Anal. Chem. 2009, 81, 1426–1432
Quantitative Fluorescence Correction Incorporating Fo ¨ rster Resonance Energy Transfer and Its Use for Measurement of Hybridization Efficiency on Microarrays Jiang Zhu,†,‡ Cheng Deng,†,‡ Guoliang Huang,*,†,‡,§ Shukuan Xu,†,‡ Keith Mitchelson,†,‡ and Jing Cheng*,†,‡,§,| Medical Systems Biology Research Center, Tsinghua University School of Medicine, Haidian District, Beijing 100084, P. R. China, Department of Biomedical Engineering, Tsinghua University, Haidian District, Beijing 100084, P. R. China, National Engineering Research Center for Beijing Biochip Technology, 18 Life Science Parkway, Changping District, Beijing 102206, P. R. China, and State Key Laboratory for Biomembrane and Membrane Biotechnology, Tsinghua University, Haidian District, Beijing 100084, P. R. China Fluorescence detection using two spectrally distinct fluorophores has long been used for the determination of the relative abundance of biomolecules, but overlap between the fluorescence spectra of each fluorophore can result in nonradiative Fo ¨rster resonance energy transfer (FRET) and distorting the signals detected by fluorescence channels. Thus conventional methods for quantifying the relative abundance of fluorophores by fluorescence emission will not be accurate if FRET can occur. In this paper we report the development of a quantitative fluorescence correction method incorporating FRET to measure the relative abundance of fluorophores in dual-labeling experiments. The quantitative fluorescence correction method incorporating FRET is accurate, comprehensive, and convenient for the measurement of the relative abundance of fluorophores in dual-labeling experiments and can also correct the FRET distortion and provide accurate, quantitative, and convenient measurement of the hybridization efficiencies on microarrays. When different molecules or different parts of a molecule are coupled, respectively, with spectrally distinct fluorophores, a higher accuracy of detection of interactions between the molecules1-3 and changes in the structure of the molecules4,5 can be obtained through FRET between one fluorophore (shorter excitation wavelength fluorophore, donor) and the other (longer * To whom correspondence should be addressed. Jing Cheng, fax: +86 10 80726898. E-mail:
[email protected]. Guoliang Huang, fax: +86 10 80726769. E-mail:
[email protected]. † Medical Systems Biology Research Center, Tsinghua University School of Medicine. ‡ National Engineering Research Center for Beijing Biochip Technology. § Department of Biomedical Engineering, Tsinghua University. | State Key Laboratory for Biomembrane and Membrane Biotechnology, Tsinghua University. (1) Sako, Y.; Minoguchi, S.; Yanagida, T. Nat. Cell Biol. 2000, 2, 168–172. (2) Zal, T.; Gascoigne, N. R. Curr. Opin. Immunol. 2004, 16, 674–683. (3) Lippincott-Schwartz, J.; Snapp, E.; Kenworthy, A. Nat. Rev. Mol. Cell Biol. 2001, 2, 444–456. (4) Weiss, S. Science 1999, 283, 1676–1683. (5) Weiss, S. Nat. Struct. Biol. 2000, 7, 724–729.
