Articles Anal. Chem. 1995, 67,247-251
Synthesis, Chromatographic Separation, and Characterization of Nearmlnfrared-Labeled DNA Oligomers for Use in DNA Sequencing Dana B. Shealy,t MalgOMta Lipowska,t Jacek Lipowski,+Narasimhachari Narayanan,t Scott Sutter,* Lucjan Strekowski,t and Gabor Patonay*Bt Department of Chemisty, Georgia State University, University Plaza, Atlanta, Georgia 30303, and LI-COR, Inc., 4421 Superior Street, Lincoln, Nebraska 68504
Traditionally, visible fluorophores have been used as labels in DNA sequencing. They absorb and fluoresce in a region of the spectrum that is susceptible to biological interferences in sequencingsamples. The increasednoise level due to autofluorescence of glass, solvent, or impurities can greatly reduce the potential sensitivity of the analysis. In an attempt to increase the sensitivity,we have investigated the use of near-Mared (near-IR) fluorophores as labels in DNA sequencing. Near-IR fluorophores possess spectral properties which are observed between 700 and 1200 nm,a region with characteristically little interference by biomolecules. Therefore, the detection of near-IR dyes is not limited by noise levels, but rather by detector capabilities. Near-IR dyes are also suitable for selective excitation with commercially available laser diodes which can further enhance the observed fluorescence signal. We have covalently linked functionalized near-IR dyes to modified DNA oligomers for use in DNA sequencing. We report the synthesis and chromatographic puri6cation of near-IR-labeledDNA oligomers. We further discuss the inherent properties of the conjugates and their use in DNA sequencing procedures. Fluorescent labels are being used increasingly in bioanalytical They have been attached successfullyto a variety of biomolecules such as enzymes, antibodies, and nucleic acids for use in immunoassay, flow cytometry, and sequencing
procedure^.^^^^^-'^ Visible fluorophores such as fluorescein, rhodamine, and Texas Red have been the most commonly used fluorescence labels.3~839J1-15 However, due to inherent interferences by biomolecules in the spectral region of visible dyes, we have investigated the use of near-infrared (near-IR) dyes in bioanalytical applications. Near-IR dyes have absorbance and emission bands in a region with characteristically little biological interference. These dyes generally absorb light between 700 and 1200 nm, making them suitable for excitation with inexpensive laser diodes. Near-IR fluorophores possess large molar absorptivities, usually between 100 000 and 200 000 M-l cm-l, and acceptable quantum yields. Near-IR dye Stokes shifts of around 20 nm allow the discrimination of scattered excitation light from fluorescence when standard interference filters are used. When near-IR dyes are suitably functionalized, they can be covalently linked to biomolecules. Like their visible counterparts, the conjugates can be purified using reversed-phase high-performance liquid chromatography (RPHPLC) or electrophoresis. We have successfully attached functionalized near-IR dyes with absorbance maxima near 780 nm to modified DNA oligomers for use in DNA sequencing. We discuss the actual conjugation conditions and separation criteria for this process. We further discuss the spectral properties of the conjugates and their utility in automated DNA sequencing. EXPERIMENTAL SECTION Sodium taurocholate was purchased from Aldrich Chemical Co. (Milwaukee, WI). A modified thymine phosphoramidite
~~
Georgia State University. + L.I-COR$Inc. (1) Zen, J.; Patonay, G. Anal. Chem. 1991,63,2934-2938. (2) Bhattacharyya, A Indian j . Biochem. Biophys. 1986,23, 171-173. (3) Lee, L.G.; Connell, C. R; Woo, S. L.; Cheng, R D.; McArdle, B. F.; Fuller, C. W.; Halloran, N. D.; Wilson, R K Nucleic Acids Res. 1992,20, 24712483. (4) Mujumdar, R B.; Emst, L. A; Mujumdar, S. W.; Waggoner, A.S. Cytometry 1989,10, 11-19. (5) Casay, G. A; Czuppon, T.; Lipowski, J.; Patonay, G. Proceedings of SPIE International Symposium OE/Biomedical Optics; Los Angeles, CA 1993. (6) Williams, R J.; Lipowska, M.; Patonay, G.; Strekowski, L.Anal. Chem. 1993, 65,601-605. (7) Middendorf, L. R;Bruce, J. C.; Bruce, R C.; Eckles, R D.; Grone, D. L.; Roemer, S. C.; Sloniker, G. D.; Steffens, D. L.; Sutter, S. L.; Brumbaugh, J. A; Patonay, G. Electrophoresis 1992,13,487-494. +
0003-2700/95/0367-0247$9.00/0 0 1995 American Chemical Society
(8) Chen, E. Y.; Kuang, W.; Lee, A L. Methods 1991,3 (1). 3-19.
