A Homogeneous DNA Hybridization System by Using A New

Feb 22, 2002 - In the system, two DNA probes whose sequences are complementary to the two different consecutive sequences of a target DNA are used; on...
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Bioconjugate Chem. 2002, 13, 200−205

A Homogeneous DNA Hybridization System by Using A New Luminescence Terbium Chelate Shinji Sueda,† Jingli Yuan,‡ and Kazuko Matsumoto*,† Department of Chemistry, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan, and Department of Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China. Received April 16, 2001; Revised Manuscript Received December 7, 2001

Homogeneous DNA hybridization assay based on the luminescence resonance energy transfer (LRET) from a new luminescence terbium chelate, N,N,N1,N1-[2,6-bis(3′-aminomethyl-1′-pyrazolyl)-4-phenylpyridine]tetrakis(acetic acid) (BPTA)-Tb3+ (λex ) 325 nm and λem ) 545 nm) to an organic dye, Cy3 (λex ) 548 nm and λem ) 565 nm), has been developed. In the system, two DNA probes whose sequences are complementary to the two different consecutive sequences of a target DNA are used; one of the probes is labeled with the Tb3+ chelate at the 3′-end, and the other is with Cy3 at the 5′-end. Labeling of the Tb3+ chelate is accomplished via the linkage of a biotin-labeled DNA probe with the Tb3+ chelatelabeled streptavidin. Strong sensitized emission of Cy3 was observed upon excitation of the Tb3+ chelate at 325 nm, when the two probe DNAs were hybridized with the target DNA. The sensitivity of the assay was very high compared with those of the previous homogeneous-format assays using the conventional organic dyes; the detection limit of the present assay is about 30 pM of the target DNA strand.

INTRODUCTION

Several homogeneous DNA hybridization assay systems have been reported (1-4), in which fluorescence resonance energy transfer (FRET) is used, and the target DNAs can be detected in a homogeneous solution without time-consuming and tedious immobilization of the DNAs or separation of the reaction solution (5). In the FRET assay, two DNA probes, each being complementary to the two different consecutive regions of a target DNA, are used. One probe has a donor dye while the other an acceptor dye. The target DNA is detected in a homogeneous manner by monitoring the sensitized emission of the acceptor resulting from the hybridization reaction. In most of the systems developed so far, organic dyes are employed as the donor-acceptor pair. However, the sensitivities of such systems are insufficient for practical use. Such low sensitivity is mostly due to the existence of the substantial background caused by the donor emission and the directly excited acceptor emission. To circumvent this problem, we have directed our attention to the lanthanide chelates as the energy donor and have already developed a homogeneous DNA hybridization system using the BHHCT-Eu3+ chelate as the donor and Cy5 as the acceptor (6-8). Some of the lanthanide chelates exhibit marked distinct luminescence properties (9-14): the luminescence emission profiles of the lanthanide chelates are very sharp, usually with the fwhm of ca. 10 nm; the Stokes shift is large, usually in the range of several hundreds of nanometers; the excitation is 300-350 nm and the emission is 615 nm for the Eu3+ chelate and 545 nm for the Tb3+ chelate. Therefore, the sensitized emission band of the organic dye acceptor * To whom correspondence should be addressed. Phone: +813-5286-3108. Fax: +81-3-5273-3489. E-mail: kmatsu@ mn.waseda.ac.jp. † Waseda University. ‡ Chinese Academy of Sciences.

Figure 1. Structure of the BPTA ligand.

can easily be distinguished from the excitation light and the emission of the lanthanide chelate donor. Also, the directly excited acceptor emission is effectively suppressed to almost zero, because the absorption spectra of the acceptor organic dyes hardly overlap on those of the donor lanthanide chelates and therefore the excitation wavelength, owing to the large Stokes shift of the lanthanide chelates. Moreover, the lifetimes of the lanthanide chelates are very long (∼1 ms), and as a result, the sensitized emission of the acceptor also becomes longlived. Thus, by employing time-resolved luminescence measurement, luminescence of the short-lived species including the directly excited acceptor emission can be eliminated (11-14). Recently, we have developed a new luminescent terbium chelate, N,N,N1,N1-[2,6-bis(3′-aminomethyl-1′-pyrazolyl)-4-phenylpyridine]tetrakis(acetic acid), (BPTA)-Tb3+ (Figure 1). The synthesis and the fluorescence properties of the chelate were detailed in the previous paper (15). The Tb3+ chelate exhibits strong luminescence characteristic of Tb3+ ion at 545 nm by the excitation of the ligand (325 nm), whose lifetime is over 2 ms. In the present manuscript, a homogeneous DNA hybridization assay system based on the luminescence resonance