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excitation wavelength fluorophore, acceptor) when the emission spectrum of the donor and the excitation spectrum of the acceptor overlap and the distance between the donor and acceptor fluorophores is within the range of 1-10 nm.6-8 In particular, the FRETbased microarray platforms allow mutations9,10 and DNA targets11,12 to be specifically and conveniently detected in parallel using the molecular beacon or TaqMan probe and are also applicable to high-throughput and sensitive analysis of protein-protein interactions using fluorescence lifetime imaging.13 However, when the relative abundance or the localization of targets is determined by the dual-labeling methods widely used for dual-color imaging,14-17 two-color DNA microarray analysis18,19 and double labeling flow cytometry,20 FRET between spectrally distinct fluorophores may result in an underestimation of the amount of the donor. Because under conditions whereby FRET can occur, the energy held by the donor in an excited-state can be either emitted by the radiative pathway or via FRET transferred (6) Stryer, L.; Haugland, R. P. Proc. Natl. Acad. Sci. U.S.A. 1967, 58, 719– 726. (7) Jares-Erijman, E. A.; Jovin, T. M. Nat. Biotechnol. 2003, 21, 1387–1395. (8) Selvin, P. R. Nat. Struct. Biol. 2000, 7, 730–734. (9) Fang, X.; Liu, X.; Schuster, S.; Tan, W. J. Am. Chem. Soc. 1999, 121, 2921– 2922. (10) Frutos, A. G.; Pal, S.; Quesada, M.; Lahiri, J. J. Am. Chem. Soc. 2002, 124, 2396–2397. (11) Kim, H.; Kane, M. D.; Kim, S.; Dominguez, W.; Applegate, B. M.; Savikhin, S. Biosens. Bioelectron. 2007, 22, 1041–1047. (12) Liu, H.; Wang, H.; Shi, Z.; Wang, H.; Yang, C.; Silke, S.; Tan, W.; Lu, Z. Nucleic Acids Res. 2006, 34, e4. (13) Nagl, S.; Bauer, R.; Sauer, U.; Preininger, C.; Bogner, U.; Schaeferling, M. Biosens. Bioelectron. 2008, 24, 397–402. (14) Shu, D.; Zhang, H.; Jin, J.; Guo, P. EMBO J. 2007, 26, 527–537. (15) Gerlich, D.; Beaudouin, J.; Gebhard, M.; Ellenberg, J.; Eils, R. Nat. Cell Biol. 2001, 3, 852–855. (16) Merrifield, C. J.; Feldman, M. E.; Wan, L.; Almers, W. Nat. Cell Biol. 2002, 4, 691–698. (17) Stephens, D. J.; Allan, V. J. Science 2003, 300, 82–86. (18) Patterson, T. A.; Lobenhofer, E. K.; Fulmer-Smentek, S. B.; Collins, P. J.; Chu, T. M.; Bao, W.; Fang, H.; Kawasaki, E. S.; Hager, J.; Tikhonova, I. R.; Walker, S. J.; Zhang, L.; Hurban, P.; de Longueville, F.; Fuscoe, J. C.; Tong, W.; Shi, L.; Wolfinger, R. D. Nat. Biotechnol. 2006, 24, 1140–1150. (19) Duggan, D. J.; Bittner, M.; Chen, Y.; Meltzer, P.; Trent, J. M. Nat. Genet. 1999, 21 (Suppl.), 10–14. (20) Bartkowiak, D.; Högner, S.; Baust, H.; Nothdurft, W.; Röttinger, E. M. Cytometry 1999, 37, 191–196. 10.1021/ac802203r CCC: $40.75 2009 American Chemical Society Published on Web 01/22/2009
nonradiatively to the acceptor, and only the radiative pathway signal can be detected by the donor channel of fluorescence detectors. In dual-labeling experiments, the emission and excitation spectra of most of the fluorophores commonly used have an overlap and the distance between fluorophores cannot be rigidly controlled, so the possibility of FRET cannot be readily excluded entirely. For the accurate determination of the relative abundance of the fluorophores, a more reasonable approach would be to apply a correction of the donor emission in dual-labeling experiments using fluorescence microscopy, microarray analysis, spectroscopic analysis, and flow cytometry. Spectral imaging has been previously described as a method for the FRET correction.21 However it is difficult for the conventional filter technology (e.g., conventional fluorescence microscopy, microarray analysis, and flow cytometry) to acquire the spectral signals. Hoppe et al.22 described a method to measure the concentration ratio of fluorophores in the presence of FRET using three microscopic fluorescence images, which requires the measurement of fluorescence lifetime for the determination of the FRET efficiency. Surface-immobilized DNA hybridization is the foundation of the microarray technique and plays a key role in gene expression profiling, drug discovery, disease diagnosis, and other applications based on the genomic detection. The measurement of hybridization efficiencies is the important event for the studies of DNA hybridization and can provide accurate and quantitative estimations for the hybridization reactions. The previous methods for the measurement of surface binding suffer from various shortcomings, such as the need for specialized techniques (e.g., the electrochemical analysis,23 surface plasmon resonance spectroscopy,24 and reflectometric interference spectroscopy25), the construction of the standard curves,26 or the performance of the control experiment. In this paper we have developed a quantitative fluorescence correction method incorporating FRET to overcome these limitations. We have demonstrated that our correction method could be used for the accurate, comprehensive, and convenient measurement of the relative abundance of fluorophores without the need for complex spectral analysis or the measurement of FRET efficiencies, taking into account both the contributions of unpaired donor and acceptor fluorophores, and the influence of different degrees of FRET interactions. We have also confirmed that our correction method was applicable for the accurate, quantitative, and convenient measurement of the DNA hybridization efficiency on the surface and the correction of the FRET distortion in twocolor DNA microarray analysis such as differential gene expression profiling. MATERIALS AND METHODS Oligonucleotides Synthesis. All oligonucleotides were synthesized using basic phosphoramidite chemistry and purified by (21) Thaler, C.; Koushik, S. V.; Blank, P. S.; Vogel, S. S. Biophys. J. 2005, 89, 2736–2749. (22) Hoppe, A.; Christensen, K.; Swanson, J. A. Biophys. J. 2002, 83, 3652– 3664. (23) Steel, A. B.; Herne, T. M.; Tarlov, M. J. Anal. Chem. 1998, 70, 4670–4677. (24) Peterlinz, K. A.; Georgiadis, R. M.; Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 3401–3402. (25) Piehler, J.; Brecht, A.; Gauglitz, G.; Zerlin, M.; Maul, C.; Thiericke, R.; Grabley, S. Anal. Biochem. 1997, 249, 94–102. (26) Riccelli, P. V.; Merante, F.; Leung, K. T.; Bortolin, S.; Zastawny, R. L.; Janeczko, R.; Benight, A. S. Nucleic Acids Res. 2001, 29, 996–1004.
