Anal. Chem. 1997, 69, 500-506
Temperature and Quenching Studies of Fluorescence Polarization Detection of DNA Hybridization Michael U. Kumke, Luchuan Shu, and Linda B. McGown*
P. M. Gross Chemical Laboratory, Department of Chemistry, Duke University, Box 90346, Durham, North Carolina 27708-0346 G. Terrance Walker,* J. Bruce Pitner, and C. Preston Linn
Becton Dickinson and Company Research Center, Research Triangle Park, North Carolina 27709
The effects of temperature and collisional quenching on fluorescence polarization detection of DNA hybridization were studied using measurements of fluorescence intensity and anisotropy and the dynamic decay of these properties. Three different tethers, 3, 6, and 12 carbons in length, were used to attach fluorescein label to the 5′ end of the 33-base oligomers. Perrin plots showed that the effective rotating volume decreases with increasing tether length and approximately doubles upon hybridization. Hybridization increases the association between the tethered dye and the DNA for the shorter tethers but displaces the fluorescein on the 12C tether from the DNA, forcing it into greater contact with the bulk solution. The 6C tether appears to promote sequence-specific interaction between fluorescein label and the oligomer, which causes unexpectedly high anisotropy at higher temperatures and increased protection from collisional quenching. In all cases, there appear to exist several possible conformations for the tethered fluorescein. As temperature is increased, these conformations tend to collapse into a single, average or preferred, conformation. The results demonstrate the importance of the selection of tether, dye, and DNA probe in designing a polarization strategy for detection of DNA hybridization, particularly with respect to tether length and DNA probe sequence. The identification of DNA sequences is becoming increasingly important in clinical diagnostics. Direct, homogeneous (nonseparation) techniques are desirable for rapid and specific detection of DNA sequences.1-4 In a previous paper,5 we described the use of fluorescence polarization for nonseparation, specific detection of DNA through its hybridization with a fluorescein-labeled DNA probe. Static and dynamic fluorescence anisotropy were used to (1) Mathies, R. A.; Zhu, H.; Clark, S. M.; Benson, S. C.; Rye, H. S.; Glazer, A. N. Anal. Chem. 1994, 66, 1941-1948. (2) Seeger, S.; Sauer, M.; Han, K.-T.; Mueller, R.; Schulz, A.; Tadday, R.; Wolfrum, J.; Arden-Jacob, J.; Deltau, G.; Marx, N. J.; Drexhage, K. H. J. Fluoresc. 1993, 3, 131-139. (3) Seeger, S.; Galla, K.; Arden-Jacob, J.; Deltau, G.; Drexhage, K. H.; Martin, M.; Sauer, M.; Wolfrum, J. J. Fluoresc. 1994, 4, 111-115. (4) Vo-Dinh, T.; Houck, K.; Stokes, D. L. Anal. Chem. 1994, 66, 3379-3383. (5) Kumke, M. U.; Li, G.; McGown, L. B.; Walker, G. T.; Linn, C. P. Anal. Chem. 1995, 67, 3945-3951.
500 Analytical Chemistry, Vol. 69, No. 3, February 1, 1997
monitor the rotational motion of the single-stranded and doublestranded labeled oligomers. Binding of a restriction enzyme to the double strand, thereby increasing its effective volume, provided enhanced detection of hybridization. Variations in both the length of the carbon tether which links the fluorescein to the 5′ end of the oligonucleotide and the location of the enzyme binding site were used to investigate the hybridization process and optimize its detection. It was found that the enzyme binding site must be near the fluorescein label in order to provide significant enhancement and that a 6-carbon tether provides the most sensitive detection of hybridization. In the present work, the effects of temperature and quenching on the polarization detection of DNA hybridization were investigated. Fluorescence intensity, anisotropy, lifetime, and anisotropy decay of the single-stranded and double-stranded DNA were measured as a function of temperature and as a function of KI concentration at room temperature. These studies employed a 33-mer from the previous study,5 to which fluorescein was attached at the 5′ end using a tether of 3, 6, or 12 carbon atoms. The unusually strong affinity of the 6-carbon tethered fluorescein for the DNA which was observed in the previous study was further investigated in this work through comparisons with two other DNA oligomers of different length and sequence.