10.1021/bc010049+ CCC: $22.00 © 2002 American Chemical Society Published on Web 02/22/2002

Homogeneous Hybridization Assay Using a Tb Chelate

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Figure 2. Principle of the homogeneous DNA hybridization assay in the present work.

energy transfer (LRET) from the BPTA-Tb3+ chelate to an organic dye, Cy3, is reported. The principle of our method is illustrated in Figure 2. The acceptor Cy3 was directly introduced into the 5′-end of the probe DNA. On the other hand, the donor Tb3+ chelate was indirectly introduced into the probe DNA via the biotin-streptavidin bridge: the donor probe DNA was prepared by addition of the Tb3+ chelate-labeled streptavidin to the biotin-labeled probe DNA. In the system, the sensitized emission of Cy3 is observed via LRET in the presence of the target DNA, but not in the absence of the target DNA. Therefore, the target DNA can be detected by monitoring the sensitized emission of Cy3, in a homogeneous solution without the conventional immobilization and separation steps. In the present work, two kinds of donor probe DNAs were used, which differ in the biotin labeling position: one has a biotin label at the 3′-end (3′-end donor probe), and the other has in the internal position (internal donor probe). These two donor probe DNAs serve to examine the effect of the distance between the donor and acceptor on the energy transfer efficiency. EXPERIMENTAL PROCEDURES

The Labeling of Streptavidin with the BPTATb3+ Chelate. The synthesis of BPTA was reported in the previous paper (15). Streptavidin (SA) was labeled with BPTA by using the succinimidyl monoester of BPTA (NHS-BPTA) according to the procedure slightly modified from that of our previous report (15) as follows. Ten milligrams (1.5 × 10-2 mmol) of NHS-BPTA and 5 mg (8.3 × 10-5 mmol) of SA were dissolved in 0.1 M carbonate buffer (pH 9.1) (10 mL). After the solution was stirred for 4 h at room temperature, the solution was dialyzed three times against 3 L of 0.1 M NaHCO3 containing 0.25 g of NaN3, each time for 24 h at 4 °C. The labeling ratio of BPTA to SA was determined as SA(BPTA)26 by using the absorbance of the labeled SA solution and the molar extinction coefficient of BPTA. The complexation with Tb3+ was achieved by dilution of the labeled SA solution (1 mL) with 50 mM Tris-HCl buffer (pH 7.8) (24 mL) containing equimolar amount of TbCl3

and incubation for a few minutes at room temperature. The luminescence of the Tb complex appeared immediately. Dilution of a nonlabeled SA solution with the same TbCl3 buffer solution did not give luminescence, which confirmed that Tb3+ binds to BPTA and gives the luminescence. The high stability of the BPTA-Tb3+ complex was confirmed by the two experiments: First, the competitive binding assay with EDTA showed that the binding constant of the free BPTA with Tb3+ is higher than that of EDTA (Ka > 1018). Second, the Tb3+ titration with BPTA labeled to SA by monitoring the Tb3+ luminescence showed that the chelate formation is stoichiometric in the concentration range less than that of the presence work. Preparation of the Probe DNAs and the Target DNAs. Figure 3 shows the probe DNAs and the target DNAs used in the present work. The 31mer and 34mer were used as the target DNAs in the 3′-end biotin probe system and the internal biotin probe system, respectively. All DNAs (oligonucleotides) were prepared on a fully automated DNA synthesizer according to the standard phosphoramide coupling method. Biotin was introduced into the 3′-end and the internal position of the oligonucleotides by using biotin TEG CPG and biotin TEG phosphoramidite (Glen Research), respectively, whereas Cy3 was introduced with Cy3 phosphoramidite (Glen Research). The purification of the oligonucleotides was carried out with reversed-phase HPLC. Hybridization Condition. The target oligonucleotides and probe oligonucleotides were mixed at a desired ratio in the buffer containing 10 mM Tris-HCl (pH 7.8) and 0.5 M NaCl. Oligonucleotides were annealed by heating at 50 °C for 15 min and cooling to room temperature. Luminescence Energy Transfer Measurement. To a solution of the hybridized oligonucleotides was added the solution of BPTA-Tb3+-labeled SA at an 1:1 molar ratio of SA to the biotinylated DNA. The final concentrations of the probe DNAs and labeled SA were 50 nM or 10 nM. After the solution was incubated for 1 h at room temperature, the luminescence was measured with the excitation at 325 nm. The luminescence was measured