high-performance liquid chromatography (TaKaRa Biotechnology, Dalian, China). The full details are provided in Table S1 of the Supporting Information. For quantitative fluorescence correction studies, a carboxytetramethylrhodamine (TMR) donor fluorophore and a cyanine 5 (Cy5) acceptor fluorophore were used. The set of four oligonucleotides (oligo-1) with the sequence 5′-TCCGTCATCGCTCAAG-3′ that differed with respect to the attached fluorophore were synthesized. These were respectively, Cy5-(oligo-1) with a Cy5 attached to the 5′ terminal; (oligo-1)TMR with a TMR attached to the 3′ terminal; (oligo-1) without an attached fluorophore; and Cy5-(12)(oligo-1)-TMR with a TMR attached to the 3′ terminal and a Cy5 attached at base 12, close to the 3′ terminal. The second set of oligonucleotides (oligo-2) complementary to oligo-1 were also synthesized with the sequence 5′-CTTGAGCGATGACGGA-3′. These also differed with respect to the attached fluorophore and were, respectively, TMR-(oligo-2) with a TMR attached to the 5′ terminal, Cy5-(oligo-2) with a Cy5 attached to the 5′ terminal, and (oligo-2) which lacked a fluorophore. A third set of oligonucleotides (oligo-3) with the sequence 5′CTTGAGCGATGACGGATTTTTTTTTTTTTTTT-NH2-3′, in which the 5′-half-was complementary to the oligo-1 set of oligonucleotides, could be coupled to an aldehyde-activated glass surface by the 3′ terminal amidocyanogen. The poly T tail of the 3′half-was used to hold the oligonucleotide sequence away from the chip surface so that it was able to freely hybridize with complementary oligonucleotides. Either of the two capture oligonucleotides was employed: TMR-(oligo-3)-NH2 in which the TMR was attached to the 5′ terminal or (oligo-3)-NH2 which lacked an attached fluorophore. Fluorescence Measurements in Solution. To study the quantitative fluorescence correction method, seven solutions were prepared in 450 µL of hybridization buffer (10 mM TrisHCl, 50 mM NaCl, and 1 mM EDTA in ultrapure water), including the mixture of Cy5-(oligo-2) and (oligo-1), the mixture of TMR(oligo-2) and (oligo-1), the mixture of Cy5-(12)(oligo-1)-TMR and (oligo-2), the mixture of TMR-(oligo-2) and Cy5-(oligo-2), and three mixtures of Cy5-(oligo-1) and TMR-(oligo-2). Each of Cy5(oligo-2) and TMR-(oligo-2) was mixed, respectively, with complementary (oligo-1) in the same concentration of 0.5 µM, which was called Cy5-dsDNA and TMR-dsDNA. Cy5-(12)(oligo-1)-TMR was mixed with its complementary (oligo-2) in the same concentration of 0.5 µM, which was called Cy5-dsDNA12-TMR. In the mixture of Cy5-(oligo-2) and TMR-(oligo-2), Cy5-(oligo-2) and TMR(oligo-2) were in the same concentration of 0.5 µM, which was called Cy5/TMR-ssDNA. In three mixtures of Cy5-(oligo-1) and TMR-(oligo-2), the concentrations of Cy5-(oligo-1) were each 0.5 µM and the concentrations of TMR-(oligo-2) were 0.25, 0.5, and 1 µM, respectively, that were at Cy5-to-TMR ratios of 2:1, 1:1, and 1:2, which were called Cy5(2)-dsDNA16-TMR(1), Cy5(1)-dsDNA16-TMR(1), and Cy5(1)-dsDNA16-TMR(2). The seven mixtures were incubated in the hybridization buffer at 96 °C for 2 min and then cooled slowly down to 20 °C. Each of the seven solutions was measured by a LS50B luminescence spectrometer (Perkin-Elmer, Wellesley, MA) at 20 °C in a quartz cuvette. Each solution was excited at 532 and 640 nm, respectively, and emission spectra were recorded in the range of 550-750 nm at a scan rate of 2 nm/s. An excitation slit width Analytical Chemistry, Vol. 81, No. 4, February 15, 2009
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Figure 1. The pattern of two microarrays for oligonucleotides (oligo3)-NH2 and TMR-(oligo-3)-NH2 printed in six subarrays. H1, 4 µM; H2, 2 µM; H3, 1 µM; H4, 0.5 µM; H5, 0.25 µM; H6, 0.125 µM.