EXPERIMENTAL SECTION Materials. Details of the synthesis, purification, and preparation of the DNA oligomers have been described.5 The oligomers are 33-mers of sequence 5′-GGAATTCATCCGTATGGTGGATAACGTCTTTCA. Fluorescein (F) is attached at the 5′ end by a tether that is 3, 6, or 12 carbon atoms in length (3C, 6C, or 12C, respectively). Chemical structures of the tethers are as follows: 3C, FCONH(CH2)3OPO2DNA(5′-3′); 6C, FCONH(CH2)6OPO2DNA(5′-3′); and 12C, FCONH(CH2)5CONH(CH2)6OPO2DNA(5′-3′). The corresponding, labeled oligomers are referred to as DNA33-3C, DNA33-6C, and DNA33-12C. A 28-mer of sequence 5′-GGAATTCAGTTATCCACCATACGGATAG and a 34mer of sequence 5′-TAGAGTCTTCAAATATCAGAGCTTTACCTAACAA, both having 6C-tethered fluorescein at the 5′ end, were also used in the temperature studies. Unless otherwise noted, the concentration of oligomer in the samples was 1 × 10-7 M. S0003-2700(96)00823-2 CCC: $14.00
© 1997 American Chemical Society
Temperature and quenching studies were performed in a buffer solution containing 100 mM TRIS-HCl (pH 7.5), 0.6 mM K2HPO4 (pH 7.5), 50 mM NaCl, 6% glycerol, 1 mM EDTA, 24 µg/mL bovine serum albumin, 0.02% Triton X-100, and 0.6 mM β-mercaptoethanol. This is the same buffer that was used in the previous studies.5 KI (Aldrich) was used as a fluorescence quencher. In the experiment in which sample viscosity was varied, a phosphate buffer (pH 7.0) was used as the solvent and viscosity was adjusted by addition of a sucrose stock solution (18.52 g of sucrose in 10 mL of H2O). In a first approximation, in both the Perrin plots and the viscosity studies, the temperature dependence of the viscosity of water was used to calculate η for the data analysis. The highest viscosity corresponded to a concentration of 57.7% w/w sucrose in the samples. Methods. The temperature of the sample compartment of the spectrofluorometer was maintained by a circulating water bath at 25 ( 0.1 °C for the fluorescence quenching experiments and varied in the range of 5-50 °C for the temperature studies. Fluorescence intensity and anisotropy were measured using a phase modulation spectrofluorometer (Model SLM48000S, Spectronics Instruments, Inc.) with a 450 W xenon arc lamp for excitation. The excitation monochromator was set to 488 nm, with a bandpass of 4 nm at the entrance and exit slits. Emission wavelength selection was achieved using a combination of a 520 nm long-pass filter (Oriel) and a 560 nm short-pass filter (CVI Laser Corp.). The fluorescence anisotropy (r) was measured using the L-format instrumental configuration6 according to
r)
Ivv - IvhG Ivv + 2IvhG
(1)
where I is the measured intensity and the subscripts v and h refer to the orientation (vertical or horizontal) of the polarizers in the excitation beam (first subscript) and the emission beam (second subscript). The instrumental correction factor, G, is equal to the ratio of Ihv to Ihh. Fluorescence lifetime and dynamic fluorescence anisotropy measurements were performed in the frequency domain, using a multiharmonic Fourier transform spectrofluorometer (Model SLM 4850MHF, Spectronics Instruments, Inc.). An argon ion laser (aircooled, Model 543, Omnichrome, or water-cooled, Model 307, Coherent) was used for excitation at 488 nm. Emission wavelength selection was achieved as described above. To avoid artifacts due to photoselection effects in the lifetime measurements, an emission polarizer was set to the magic angle of 54.7°. A base frequency of 4.1 MHz and a correlation frequency of 7.292 Hz were used in all dynamic experiments. Data at 60 modulation frequencies ranging from 4.1 to 246 MHz were analyzed. Each measurement consisted of 15 pairs of sample-reference measurements, each of which was the internal average of 100 samplings. The fluorescence lifetime data were evaluated using either standard nonlinear least-squares (NLLS) or the maximum entropy method (MEM, Maximum Entropy Data Consultant Ltd., Cambridge, U.K.), which is a self-modeling technique that provides a unique solution without assumption of an a priori model.7 In the NLLS analysis, the fluorescence lifetime data were best repre(6) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum Press: New York, 1983. (7) Brochon, J. C.; Livesey, A. K. Chem. Phys. Lett. 1990, 174, 517-522.