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Figure 4. Spectra of the BPTA-Tb3+ chelate bound to SA, and Cy3 bound to the oligonucleotide. The solid line is the emission spectrum (λex ) 325 nm) of the BPTA-Tb3+ chelate, and the dashed line and the solid line with dots are the emission spectrum (λex ) 545 nm) and absorption spectrum of Cy3, respectively. Figure 3. Sequences of the target DNAs and the probe DNAs used in the present study. The hybridization sites on the target DNAs are underlined.

on a LS-50B (Perkin-Elmer) fluorometer equipped with a Xenon flash lamp (10 µs-pulse width and repetition rate of 50 Hz). A 420 nm cut off filter was employed to eliminate the second-order scattering light. The spectra of LRET were measured in two modes. One is a normal luminescence mode, in which all the luminescence was recorded over a gate period of 80 µs after pulse excitation without delay time. The other is the time-resolved luminescence mode, in which luminescence is recorded over a gate period of 500 µs with 40 µs of delay time after pulse excitation. In this time-resolved mode, the luminescence of only the long-lived species can be detected; the fluorescence of the short-lived species are eliminated. Luminescence decay curves were obtained by measuring the luminescence intensity at 565 nm with the gate period of 10 µs gate period after the appropriate delay time. By varying the delay time, the luminescence decay curves were constructed. RESULTS

Spectral Characteristics of the Donor and Acceptor. The emission spectrum of the BPTA-Tb3+ chelate bound to SA, and the emission and absorption spectra of Cy3 bound to the oligonucleotide are shown in Figure 4. The emission peak of the Tb3+ chelate overlaps on the absorption spectrum of Cy3, satisfying the requirement for luminescence resonance energy transfer. It should also be noted that the emission intensity of the Tb3+ chelate at the Cy3 emission maximum of 565 nm is very weak; the emission intensity of the Tb3+ chelate at 565 nm is 250 times lower than that at the Tb3+ chelate maximum of 545 nm. Therefore, high signal/background ratio is obtained when the sensitized emission of the acceptor is monitored at 565 nm, resulting in the highly sensitive detection of the target DNA. Measurement of the Emission Spectra in the Normal Luminescence Mode. Figure 5 shows the

Figure 5. Comparison of the emission spectra obtained in the normal luminescence detection mode. Solid line represents the emission spectrum obtained for the 3′-end donor probe DNAs in the presence of its target DNA (31mer). Dashed line represents the emission spectrum obtained for the internal donor probe DNAs in the presence of its target DNA (34mer). Solid line with dots represents the emission spectrum observed in the absence of the target DNAs. Both of the 3′-end donor system and the internal donor system gave the identical spectrum in the absence of the target DNAs. The concentrations of the probe DNAs, the target DNAs, and SA were 50 nM.

emission spectra obtained in the normal luminescence mode, i.e., the spectra measured without delay time. The comparison of the emission spectra in the presence and absence of the target DNAs shows that the sensitized emission of Cy3 is observed only in the presence of the target DNAs. The sensitized emission is stronger in the 3′-end donor probe system than the internal donor probe system, and this is due to the well-known relation that the energy transfer efficiency decreases with R-6, where R is the distance between the donor and the acceptor (16). The biotin is separated from Cy3 by 11 base pairs (ca.

Homogeneous Hybridization Assay Using a Tb Chelate

Figure 6. The luminescence decay curves of Cy3 observed at 565 nm. Closed circles are for the 3′-end donor probe DNA, and open circles are for the internal donor probe DNA. Both of the two curves were obtained in the presence of the target DNAs. The curve with triangles was obtained in the absence of the donor probe. The concentrations of the probe DNAs, the target DNAs, and SA were 50 nM.