(9) Brumbaugh, J. A; Middendorf, L. R; Grone, D. L.; Ruth, J. L Proc. Natl. Acad. Sci. U.S.A. 1988,85, 5610-5614. (10) Boyer, A E.; Lipowska, M.; Zen, J.-M.; Patonay, G.; Tsang, V. C. W. Anal. k n . 1 9 9 2 ,(3), ~ 415-428. (11) Schubert, F.; Ahlert, K; Cech, D.; Rosenthal, A Nucleic Acids Res. 1990, 18 ( l l ) , 3427. (12) Voss, H.; Schwager, C.; Wirkner, U.; Zmmerman, J.; Ertle, H.; Hewitt, N.; Rupp, T.; Stegemann, J.; Ansorge, W. Methods Mol. Cellular Biol. 1992,3, 30-34. (13) Freeman, M.; Baehler, C.; Spotts, S. Biotechnology 1990,8, 147-148. (14) Prober, J. M.; Trainor, G. L.; Dam, R J.; Hobbs, F. W.; Robertson, C. W.; Zagursky, R J.; Cocuzza, A J.; Jensen, M. A; Baumeister, K Science 1987, 238,336-341. (15) Smith, L. M.; Sanders, J. Z.; Kaiser, R J.; Hughes, P.; Dodd, C.; Connell, C. R; Heiner, C.; Kent, S. B. H.;Hood, L. E. Nature 1986,321,674-679.
Analytical Chemisfty, Vol. 67,No. 2,January 75, 7995 247
A)
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Figure I. (A) Modified thymine base with a terminal amino linker arm that was incorporatedin position eight of the synthetic M13 (-29) forward primer as a substitute for thymine to allow an attachment site for the dye. Alternatively, a six-carbon linker with a terminal amino group (B) was attached to the 5' end of the primer. The isothiocyanate moieties on the dyes were reacted with the primary amino group (*) on the linkers.
(amino modifer dT) and a 5' amino linker (5'amino modifier C6) were obtained from Glen Research, Inc. (Sterling,VA). Standard nucleoside phosphoramidites and DNA sjllthesis reagents were purchased from Cruachem, Inc. (Sterling,VA). All solvents were HPLC grade and were purchased from Fisher Scientific Co. (Pittsburgh, PA). Chemicals and reagents were used without further purification. Ultrapure water (UPH20) was used in the preparation of all reagents and samples. UPBzO was prepared by treating deionized water in a NanoPure water purification system (Barnstead/Thermolyne Corp., Dubuque, IA) equipped with ultrapure, macropure, and organic-free cartridges and a 0.2 pm final filter to provide bioanalytical grade I water. Nineteen-base DNA oligomers [M13 (-29) forward primer] were made on a PS250 DNA synthesizer (Cruachem) using standard ,L?-cyanoethylphosphoramiditechemistry and incorporating either a modified thymine base with a terminal amino linker arm in position eight or an amino modifer at the 5' terminus of the primer (Figure 1). An SSI HPLC system (Scientific Systems, Inc., State College, PA) was used for separation of samples. The HPLC system was equipped with a 222D HPLC pump and 232D gradient mixer. Dual wavelength detection was achieved by connecting in tandem an SSI Model 500 variable UV/vis absorbance detector with a tungsten lamp and a Linear UV-106 tixed wavelength UV detector with a mercury lamp. The HPLC system was interfaced to a computer supplied with Visions IV chromatog-iaphydata systems software. The column system consisted of SGE 10 cm x 4 mm CS reversed-phase columns (Fisher Scientific). The collected HPLC fractions were concentrated in a SpeedVac centrifugal concentrator (Savant Instruments, Inc., Farmingdale, Ny). A Lambda-2 UV/vis spectrophotometer (Perkin Elmer, Norwalk, CT) interfaced with a Zenith data system supplied with P E C S operating software was used to obtain absorbance spectra. Fluorescence spectra were obtained on an SLM8000 spectrofluorometer (SLM Aminco, Urbana, IL) equipped ~ t a hxenon arc lamp and modified to include a 780 nm laser diode (Sharp Instruments, Mahwah, NJ) excitation source. Other fluorescence data were acquired using the laser microscope optics assembly 248 Analytical Chemistry, Vol. 67, No. 2, January 15, 1995
Figure 2. Chemical structure of the heptamethine cyanine dyes used in this study.