sented by a biexponential decay model; an increase in the number of decay components from two to three did not improve the goodness-of-fit, as indicated by the χ2 value and the randomness of the residuals. In the MEM analysis, a lifetime window of 0.120 ns that was divided logarithmically into 200 lifetime cells was used. A solution of fluorescein in phosphate buffer (pH 7.5) served as the reference fluorophore. The reference lifetime was determined to be 4.02 ( 0.05 ns. In the dynamic fluorescence anisotropy (DFA) measurements, the time-dependent anisotropy decay, r(t), was calculated from
r(t) )
I(t)v - I(t)h
(2)
I(t)v + 2I(t)h
where I(t)v and I(t)h represent the fluorescence intensity decays of the vertical and horizontal components of the emission beam that is excited with vertically polarized light. The DFA data were fitted by NLLS. Best results were generally obtained for fits of the data to the anisotropic rotator model, in which the anisotropy decay is described by n
r(t) ) r0
∑R exp(-t/Φ ), i
i
n)2
(3)
i)1
where r0 is the limiting anisotropy at t ) 0, i.e., the intrinsic anisotropy in the absence of rotational diffusion, and Φi and Ri are the rotational correlation time and the associated amplitude, respectively, of the ith decay component. The anisotropic rotator model is used to describe the multiexponential anisotropy decay of a molecule which does not exhibit equal rotational rates in all directions.6 In the analysis of the DFA data, the fluorescence lifetimes were fixed to values recovered in the lifetime experiments. The limiting anisotropy r0 of fluorescein was fixed to 0.40.8,9 This value was confirmed by allowing r0 to vary in several of the fits. RESULTS Temperature Studies. Steady State Anisotropy. The fluorescence anisotropy (r) of the single-stranded (ss) and doublestranded (ds) oligomers was measured in the temperature range of 5-40 °C. Results are shown in Figure 1. In general, anisotropy decreases with increasing tether length, indicating the increased freedom of rotation afforded by a longer tether, and increases upon hybridization, reflecting slower, more hindered rotation of the dye in the double-stranded complex. Anisotropy is expected to decrease with increasing temperature due to faster rotation, decreased viscosity, and decreased excited state lifetime. Fraying of the edges of the double strand, which would also lead to increased rotational freedom at higher temperatures, is not observed in this temperature range. As can be seen in Figure 1, anisotropy generally does decrease with increasing temperature, with several notable exceptions. For the 6C tether, the anisotropy decreases as expected up to 25 °C but then begins to increase, most dramatically for the single strand. For comparison, r vs temperature was investigated for two other oligomers with the same 6C tether to fluorescein. A 28-mer with (8) Johansson, L. B.-A. J. Chem. Soc., Faraday Trans. 1990, 86, 2103-2107. (9) Fleming, G. R.; Morris, J. M.; Robinson, G. W. Chem. Phys. 1976, 17, 91100.
