrate again the formation of weak or nonfluorescent ground-state complexes between the dyes and the terminal Trp residue (Figure 4). The fluorescence lifetimes are slightly reduced and can be fitted satisfactorily using a monoexponential model. Only in the case of the MR 121 conjugate does a second shorter decay component of 500 ps with an amplitude of 0.07 appear. For the Bodipy-FL conjugate the quantum yield increases substantially from 0.02 for the Trp conjugate to 0.27 for the peptide conjugate (Table 4). In addition, the fluorescence lifetime increases from 440 ps to 2.52 ns. This indicates that static quenching within groundstate complexes plays only a minor role in these peptides. For fluorescein, the fluorescence quantum yield and lifetime increase while Cy5 is only slightly quenched in all conjugates investigated. Assuming quenching electron transfer from the Trp residue to the dye, at least two mechanisms have to be considered (30): through-space and through-bond. Due to the flexibility of the peptide used in our studies, a clear answer cannot be given. However, our data indicate that contact formation between the Trp residue and the chromophore is required for efficient quenching. This implies that electron transfer through-bond is only of minor importance. Hence, the distinct dynamic quenching measured for dyes such as Bodipy-FL might reflect
1138 Bioconjugate Chem., Vol. 14, No. 6, 2003
collisions between the terminal Trp residue and the dye due to the flexibility of the peptide. That is, the fluorescence lifetime of Bodipy-FL-labeled peptides that contain a Trp residue might eventually be used to reveal contact formation rates in peptides (28, 31). The results demonstrate that knowledge of the spectroscopic behavior of the dyes used to label peptides or proteins is crucial for the design of biological relevant applications. Because of the importance of the biotinstreptavidin techniques, dyes were covalently attached to streptavidin and the effect of biotin binding was investigated (Table 4). The strong binding of biotin to the 60 kDa protein steptavadin (KD ) 10-15 M) has become a standard tool in molecular biology (32, 33). Each hydrophobic binding pocket of streptavidin contains four tryptophan residues that are involved in strong biotin binding (22). With the exception of Cy5, the fluorescence of all streptavidin conjugates is substantially quenched (Table 4). While the increase in fluorescence quantum yield is less pronounced in the case of the fluorescein conjugate, Φf,rel of the MR 121, ATTO 655, ATTO 680, and Bodipy-FL conjugate shows a dramatic increase upon addition of an excess biotin (Table 4). As in the case of the peptide and Trp conjugates, it is likely that the strong reduction in fluorescence intensity is caused by reversible quenching of the dye with tryptophan residues of streptavidin. The strong increase in fluorescence intensity of the MR121, ATTO 655, ATTO 680, and Bodipy-FL streptavidin conjugate upon binding to biotin is advantageous for biological applications where washing steps have to be avoided. CONCLUSIONS
The fluorescence quenching of various fluorescent dyes by amino acids, and in particular by tryptophan, is studied and compared using steady-state and timeresolved fluorescence spectroscopy. The red-absorbing oxazine derivative MR121 and the two ATTO dyes ATTO 655 and ATTO 680 are almost exclusively quenched by tryptophan while rhodamine and bora-diaza-indacene derivatives that absorb at shorter wavelengths are also quenched substantially by tyrosine residues. The quenching of the carbocyanine dye Cy5 by amino acids is generally very low. Fluorescence quenching of the redabsorbing dyes MR 121, ATTO 655, and ATTO 680 by tryptophan is dominated by the formation of nonfluorescent ground-state complexes, i.e., static quenching, with association constants, Kass., ranging from of 96-206 M-1. On the other hand, quenching of various rhodamine derivatives and fluorescein comprises static and dynamic quenching with comparable efficiency. For the two boradiaza-indacene dyes Bodipy-FL and Bodipy630/650, dynamic quenching is more efficient than static quenching. Selective fluorescence quenching of dyes by tryptophan residues in proteins has great potential for the development of diagnostic applications (21, 27), and new methods for the direct measurement of submicrosecond intramolecular contact formation in biopolymers at the single molecule level (28). Furthermore, the data show that changes in the fluorescence quantum yield of dye-labeled peptides might be used in developing fluorescence-based assays, e.g., protease assays. ACKNOWLEDGMENT
The authors wish to thank Prof. K. H. Drexhage (Universita¨t-Gesamthochschule Siegen) for providing the oxazine derivative MR121. This work was supported
Marme´ et al.