of a Model 4000 automated fluorescenceDNA sequencer (LI-COR Inc., Lincoln, NE) equipped with a 780 nm laser diode and a silicon avalanche photodiode. Relative quantum yields (a)were calculated using IR-125 as a reference. The quantum yield of IR-125 in DMSO is 13%.16 Thermal denaturation and renaturation of the duplex oligomers were performed on a Cary 3 spectrophotometer (Varian Associates, Inc., Palo Alto, CA) equipped with a controlled variable temperature cuvette unit. The instrument was interfaced to a microcomputer supplied with operating software. The first derivative of the melting curve produced a single peak, from which the melting temperature was obtained. The heptamethine cyanine (HMC) dyes functionalized with the isothiocyanato (ITC) moiety (Figure 2) were synthesized inhouse, and their preparation and purification have been reported el~ewhere.'~-'~ Conjugation. The procedure for covalent labeling of the oligonucleotideswith near-IR dyes 1-4 was an adaptation of the protein labeling method of Bhattacharyya,2using isothiocyanato moieties to form a thiourea linkage between the label and a primary amine of the biomolecule. Conjugation of the HMC dyes 1-4 was facilitated by dissolving 10 nmol of M13 (-29) forward DNA primer in 150 pL of 50 mM carbonate buffer @H 9.2) in a microcentrifuge tube. A 75 pL aliquot of a M solution of the cyanine dye (75 nmol) in DMSO was slowly added to the primer solution. The reaction mixture was stirred for 3 h at a controlled 25 "C temperature with the exclusion of ambient light to avoid photodegradation of the dye. Under these basic conditions, the terminal amino group on the linker arm of the modified thymine base reacted cleanly with the isothiocyanate to form a stable thiourea bond. A control reaction using the unmodified DNA oligomer showed that the free amino groups on the DNA bases did not react with the isothiocyanateof the dye. This is consistent with the low nucleophilicity of these amino groups. Purification. The conjugates were purified by semipreparative RP-HPLC using a CSanalytical column. Dyes 1 and 3 were sufficiently soluble in aqueous media to successfully utilize (16)Benson, R C.; Kues, H. A J. Chem. Eng. Data 1977,22, 379. (17) Strekowski, L.; Lipowska, M.; Patonay, G. J. 0%.Chem. 1992,57, 45784580. (18) Strekowski, L.;Lipowska, M.; Patonay, G.Synth. Commun. 1992,22 (17), 2593-2598. (19)Lipowska, M.; Patonay, G.; Strekowski, L. Synth. Commun. 1993,23 (21).
n
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Figure 4. Absorbance and fluorescence spectra of a dye 1 conjugate M) in water. The fluorescence spectrum was obtained using laser diode excitation (780 nm, 5 mW) and emission detection (798 nm) using a standard photomultiplier tube. 1
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Figure 3. Near-IR absorbance chromatogram (770 nm) of a dye 1 conjugation reaction (top), exhibiting a clear separation of the three basic components. An near-IR absorbance chromatogram of the separation of a dye 2 conjugation reaction (bottom) at 770 nm shows the coelution of the unlabeled DNA with the dye conjugate. The unbound DNA peaks are not observed at 770 nm; however, their positions are indicated on the chromatograms above.