Analytical Chemistry, Vol. 69, No. 3, February 1, 1997
501
Figure 1. Fluorescence anisotropy of fluorescein-labeled oligomers as a function of temperature.
the same sequence as the 33-mer at the 5′ end showed the same anomalous increase in anisotropy at higher temperatures. Several 28-mers and a 34-mer that had different sequences at the 5′ end did not show the increase. Therefore, the anomalous behavior is attributed to a sequence-specific interaction between fluorescein and the oligomer that is facilitated by the 6C tether length. Also shown in Figure 1 is an unusually large decrease of the ss-DNA33-3C anisotropy as temperature is increased to 25 °C. The dramatic increase in rotational freedom for the 3C tether and the anomalous behavior for the 6C tether were the first indications of the complexity of the molecular motions underlying the anisotropy and its response to temperature. Anisotropy and the hydrodynamic volume, V, of a rotating molecule or molecular assembly in a homogeneous solution are related by the Perrin equation10:
1/r ) (1/r0)[1 + (τRT/ηV)]
(4)
where r0 is the limiting anisotropy of the molecule, i.e., the anisotropy in the absence of rotation, τ is the observed fluorescence lifetime, R is the gas constant, T is absolute temperature, and η is the viscosity of the solution at T. If a Perrin plot of 1/r vs T/η is linear, the y-intercept of 1/r0 and the slope of τR/Vr0 will yield the limiting anisotropy and the hydrodynamic volume, respectively. As shown in Figure 2, the Perrin plots are linear for all of the oligomers except ss-DNA33-3C in the temperature range of approximately 5-25 °C. At higher temperatures, negative deviations from linearity are observed for the single-stranded oligomers, as expected from the results in Figure 1. The Perrin plots of the double-stranded oligomers are more linear. Estimates were obtained, using the linear portions of the Perrin plots, of r0 and an overall, relative hydrodynamic volume, Vrel, as (10) Cantor, C. R.; Schimmel, P. R. Biophysical Chemistry Part II: Techniques for the Study of Biological Structure and Function; W. H. Freeman & Co.: San Francisco, CA, 1980, pp. 463-465.
502 Analytical Chemistry, Vol. 69, No. 3, February 1, 1997
Figure 2. Perrin plots for fluorescein-labeled oligomers. T/η values (K/cP) for successive points, left-to-right, are 278.15/1.519, 283.15/ 1.307, 288.15/1.139, 293.15/1.002, 298.15/0.8904, 303.15/0.7975, 308.15/0.7194, and 313.15/0.6529. Table 1. Limiting Anisotropies (r0) and Relative Hydrodynamic Volumes (Vrel) Estimated from Linear Portions of Perrin Plots for Fluorescein-Labeled Oligomers r0a
DNA33-3C DNA33-6C DNA33-12C
Vrelb
ss
ds
ss
ds
% change
-0.37 -0.69 -0.11
0.51 0.40 0.38
1.5 1 0.56
2.8 1.8 1.1
87 80 96
a Assumes an intrinsic lifetime of 4.2 ns for fluorescein, based on the observed temperature dependence of the lifetime. b Relative hydrodynamic volumes are normalized to a value of unity for ss-DNA336C. The r0 values determined for the ds-DNA are used in the calculation.
sensed by the fluorescein label. The relative volume, which represents an overall average of the effective volumes experienced by the tethered fluorescein, is used instead of a single, absolute volume V, because the rotation of the tethered labels is not governed by simple, isotropic motions. The results are shown in Table 1. For the double strands, the values of r0 are consistent with literature values of ∼0.4 for fluorescein.11 Rotation about the 3C tether is considerably more restricted than that about the longer tethers, and the largest freedom is seen for the 12C tether. For the single strands, the values of r0 are all negative and unreasonable, indicating serious deviations from the predicted temperature response. For all three oligomers, the relative volumes approximately double upon hybridization, which reflects the approximate doubling of the size of the oligomer upon hybridization. The apparent volume decreases with increasing tether length due to increased rotational freedom of the fluorescein. To see if the effects of temperature could be explained by changes in viscosity alone, another experiment was performed with the DNA33-6C, in which the temperature was held constant at 25 °C and the viscosity of the solution was varied by adding (11) Fleming, G. R.; Morris, J. M.; Robinson, J. W. Chem. Phys. 1976, 17, 91100.