by the Bundesministerium fu¨r Bildung, Wissenschaft, Forschung und Technologie (Grants 311864 and 13N8349). LITERATURE CITED (1) Haugland, R. P. (1996) Handbook of fluorescent probes and research chemicals, 6th ed., Molecular Probes, Eugene, OR. (2) Cardullo, R. A., Agrawal, S., Flores, C., Zamecnik, P. C., and Wolf, D. E. (1988) Detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer. Proc. Natl. Acad. Sci. U.S.A. 85, 8790-8794. (3) Tyagi, S., and Kramer, F. R. (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14, 303-308. (4) Tyagi, S., Marras, S. A. E., and Kramer, F. R. (2000) Wavelength-shifting molecular beacons. Nat. Biotechnol. 18, 1191-1196. (5) Marras, S. A. E., Kramer, F. R., and Tyagi, S. (2002) Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes. Nucleic Acid Res. 30, e122. (6) Fo¨rster, T. (1949) Experimentelle und theoretische Untersuchung des zwischenmolekulares U ¨ bergangs von Elektronenanregungsenergie. Zeitschrift fu¨ r Naturforschung 4a, 321-327. (7) Weiss, S. (1998) Fluorescence spectroscopy of single biomolecules. Science 283, 1676-1683. (8) Gordon, G. W., Berry, G., Liang, X. H., Levine, B., and Herman, B. (1998) Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys. J. 74, 2702-2713. (9) Schwille, P., Meyer-Almes, F. J., and Rigler, R. (1997) Dualcolor fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution. Biophys. J. 72, 1878-1886. (10) Heinze, K. G., Rarbach, M., Jahnz, M., and Schwille, P. (2002) Two-photon fluorescence coincidence analysis: Rapid measurements of enzyme kinetics. Biophys. J. 83, 1671-1681. (11) Hom, E. F. Y., and Verkman, A. S. (2002) Analysis of coupled bimolecular reaction kinetics and diffusion by twocolor fluorescence correlation spectroscopy: Enhanced resolution of kinetics by resonance energy transfer. Biophys. J. 83, 533-546. (12) Knemeyer, J. P., Marme´, N., and Sauer, M. (2000) Probes for detection of specific DNA sequences at the single molecule level. Anal. Chem. 72, 3717-3724. (13) Piestert, O., Barsch, H., Buschmann, V., Heinlein, T., Knemeyer, J. P., Weston, K. D., and Sauer, M. (2003) A single molecule sensitive DNA-hairpin system based on intramolecular electron transfer. Nano Lett. 3, 979-982. (14) Seidel, C. A. M., Schulz, A., and Sauer, M. (1996) Nucleobase-specific quenching of fluorescent dyes. 1. Nucleobase oneelectron redox potentials and their correlation with static and dynamic quenching efficiencies. J. Phys. Chem. 100, 55415553. (15) Sauer, M., Han, K. T., Ebert, V., Mu¨ller, R., Schulz, A., Seeger, S., Wolfrum, J., Arden-Jacob, J., Deltau, G., Marx, N. J., Zander, C., and Drexhage, K. H. (1995) New fluorescent dyes in the red region for biodiagnostics. J. Fluoresc. 5, 247261. (16) Edman, L., Mets, U ¨ ., and Rigler, R. (1996) Conformational transitions monitored for single molecules in solution. Proc. Natl. Acad. Sci. U.S.A. 93, 6710-6715. (17) Eggeling, C., Fries, J. R., Brand, L., Gu¨nther, R., and Seidel, C. A. M. (1998) Monitoring conformational dynamics of a single molecule by selective fluorescence spectroscopy. Proc. Natl. Acad. Sci. U.S.A. 95, 1556-1561. (18) Heinlein, T., Knemeyer, J. P., Piestert, O., and Sauer, M. (2003) Nucleobase-specific quenching of fluorescent dyes in DNA-hairpins. J. Phys. Chem. B 107, 7957-7964. (19) Kurata, S., Kanagawa, T., Yamada, K., Torimura, M., Yokomaku, T., Kamagata, Y., and Kurane, R. (2001) Fluorescent quenching-based quantitative detection of specific DNA/RNA using BODIPY FL-labeled probe or primer. Nucleic Acids Res. 29, e34.
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BC0341324