of 10 nm and an emission slit width of 10 nm were used during the experiments. As the maximum emission of TMR is at 580 nm and the maximum emission of Cy5 is at 670 nm, we used three channels (no complete spectra data) for the measurements. These channels were a donor (TMR) channel (excitation ) 532 nm, emission ) 580 nm), an acceptor (Cy5) channel (excitation ) 640 nm, emission ) 670 nm), and a FRET channel (excitation ) 532 nm, emission ) 670 nm). To ensure the reproducibility, four experiments were performed. Fluorescence Measurements on Microarrays. The oligonucleotides of (oligo-3)-NH2 and TMR-(oligo-3)-NH2 were diluted in DNA spotting buffer (CapitalBio, Beijing, China) to fabricate two types of microarrays at concentrations ranging from 4 to 0.125 µM each with six subarrays. The printing patterns of oligonucleotide spots on the two microarrays are the same (Figure 1). These microarrays were called the (oligo-3)-NH2 and the TMR-(oligo-3)-NH2 arrays. The microarrays were spotted using the SmartArrayer-48 microarray spotter (CapitalBio), and the oligonucleotides on the microarrays were covalently immobilized on the aldehyde-activated glass slides (CapitalBio) by the mediation of an amino group at their ends. Both Cy5-(oligo-1) and (oligo-1)-TMR were diluted in microarray hybridization buffer (5× Denhardt’s solution, 0.2% SDS, 3× SSC in ultrapure water) to 1 µM, respectively, and 12 µL of each solution was then hybridized with the (oligo-3)-NH2 array to fabricate the Cy5-dsDNA-PolyT array and the TMR-dsDNAPolyT array. Equal concentrations (final at 0.5 µM) of Cy5(oligo-1) and (oligo-1)-TMR were also mixed together in microarray hybridization buffer, and 12 µL of this solution was hybridized to the (oligo-3)-NH2 array to fabricate the Cy5/TMRdsDNA-PolyT array. The TMR-(oligo-3)-NH2 arrays were hybridized with 12 µL of hybridization solution containing either Cy5-(oligo-1), (oligo-1), or Cy5-(oligo-2) (each at 1 µM) to fabricate the TMR-dsDNA-Cy5-PolyT array, the TMR-dsDNAnofluo-PolyTarray,andtheTMR-ssDNA-PolyTarray,respectively. After hybridization at 42 °C for 2 h, these microarrays were washed for 4 min, first in washing buffer I (0.2% SDS, 2× SSC) and then with washing buffer II (0.2× SSC) at 42 °C, and then were dried by centrifugation at 1600 rpm for 1 min. The microarrays were then scanned by a modified LuxScan-10K/A dual-channel laser confocal microarray scanner (CapitalBio), which was equipped with a third channel for the detection of FRET signals. This was achieved by inclusion of a new set of laser and emission filter. The fluorescence signals were collected from three 1428
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channels, including a donor (TMR) channel (excitation ) 532 nm, emission ) 570 nm), an acceptor (Cy5) channel (excitation ) 640 nm, emission ) 675 nm), and a FRET channel (excitation ) 532 nm, emission ) 675 nm). The excitation power was set to 80% and the PMT gain was set to 65% for the TMR channel, the excitation power 82% and the PMT gain 65% for the Cy5 channel, and the excitation power 80% and the PMT gain 65% for the FRET channel. The microarray images were generated by LuxScan 2.1 software (CapitalBio) and were analyzed by SpotData Pro software (CapitalBio). This processing converted the image of the microarray into a matrix of spot intensities. Method for Quantitative Fluorescence Correction Incorporating FRET and Crosstalk. With the presence of FRET, the direct acceptor emission obtained from the acceptor channel (excitation, acceptor excitation wavelength; emission, acceptor emission wavelength), which is represented by IDirectAcceptor, will be the same as the