Table 2. Fluorescence Lifetime (τ), Peak Width at Half-Height (w), and Fractional Intensity Contribution (r) of the Major Lifetime Component of Fluorescein-Labeled Oligomers as a Function of Temperature
ss-DNA33-3C
ds-DNA33-3C
ss-DNA33-6C
ds-DNA33-6C
ss-DNA33-12C
ds-DNA33-12C
T (°C)
τ (ns)a
w (ns)
Rb
5.0 15.0 35.0 45.0 5.0 15.0 35.0 45.0 5.0 15.0 35.0 45.0 5.0 15.0 35.0 45.0 5.0 15.0 35.0 45.0 5.0 15.0 35.0 45.0
4.2 4.1 3.5 3.4 4.1 4.0 3.8 3.5 4.2 4.2 3.8 3.7 4.2 4.2 4.0 3.9 4.3 4.2 3.8 3.7 4.1 4.1 4.0 3.8
3.6 3.9 2.9 2.6 3.6 5.4 2.6 5.1 3.9 2.1 0.6 1.1 1.6 1.9 1.1 0.8 1.7 5.3 0.7 0.5 3.0 2.7 1.6 0.9
0.94 0.97 0.97 0.96 0.77 0.81 0.81 0.92 0.96 0.95 0.96 0.96 0.98 0.98 0.97 0.97 0.94 1.00 1.00 1.00 0.96 0.98 0.88 0.99
a Lifetime τ is taken as the maximum of the lifetime peak of width w recovered by MEM analysis. b Fractional intensity may be less than 100% due to contributions from minor lifetime components (see text), scattered light, and background noise.
sucrose. The resulting viscosity ranged from 0.89 cP for pure water to 38.01 cP for 57.7 wt % sucrose,12 which corresponds to the linear range that was observed in the temperature studies. The Perrin plots obtained by increasing viscosity were essentially identical to the plots obtained by increasing temperature for the double-stranded DNA, while the plots for single-stranded DNA had smaller slopes and higher intercepts for the viscosity curve. This indicates that decreasing solvent viscosity is responsible for the decreases in anisotropy with increasing temperature over the linear range for the double strands. For the single strands, there is less freedom of rotation than would be predicted by the temperature-dependence of viscosity alone. Fluorescence Lifetime. Fluorescence lifetime was studied in the temperature range of 5-45 °C (Table 2). Both NLLS and MEM analyses recovered essentially monoexponential decays for all three single-stranded oligomers and for the double-stranded forms of DNA33-6C and DNA33-12C. A very small contribution from a second, subnanosecond component in some of the fits for these oligomers can be attributed to experimental noise, as indicated by changes in the lifetime of the second component with changing lifetime windows used in the MEM analysis.13 For ds-DNA333C, a second, real component of 1-2 ns was recovered. This component may be due to a second conformation or local configuration experienced by the tethered dye. Fluorescence lifetime decreases at the higher temperatures, probably due to increased motions of the oligomers. A second trend revealed by the MEM plots is a decrease in the widths of (12) Handbook of Chemistry and Physics, 63rd ed.; CRC Press: Boca Raton, FL, 1982. (13) Shaver, J. M.; McGown, L. B. Anal. Chem. 1996, 68, 9-17.