35 Å) in the internal donor probe system, whereas the separation is only a single base in the 3′-end donor probe system. It is notable that the intensity of the sensitized emission obtained in the 3′-end donor probe system is significantly strong; the signal/background ratio at 565 nm is approximately 30:1 (the signal/background ratio represents the ratio of the emission intensities at 565 nm in the presence and absence of the target DNA). Measurement of the Luminescence Decay Curves. Since the lifetime of the BPTA-Tb3+ chelate is very long (>2 ms), it is expected that the sensitized emission of the acceptor, Cy3, also becomes long-lived. The luminescence decay curves were measured for the sensitized emission by monitoring the intensity at 565 nm (Figure 6). Clearly, the lifetimes of the sensitized emission in both of the donor probe systems are longer-lived than that obtained in the absence of the donor probe DNAs. While exact quantitative determination of the lifetime is not possible in the present LRET, where multiple donor molecules with various distances to the acceptor molecule transfer the energy, the lifetimes were roughly estimated in the range of a few tens to a few hundreds microseconds. This long-lived property clearly demonstrates that the observed emission of Cy3 in the presence of the target DNAs is due to LRET. In Figure 6, while the luminescence intensity is stronger during the initial 0.1 ms for the 3′-end biotin probe system than for the internal biotin probe system, this relation is reversed after 0.1 ms. This means that the lifetime of the sensitized emission of Cy3 becomes longer as the distance between the donor and the acceptor increases. This behavior is explained by the mechanism of LRET as follows: when the distance is short, the efficiency of the energy transfer increases, and the lifetime of the Tb3+ chelate becomes short. At the same time, the lifetime of the acceptor, Cy3 decreases, since the donor Tb3+ chelate becomes short-lived. Therefore, the lifetime of the sensitized emission is shorter in the 3′-end biotin probe system, in which energy transfer occurs more effectively. Measurement of the Emission Spectra in the Time-Resolved Luminescence Mode. It was found in

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Figure 7. Comparison of the emission spectra observed in the time-resolved luminescence mode. The spectra were obtained with 40 µs delay time and 500 µs gate time. Solid line represents the emission spectrum obtained for the 3′-end donor probe DNA in the presence of its target DNA (31mer). Dashed line represents the emission spectrum obtained for the internal donor probe DNA in the presence of its target DNA (34mer). Solid line with dots represents the emission spectrum observed in the absence of the target DNAs (an identical spectrum was obtained for the both donor probe DNAs systems). The concentrations of the probe DNAs, the target DNAs, and SA were 50 nM.

the above decay curves that the sensitized emission of Cy3 is moderately long-lived (microsecond order) and therefore can be monitored in the time-resolved luminescence mode. Figure 7 shows the observed timeresolved luminescence spectra in the presence and absence of the target DNAs. In this detection mode, the intensity of the sensitized emission at 565 nm is almost the same in both of the two donor probe DNA systems. As shown in the decay curve experiment, the intensity of the sensitized emission is stronger during the initial 0.1 ms for the 3′-end biotin probe system than for the internal biotin probe system; however, this relation is reversed after 0.1 ms. In this work, the time-resolved spectra were obtained with the gate period of 0.5 ms after 0.04 ms of the delay time. It seems that the intensities of the sensitized emission of the Cy3 happened to be the same in both of the two probe DNA systems under the present condition. Contrary to our expectation, the signal/background ratio at 565 nm in the time-resolved mode is lower (approximately 10:1) than that in the normal luminescence mode (approximately 30:1). In the present LRET assay, multiple Tb3+ chelates labeled to a large streptavidin molecule act as the donor, and the energy transfer occurs between these multiple Tb3+ donors and a single Cy3 molecule. The distances of the Tb3+ chelates to the Cy3 molecule span a substantial range, since the Tb3+ chelates are bound to a large streptavidin molecule. Some of the chelates efficiently transfer the absorbed energy to Cy3, whereas others do not, depending on the distance. Under such circumstances, the lifetime of the Tb3+ chelate is still longer than that of Cy3. At 565 nm, the Tb3+ chelate has weak emission. This weak emission is really weak compared to the sensitized emission of Cy3 in the normal luminescence measurement, however becomes moderately strong and constitutes the background in the time-resolved measurement, since both of the intensities are collected after 0.04 ms of delay time.

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Figure 8. The plot of the Cy3 emission intensity at 565 nm against the concentration of the target DNA (31mer). The plot was obtained for the 3′-end donor probe DNA system. Closed circles are for the normal luminescence mode, and the open circles are for the time-resolved luminescence mode. The concentrations of the probe DNAs and SA were 10 nM.