emission of the acceptor in the absence of donor (ITotalAcceptor). However, the direct donor emission obtained from the donor channel (excitation, donor excitation wavelength; emission, donor emission wavelength), which is represented by IDirectDonor, will be less than the emission of the donor in the absence of acceptor (ITotalDonor) because of the nonradiative energy transfer. The measured signal from the donor channel therefore does not correspond in direct proportion to the quantity of the donor fluorophore and thus should be corrected by incorporating a FRET component. When both donor and acceptor fluorophores exist in the experimental system, the total donor emission and the total acceptor emission can be determined by the following equations, regardless of whether the donor fluorophores participate in FRET or not (the detailed derivation is presented in the Supporting Information), ITotalDonor ) IDirectDonor +
ISensitizedAcceptor G
ITotalAcceptor ) IDirectAcceptor
(1) (2)
where ISensitizedAcceptor is the sensitized acceptor emission due to FRET and can be obtained from the FRET channel (excitation, donor excitation wavelength; emission, acceptor emission wavelength) and G is the detection-correction factor and remains constant in a given experimental system (measuring instrument and fluorophores).27-30 Here if we define the response factor γ as the ratio of the acceptor emission (in the absence of the donor) to the donor emission (in the absence of the acceptor) when the donor and the acceptor are equimolar, the factor G can be accurately determined using the sample, in which the quantity of the donor equals that of the acceptor and FRET is present between them, by the following equation (the detailed derivation is presented in the Supporting Information), G)
ISensitizedAcceptor IDirectAcceptor - IDirectDonor γ
(3)
(27) Gordon, G. W.; Berry, G.; Liang, X. H.; Levine, B.; Herman, B. Biophys. J. 1998, 74, 2702–2713. (28) Ha, T.; Ting, A. Y.; Liang, J.; Caldwell, W. B.; Deniz, A. A.; Chemla, D. S.; Schultz, P. G.; Weiss, S. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 893–898.
Figure 2. Quantitative fluorescence correction in solution. (A) Fluorescence spectra of TMR-dsDNA and Cy5-dsDNA excited at 532 and 640 nm. (B) Fluorescence spectra of the dual-labeled samples excited at 532 nm.
Thus the quantity ratio E of the acceptor to the donor can be determined by combining eqs 1 and 2. E)
IDirectAcceptor ISensitizedAcceptor γ IDirectDonor + G
(
)
(4)
For the correction of fluorescence emission, which incorporates the effect of crosstalk due to the spectral overlap of fluorophores, ISensitizedAcceptor, IDirectDonor, and IDirectAcceptor should be corrected using the method previously described,27 ISensitizedAcceptor ) MDA
1 - Rβφφ R - MDD (1 - Rφ)(1 - βφ) 1 - Rφ β (5) MAA 1 - βφ
IDirectDonor ) MDD
1 φ - MDA 1 - Rφ 1 - Rφ
(6)
1 φ - MDA 1 - βφ 1 - βφ
(7)
IDirectAcceptor ) MAA
Here, R equals the ratio of the signal obtained from the FRET channel to the signal obtained from the donor channel in a sample containing only the donor, β equals the ratio of the signal obtained from the FRET channel to the signal obtained from the acceptor channel in a sample containing only the acceptor, φ equals the ratio of the signal obtained from the donor channel to the signal obtained from the FRET channel in a sample containing only the acceptor, and φ equals the ratio of the signal obtained from the acceptor channel to the signal obtained from the FRET channel in a sample containing only the donor. All of these values remain constant in a given experimental system (measuring instrument and fluorophores). MDA, MDD, and MAA are the measured signals from the FRET channel, the donor channel, and the acceptor channel, respectively. RESULTS AND DISCUSSION Validation of Quantitative Fluorescence Correction Incorporating FRET. Having developed the quantitative fluorescence correction method theoretically, we then validated this correction method by experimental measurement in solution. We first determined the system factors, then compared and discussed