Table 3. Rotational Correlation Times (Φ1 and Φ2) and Fractional Contribution of Φ1 to the Total Anisotropy Decay (r1), Recovered from NLLS Analysis Using a Biexponential Anisotropic Rotator Model, of Fluorescein-Labeled Oligomers as a Function of Temperature
ss-DNA33-3C
ds-DNA33-3C
ss-DNA33-6C
ds-DNA33-6C
ss-DNA33-12C
ds-DNA33-12C
T (°C)
Φ1 (ns)
R1
5.0 15.0 25.0 35.0 45.0 5.0 15.0 25.0 35.0 45.0 5.0 15.0 25.0 35.0 45.0 5.0 15.0 25.0 35.0 45.0 5.0 15.0 25.0 35.0 45.0 5.0 15.0 25.0 35.0 45.0
0.7 0.6 0.5 0.4 0.4 1.2 1.2 0.7 1.0 0.8 1.1 0.8 0.6 0.7 0.7 1.0 0.9 0.8 0.8 0.7 0.5 0.4 0.3 0.3 0.3 0.6 0.5 0.3 0.3 0.3
0.69 0.81 0.93 0.99 1.00 0.81 0.90 0.84 0.98 1.00 0.94 0.96 0.97 1.00 1.00 0.87 0.95 0.94 1.00 1.00 0.83 0.90 0.93 1.00 1.00 0.87 0.92 0.92 0.98 1.00
Φ2 (ns) 15 13 11 15 2200 35 7 160 8a 110 100 400 19 52 670 6 5 7 89a 6 5 6 6
a A correlation time Φ was recovered even though R is 1.00. The 2 1 contribution of Φ2 is, therefore, less than 0.01 (i.e., less than 1% contribution to the total anisotropy decay).
the recovered lifetime peaks, toward more discrete lifetimes, as temperature is increased. This is attributed to increasingly rapid motion of the tethered fluorescein, resulting in the collapse of different conformational motions into a single, average motion. Again, the exception is ds-DNA33-3C, which showed no conclusive change in the substantial width of the longer lifetime component with temperature. Instead, increasing temperature caused a decrease in the fractional contribution of the second, shorter lifetime component from 20 ( 3% at 5 °C to 4 ( 3% at 45 °C. Additional contributions from scattered light and background noise account for the remaining fractional intensity. Dynamic Fluorescence Anisotropy. The DFA results (Table 3) were obtained by fitting the data to the model of the anisotropic rotator with two rotational correlation times. This model generally provided the best fits here and in previous studies.9 In the DFA analysis, the lifetime was fixed to the dominant (and in most cases, the only) fluorescence lifetime component recovered from the MEM analysis (see Table 2). As shown in Table 3, the short correlation time, Φ1, decreases with increasing temperature for all of the oligomers in both the single and double strands. For the 6C and 12C tethers, hybridization has no significant effect on the values or temperature dependence of Φ1. For the 3C tether, Φ1 of the double strand is approximately twice that of the single strand over the entire temperature range. The value of Φ1 is lowest for DNA33-12C (both single- and double-stranded) and largest for the ds-DNA33Analytical Chemistry, Vol. 69, No. 3, February 1, 1997
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Table 4. Stern-Volmer Constants (kq) for KI Quenching of DNA Oligomers at 25 °C kq (109 M-1 s-1)
DNA33-3C DNA33-6C DNA33-12C
ss
ds
% change
1.57 1.20 1.52
1.46 1.11 1.73
-7 -8 14
3C and the single- and double-stranded DNA33-6C. For all of the oligomers, the fractional contribution of Φ1 to the total anisotropy decay (R1) increases with increasing temperature, accounting for 100% of the decay as the temperature approaches 45 °C. The value of R1 at the low temperatures is lowest for the 3C tether and highest for the 6C tether. For an isotropic rotator, the steady-state anisotropy should decrease as the rotational correlation time decreases because faster rotation causes faster depolarization. For the oligomers, Φ1 dominates the anisotropy decay. Therefore, the lowest anisotropy in Figure 1 corresponds to the shortest Φ1, which is observed for the 12C tether. The highest anisotropy corresponds to the longest Φ1, which is observed for the 3C tether in the dsDNA. No definite trends could be discerned for the long correlation time, Φ2, which generally accounted for less than 10% of the anisotropy decay at all but the lowest temperatures. Determination of Φ2 is subject to large uncertainties because it is such a minor contribution and is slow compared to the short fluorescence lifetime of the fluorescein label. The fluorescence lifetime determines an optimal time window for the detection of the fluorescence anisotropy decay; it is difficult to resolve a rotational motion that occurs on a time scale much above or