This is the reason for the low signal/background ratio at 565 nm in Figure 7 than in Figure 5. The Sensitivities for DNA Detection in the Two Detection Methods. The sensitivity of the present method was examined by using the 3′-end biotin probe system. Figure 8 shows the plot of the Cy3 emission intensity at 565 nm against the concentration of the target DNA. Figure 8 shows both of the normal mode and the time-resolved mode. The detection limit was determined as the concentration corresponding to the twice amount of the background standard deviation. Detection limits in the normal mode and the time-resolved mode are about 30 pM and 50 pM, respectively, when 10 nM of the probe DNA was used. These detection limits are 2 orders of magnitude superior to that obtained with the previous homogeneous hybridization assay (17). DISCUSSION

In the present work, a homogeneous DNA hybridization assay has been developed based on the luminescence resonance energy transfer from the BPTA-Tb3+ chelate to Cy3. Two systems differing in the distance between the donor and the acceptor were constructed by using two donor probe DNAs, one having the biotin label at the 3′end and the other at the internal position. As expected, the emission was more effectively sensitized in the system of the 3′-end biotin probe. By using the 3′-end biotin system, the high signal/background ratio (30:1) was obtained at 565 nm in the normal luminescence mode. Such a high signal/background ratio cannot be achieved in the systems using organic dyes as the donor-acceptor pairs. The emission and absorption peak profiles of the organic dye pairs are broad and overlap to each other to some extent; as a result, the emission of the donor and the directly excited emission of the acceptor cause substantial background to the sensitized emission. However, in our system using the Tb3+ chelate as the donor, the emission of the donor hardly interferes with the sensitized emission, because the emission profile of the Tb3+ chelate is very sharp and the emission intensity is very weak at the maximum emission wavelength of Cy3.

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The directly excited emission of the acceptor is also very effectively suppressed, since Cy3 is scarcely excited at the excitation wavelength for the Tb3+ chelate, 325 nm (the molar extinction coefficient of Cy3 at 325 nm is about one-eightieth of that at its maximum absorption wavelength, 548 nm). The detection limit of the present method is excellent, due to the high signal/background ratio and the very effective energy transfer. The Tb3+ chelate is multiply labeled onto the probe DNA by using the streptavidinbiotin interaction, and causes strong sensitized emission of Cy3. Such multiple labeling strategy was not effective for enhancing the fluorescence of the acceptor in the conventional donor organic dye system, due to π-stacking and reabsorption of the emitted fluorescence of the donor (18). The Tb3+ chelate molecules do not stack to each other and do not quench their emission since they have a large Stokes shift. Under the time-resolved luminescence measurement condition (delay time 40 µs, gate time 500 µs), the intensity of the sensitized emission of the acceptor was almost the same between the 3′-end donor probe system and the internal donor probe system. This behavior is explained by the LRET property that as the distance between the donor and the acceptor increases, the efficiency of the energy transfer is decreased, whereas the lifetime of the sensitized emission is increased. In the time-resolved mode, the signal/background ratio at 565 nm is markedly low, as compared with that in the normal luminescence mode, though the emission in the latter mode contains a considerable contribution from the direct excitation of the Cy3 acceptor. Consequently, the detection limit is better in the normal luminescence mode than in the time-resolved mode. However, the time-resolved detection mode is still attractive, especially for the samples containing many impurities or coexisting materials, since the background emission caused by the short-lived species can be eliminated by the time-resolved mode. Considering these LRET characteristics, the optimal distance between the donor and the acceptor and the optimal measurement conditions must be found, and such parameters are expected to depend on the nature of the samples. ACKNOWLEDGMENT

This work has been supported by CREST (Core Research for Evolutional Science and Technology) of Japan Science and Technology Corporation (JST) and the Grantin-Aid for COE Research, Ministry of Education, Culture, Sports, Science and Technology (MEXT). LITERATURE CITED (1) Heller, M. J., and Morrison, L. E. (1985) Chemiluminescent and Fluorescent Probes for DNA Hybridization Systems. Rapid Detection and Identification of Infectious Agents (Kingsbury, D. T., and Falkow, S., Eds.) pp 245-256, Academic Press, New York. (2) Matthews, J. A., and Kricka, L. J. (1988) Analytical Strategies for the Use of DNA Probes. Anal. Biochem. 169, 1-25. (3) 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. (4) Mergny, J.-L., Boutorine A. S., Garestier, T., Belloc, F., Rouge´e, M., Bulychev, N. V., Koshikin, A. A., Bourson, J., Lebedev, A. V., Valeur, B., Thuong, N. T., and He´le`ne, C. (1994) Fluorescence Energy Transfer as a Probe for Nucleic Acid Structures and Sequences. Nucleic Acids Res. 22, 920928.

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