the expected values (known concentration ratio present in our experiment), observed values (calculated concentration ratio without FRET correction), and corrected values (calculated concentration ratio with FRET correction) of Cy5-to-TMR ratios. In solution only intramolecular FRET is present, that is the interaction between the donor and acceptor both coupled in the same double-stranded DNA molecule. From Figure 2A, no emission from the Cy5 channel with the TMR-dsDNA solution or from the TMR channel with the Cy5dsDNA solution is detectable, so the crosstalk factors φ and φ equal zero. The crosstalk factor R equals 0.075 and the crosstalk factor β equals 0.039 in this experimental system. The transfer factor γ can be calculated from the ratio of the emission of Cy5dsDNA solution (to the Cy5 channel) to the emission of TMR-dsDNA solution (to the TMR channel), which equals 6.680 ± 0.091. In order to determine the detection-correction factor G, equimolar concentrations of Cy5-(oligo-1) and TMR-(oligo-2) were hybridized to form double-stranded Cy5(1)-dsDNA16-TMR(1), giving a Cy5-to-TMR ratio of 1:1. According to the DNA cylindrical model,31 the distance between the Cy5 and TMR fluorophores in this hybrid is within the range of 1-10 nm, hence there is a FRET interaction between them. The detection-correction factor G equals 0.602 ± 0.088 for this experimental system. For the validation of the FRET correction method in solution, five dual-labeled samples were measured, including the solutions of Cy5(2)-dsDNA16-TMR(1), Cy5(1)-dsDNA16-TMR(2), Cy5(1)dsDNA16-TMR(1), Cy5-dsDNA12-TMR, and Cy5/TMR-ssDNA. Figure 2B shows the spectra of these five samples when they were excited at 532 nm. Because the Cy5 fluorophores are in equimolar concentrations, the signals from the Cy5 channel in the five dual-labeled samples approximately equal that in the Cy5-dsDNA solution. The expected values, observed values, and corrected values of Cy5-to-TMR ratios in these samples are shown in Table 1. In the solutions of Cy5(1)-dsDNA16-TMR(1), Cy5(2)-dsDNA16TMR(1), and Cy5(1)-dsDNA16-TMR(2), the concentration ratios of Cy5 to TMR are 1.000, 2.000, and 0.500, respectively. The (29) Zal, T.; Gascoigne, N. R. Biophys. J. 2004, 86, 3923–3939. (30) Lee, N. K.; Kapanidis, A. N.; Wang, Y.; Michalet, X.; Mukhopadhyay, J.; Ebright, R. H.; Weiss, S. Biophys. J. 2005, 88, 2939–2953. (31) Deniz, A. A.; Dahan, M.; Grunwell, J. R.; Ha, T.; Faulhaber, A. E.; Chemla, D. S.; Weiss, S.; Schultz, P. G. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 3670– 3675.
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Table 1. Concentration Ratio of Cy5 to TMR in Solutiona sample in solution
expected ratio
observed ratio
corrected ratio
Cy5(2)-dsDNA16-TMR(1) Cy5(1)-dsDNA16-TMR(2) Cy5(1)-dsDNA16-TMR(1) Cy5-dsDNA12-TMR Cy5/TMR-ssDNA
2.000 0.500 1.000 1.000 1.000
5.142 ± 1.203 0.802 ± 0.225 2.536 ± 0.348 5.334 ± 0.732 0.903 ± 0.022
1.921 ± 0.221 0.484 ± 0.061 1.012 ± 0.090 0.837 ± 0.060 0.891 ± 0.020
a The expected ratio, observed ratio, and corrected ratio represent the known concentration ratio present in our experiment, the calculated value without FRET correction, and the calculated value with FRET correction, respectively.