below that of the excited state lifetime. The steady-state anisotropy differences between the ss-DNA and ds-DNA for a given tether arise from differences between the correlation times and their relative contributions to the total anisotropy decay. The anomalous behavior of the ss-DNA33-3C in Figure 1 can be explained on the basis of the unusually large contribution from the longer Φ2 is at low temperatures, combined with the large decrease in the value of Φ1 with increasing temperature. Quenching Studies. Quenching experiments were performed at 25 °C in which KI was added to the oligomer solutions in concentrations ranging from 0 to 0.185 M. Iodide ion is known to be a dynamic, or collisional, fluorescence quencher. Fluorescence intensity data were analyzed by standard Stern-Volmer analysis. The plots showed good linearity, and the fits had correlation coefficients of 0.98 and better. Values for the quenching rate constant, kq (Table 4), were in the range of (1.1-1.73) × 109 M-1 s-1 for fluorescein label in the oligomers and 2.5 × 109 M-1 s-1 for free fluorescein. The latter is in agreement with rate constants reported in the literature.14 The kq of DNA33-12C is most similar to that of free fluorescein, while that of DNA33-6C is smallest, indicating the most protection from iodide quenching. For the 3C and 6C tethers, the kq decreases slightly upon hybridization, while for the 12C tether, the kq is significantly higher for the double strand. (14) Desilets, D. J.; Kissinger, P. T.; Lytle, F. E. Anal. Chem. 1987, 59, 12461248.
504 Analytical Chemistry, Vol. 69, No. 3, February 1, 1997
Figure 3. Fluorescence anisotropy of fluorescein-labeled oligomers as a function of quencher KI concentration at 25 °C.
Figure 4. Fluorescence lifetime of fluorescein-labeled oligomers as a function of quencher KI concentration at 25 °C.
Iodide quenching caused large increases in the anisotropy of the oligomers (Figure 3). Assuming that the interaction between the fluorescein label and I- is a simple collision between iodide in the bulk solution and the fluorescein label, the increase in anisotropy can be attributed to a decrease in fluorescence lifetime, as predicted by the Perrin equation. Such decreases in lifetime were, in fact, observed for the oligomers upon addition of KI, as shown in Figure 4. The effect of KI on the fluorescence lifetime is largest for the double-stranded DNA with the 12C tether and smallest for the single-stranded oligomers with the 3C and 6C tethers. This is consistent with the trends in kq. The other oligomers showed similar, intermediate effects. In a control experiment to evaluate any effects of increasing ionic strength, chloride ion (as KCl) was added to DNA33-6C at the same concentrations as in the iodide experiment. Effects on the fluorescence properties were much smaller than those for iodide, which verifies that the effects of iodide were due to quenching rather than increasing ionic strength. In general, the fluorescence lifetimes in Figure 4 were adequately described by a single, discrete exponential model. Fits
Table 5. Rotational Correlation Times (Φ1 and Φ2) and Fractional Contribution of Φ1 to the Total Anisotropy Decay (r1), Recovered from NLLS Analysis Using a Biexponential Anisotropic Rotator Model, of Fluorescein-Labeled Oligomers as a Function of Quencher Concentration at 25 °C
ss-DNA33-3C
ds-DNA33-3C
ss-DNA33-6C
ds-DNA33-6C
ss-DNA33-12C
ds-DNA33-12C
[KI], M
Φ1 (ns)
R1
Φ2 (ns)
0 0.063 0.123 0.185 0 0.063 0.123 0.185 0 0.063 0.123 0.185 0 0.063 0.123 0.185 0 0.063 0.123 0.185 0 0.063 0.123 0.185
0.5 0.5 0.4 0.4 0.7 0.7 0.6 0.5 0.6 0.6 0.7 0.6 0.8 0.7 0.7 0.6 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3
0.93 0.81 0.72 0.67 0.84 0.80 0.75 0.69 0.97 0.95 0.95 0.91 0.94 0.91 0.88 0.89 0.93 0.89 0.85 0.80 0.92 0.91 0.87 0.84
11 17 27 30 7 13 24 29 400 350 420 19 670 32 19 55 7 13 15 18 6 6 8 12