unpaired TMR fluorophores are present in the Cy5(1)-dsDNA16TMR(2) solution, and the unpaired Cy5 fluorophores are present in the Cy5(2)-dsDNA16-TMR(1) solution. From Table 1, the observed concentration ratios are much higher than the expected values. These discordances are caused by the presence of intramolecular FRET, which results in the loss of donor emission and the underestimation of the donor concentrations. In the solutions of Cy5(1)-dsDNA16-TMR(1), Cy5-dsDNA12TMR, and Cy5/TMR-ssDNA, the expected concentration ratios of Cy5 to TMR are all 1.000. From Table 1, the observed concentration ratios of Cy5-dsDNA12-TMR and Cy5(1)-dsDNA16TMR(1) are both much higher than the expected values, whereas there is a greater difference between the observed value of Cy5dsDNA12-TMR and the expected value, than between the observed value of Cy5(1)-dsDNA16-TMR(1) and the expected value. There is a greater degree of intramolecular FRET interaction for Cy5-dsDNA12-TMR than for Cy5(1)-dsDNA16-TMR(1) because the Cy5 and TMR fluorophores are closer in the hybrid, and greater FRET interaction will cause lower observed concentration of donor fluorophores. In contrast, in the solution of Cy5/TMRssDNA, the Cy5-labeled oligonucleotide and the TMR-labeled oligonucleotide have identical sequence and are not able to hybridize or to form stable hairpin loops, so there is no FRET interaction between the Cy5 and TMR fluorophores. After FRET correction, the corrected concentration ratios in these five samples are close to the expected values (Table 1). These results confirm experimentally that the correction method can be used to eliminate the influence of FRET in the samples with different relative concentrations of the Cy5 and TMR fluorophores, with the presence of unpaired Cy5 and TMR fluorophores, and with different degrees of FRET interactions between them. The differences between the corrected ratio and the expected ratio may be partly caused by the differences in fluorescence properties among the samples. Hybridization Efficiency Measurement on a Microarray. In order to measure the hybridization efficiency on a microarray, it is necessary to determine both the quantity of single-stranded DNA immobilized on the microarray (called probe DNA) and the quantity of single-stranded DNA hybridized with the probe DNA on the same microarray (called sample DNA). We can label both the probe DNA and the sample DNA, ideally with two spectrally distinct fluorophores. After hybridization and removal of any unbound sample DNA, the quantitative relation between the probe DNA and the sample DNA is typically determined by the fluorescence intensities. However, FRET may occur between the 1430
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fluorophore pair frequently used in microarray experiments (e.g., TMR/Cy5 and Cy3/Cy5 pairs) under particular conditions, both when the two fluorophores are too close in a hybrid and when the densities of the fluorophores on the microarray are too high. The former case can lead to the intramolecular FRET, while the latter case can lead to the intermolecular FRET (interaction between the donor and acceptor, respectively, coupled in different adjacent double-stranded DNA molecules). In particular, the intermolecular FRET could occur in differential gene expression profiling experiments if the probe densities are too high. For the measurement of the hybridization efficiency, we undertook comprehensive FRET correction steps. The Cy5dsDNA-PolyT array, the TMR-dsDNA-PolyT array and the Cy5/ TMR-dsDNA-PolyT array were fabricated for the determination of system factors, and the scanned images of these microarrays are shown in Figure S1 of the Supporting Information. The transfer factor γ was determined from the ratio of the Cy5 signals on the Cy5-dsDNA-PolyT array to the TMR signals on the TMR-dsDNAPolyT array, which equals 2.232 ± 0.373. The crosstalk factor R equals 0.025, and the crosstalk factor β equals 0.024. The TMR signals on the Cy5-dsDNA-PolyT array and the Cy5 signals on the TMR-dsDNA-PolyT array were approximately zero, so the crosstalk factors φ and φ equal zero. On the Cy5/TMR-dsDNAPolyT array, densities of the Cy5 and TMR fluorophores were approximately equal in each spot area. After crosstalk correction, the signals obtained from the FRET channel were strong from four subarrays of this microarray corresponding to the immobilized (oligo-3)-NH2 at 4, 2, 1, and 0.5 µM. On these spots only intermolecular FRET was present, and an increase in the quantity ratio of Cy5 to TMR can be observed, which indicated greater degrees of intermolecular FRET, as the concentrations of immobilized probe increased (data not shown). The effects of intermolecular FRET were utilized to determine the factor G, which equals 0.594 ± 0.109 for this system. We measured the hybridization efficiency on the TMR-dsDNACy5-PolyT array (measurement microarray). The control microarray (TMR-dsDNA-nofluo-PolyT array) was hybridized with unlabeled (oligo-1) sample, whereas the measurement microarray was hybridized with the Cy5-labeled sample of Cy5-(oligo-1). The actual quantities of probe DNA molecules on the measurement microarray can be estimated by those on the control microarray (expected values). The scanned fluorescence images are shown in Figure 3A. From Figure 3B, there are great differences between the spot intensities from the TMR channel on the measurement microarray (observed values) and the corresponding spot values on the control microarray. When the intercept is set to zero, the slope of the fitted regression line is 0.31, and the correlation coefficient is 0.42 between them. On the TMR-dsDNA-Cy5-PolyT array, intramolecular FRET occurred due to the short distance between the fluorophores in the hybrid and intermolecular FRET was also present due to the high probe densities (similar to the intermolecular FRET on the Cy5/TMR-dsDNA-PolyT array). When the probe concentration is below 0.5 µM, there is no great intermolecular FRET and the differences between the observed TMR signals and the expected values are mainly caused by the intramolecular FRET. However when the probe concentrations are equal to or higher than 0.5 µM, these differences are more
Figure 3. Measurement of hybridization efficiency on a microarray. (A) The microarray images of the measurement microarray and the control microarray. (B) Linear regression analysis of the TMR intensity on the measurement microarray and that on the control microarray. The corrected TMR intensity and the uncorrected TMR intensity on the measurement microarray are compared with the TMR intensity on the control microarray. (C) The hybridization efficiencies on the microarray. Three curves indicate the hybridization efficiencies calculated by the TMR intensities on the control microarray, by the uncorrected TMR intensities on the measurement microarray, and by the corrected TMR intensities on the measurement microarray. The Cy5 intensities are all determined on the measurement microarray in each calculation.
significant, because higher immobilized probe densities result in greater intermolecular FRET. After FRET correction, the TMR signals on the measurement microarray are close to the values on the control microarray, and the slope of the fitted regression line is then 1.13 (close to 1.00) and the correlation coefficient is 0.99 (close to 1.00) between them. These results show that quantitative fluorescence correction method can be used to accurately correct the donor emission on a microarray, taking into account both intermolecular FRET and intramolecular FRET. Then the hybridization efficiencies calculated by different methods are shown in Figure 3C. The hybridization efficiencies calculated without FRET correction are significantly different from the values calculated by the control method mainly due to the intramolecular FRET on spots with low probe densities and mainly due to the intermolecular FRET on spots with high probe densities, whereas the values with FRET correction are close to the values by the control method. Thus, the hybridization efficiency can be measured accurately and quantitatively by our method without the need for independent control experiments. Since the amount of sample DNA in hybridization solution is typically much larger than the probe amount on a microarray, the formation of duplex DNA is efficient and the hybridization efficiency remains stable from the lower to the higher probe density. Here we determined the hybridization efficiency according to the measurement of the quantity ratio of fluorophores on a microarray. In order to validate that all the free dye-labeled sample DNA molecules were removed, we hybridized the Cy5-(oligo-2)
in hybridization buffer with the TMR-(oligo-3)-NH2 array to form the TMR-ssDNA-PolyT array. No fluorescence emission from the Cy5 channel could be detected on the TMR-ssDNA-PolyT array, since the oligo-3 and oligo-2 share common sequences and do not form duplex DNA (data not shown). This experiment confirmed that Cy5-(oligo-2) molecules did not adhere randomly to the surface of the array and any Cy5-labeled sample molecules detected on the measurement microarray were present as hybridized duplex DNA. CONCLUSIONS In this paper, we have developed a quantitative fluorescence correction method incorporating FRET, applicable both in solution and on microarrays. The results show that the corrected values are close to the expected values, thus this method is accurate. As this method is also applicable for samples with different relative concentrations, with the presence of unpaired fluorophores and with different degrees of FRET interactions, it is comprehensive for the FRET correction. Since the correction method only requires the measurement of signals from three channels, this makes the FRET correction convenient in dual-labeling experiments using common fluorescence instruments without the need for spectral analysis or FRET efficiency measurements. Moreover, the correction method is applicable to eliminate the distortions of intermolecular FRET and intramolecular FRET on a microarray and thus can accurately recover the uncontaminated fluorescence signals in two-color microarray analysis such as gene expression profiling. In summary the application of the correction method Analytical Chemistry, Vol. 81, No. 4, February 15, 2009
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for the determination of hybridization efficiency on a microarray provides a sufficient approach for accurate and quantitative analysis of the DNA hybridization on a surface. ACKNOWLEDGMENT The authors wish it to be known that, in their opinion, the first two authors contributed equally to this study. This work was supported by the National Foundation of High Technology of China (Grants 2006AA020701 and 2006AA020803), the National Program on Key Basic Research Projects 973 of China (Grant
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Analytical Chemistry, Vol. 81, No. 4, February 15, 2009
2006CB705700), and the Natural Science Foundation of Zhejiang Province (Grant 2006C21G3210005). SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review December 9, 2008. AC802203R
October
16,
2008.
Accepted