to continuous distribution lifetime models (e.g., Gaussian distributions) indicated that the lifetimes for DNA33-6C had essentially zero peak width for the double strand and slight widths for the single strand. The peak widths for the 3C and 12C tethers in both the single and double strands were all on the order of 1 ns. A significant peak width indicates some heterogeneity in the lifetime, due to either the existence of more than one microenvironment for the fluorescein yielding multiple, similar lifetimes that could not be resolved or a truly continuous distribution of microenvironments for fluorescein. Thus, homogeneity is indicated for the 6C tether, while there is some indication of a small degree of microenvironmental heterogeneity for the other tethers. Table 5 shows the DFA results for the quenching experiments, obtained from NLLS fits to a biexponential, anisotropic rotator model. Fluorescence lifetimes were fixed to the values in Figure 4. The values for Φ1 were in the 10-1 ns range for all the oligomers at all of the quencher concentrations. The value for the 12C tether was significantly lower than those for the 3C and 6C tethers and was unaffected by hybridization or quencher concentration. Only for the 3C tether was a significant increase observed in Φ1 upon hybridization. For all of the oligomers, the fractional contribution from Φ1 decreased with increasing KI, and the effect was greatest for the 3C tether and smallest for the 6C tether, again reflecting the relative accessibilities of the fluorescein to quencher in the bulk solution for the different tether lengths. As in the temperature experiments, the determination of the long rotational correlation time, Φ2, was less successful. The primary reason is that Φ2 is very slow compared to the fluorescence lifetime, which is further decreased by collisional quenching. However, Φ2 is fairly well resolved for the 3C and 12C tethers, and both its value and its contribution increase as KI is increased. Values of Φ2 for the 6C tether, which had smaller
contributions from Φ2, were less consistent and larger than those for the other oligomers. DISCUSSION Consistent with our previous study at 25 °C,5 the anisotropy decay of fluorescein tethered to the DNA oligonucleotides can be described by an anisotropic rotator model. Due to local hindrance, depolarization is not complete, and the remaining anisotropy decays by a much slower motion which is attributed to the rotation of the whole oligomer. The DFA results suggest that the decrease in steady-state anisotropy with increasing temperature is due to a combination of a decrease in the value of the short correlation time, Φ1, and an increase in its contribution to the total anisotropy decay (at the expense of the long correlation time, Φ2, associated with the rotation of the entire oligomer). Decreasing viscosity alone does not account for the temperature dependence of the anisotropy of the single strands, which must then include considerations of conformational changes. The contribution of Φ2 to the anisotropy decay increases with increasing quencher concentration. This suggests that there may exist two or more discrete conformations for the tethered fluorescein, one that is extended into the bulk solution and associated with Φ1, and another in which the fluorescein is closely associated with, or “bound” to, the oligomer and associated with Φ2. Since the extended conformation would be more susceptible to quenching, the contribution of Φ1 to the total anisotropy decay would decrease with increasing quencher concentration, resulting in a greater contribution from Φ2. Since the 3C tether shows the greatest indication of two discrete conformations in the various experiments, it makes sense that it would also show the largest decrease in Φ1 with increasing quencher concentration. The 6C tether, on the other hand, generally shows the most discrete and homogeneous conformational behavior in the various experiments, arising from tight association of the 6C tethered fluorescein with the oligomer in both the single and double strands. It therefore shows the smallest effect of quencher on the contribution from Φ1. A two-conformation model is consistent with the temperature studies. All of the oligomers displayed increasingly discrete, monoexponential fluorescence decay as temperature was increased, suggesting multiple conformations that are manifested at the lower temperatures as two separate conformations for the 3C tether and unresolved distributions for the longer tethers. In the DFA studies, there is a significant contribution from Φ2 at low temperatures (