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standard reversed-phase chromatographic conditions. The three components in the conjugation mixtures of dyes 1 and 3 (free dye, free DNA, and dye-DNA conjugate) were separated using a linear gradient elution from 70%water to 100%methanol over 30 min with a flow rate of 1 mWmin, monitoring absorbance simultaneously at 254 and 780 nm. The unbound DNA was not retained on the column (i.e., eluted at dead volume time). The conjugate and dye eluted at 8 and 19 min, respectively. The three components of the conjugation mixtures of dyes 2 and 4 were separated using ion-pairing or micellar chromatography with a linear gradient from water to 100%methanol, maintaining a steady concentration of 5 mM sodium taurocholate throughout, using the same flow rate and detection system. Typical chromatograms of the separations are shown in Figure 3. The conjugate fractions were collected, combined, and evaporated in vacuo. The conjugates were quantitated and characterized by UV/vis spectroscopy and fluorescence spectroscopy. A typical spectral analysis of a dye-DNA conjugate is shown in Figure 4. The excess free dye fractions were collected and recycled. Thermal denaturation and renaturation curves taken in a pH 7.0 1,4piperazinediethane sulfonic acid (PIPES) buffer (0.01 M PIPES/0.001 M EDTA/O.l M NaC1) (Figure 5) indicated no significant difference in duplex stability between the duplexes of the dyeconjugated primer and the unconjugated primer.
20
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40
60
80
100
Temperature ("C)
Figure 5. Thermal denaturation curves of the duplexes of a dye 1-labeled oligomer M) and an unmodified oligomer M) in PIPES buffer.
Sequencing. The efficacy of the HMC conjugates for DNA sequencing procedures was evaluated using the standard 7-deazadGTP sequencing reaction protocol of the Sequenase kit (United States Biochemical, Cleveland, OH), bypassing the labeling step. A Model 4000 DNA sequencer &I-COR, Inc.) was employed,with the procedure described el~ewhere.~ RESULTS AND DISCUSSION The reaction conditions dictated by each of the dyes were similar. The solvents and the percent organic composition were optimized for each dye. Although aprotic, polar solvents were preferred, the cyanine dyes could be reacted in protic solvents as well, but upon storage, hydrolysis of the ITC group of the dye was observed. The conjugations proceeded more rapidly when the minimum amount of organic solvent required for dye dissolution was used. This fact was presumably due to the increased solubility of the DNA when less organic solvent was used. Dye 1 required no more than 10%organic moditier in the reaction mixture; dyes 2-4 required approximately 30%organic solvent for complete dissolution. Analytical Chemistry, Vol. 67, No. 2, January 15, 1995
249
Table I. Spectral Properties of Near-IR Dyes and Dye Conjugates
solvent methanol water In water 2 methanol water 2" water 3 methanol water 4 methanol water 1
E
(hC1cm-l)
140 000 110 000 C
180 000 22 000 C
94 000 94 000 110 000 36 000
,A
,Iem*
(nm)
(nm)
769 768 775 764 767 774 796 795 786 787
788 788 798 782 802 801 811 825 805 822
Stokes @ shift (nm) (%) 19 20 24 18 35 27 15 30 19 35
38 15 13 30 C
13 9 C
5 C
Dye conjugated to M13 (-29) forward DNA primer. Excitation with xenon arc lamp and emission collection using PMT. Not measured.
Possible secondary interactions were observed between the oligomers and dye 4. In the absence of DNA, but still maintaining 30%organic modifier in the mixture, most of the dye remained in crystalline form. Upon addition of the DNA, complete dissolution ensued. The specific type of secondary interactions between dye 4 and the DNA is unknown; however, Rye et a1.20reported bisintercalation of asymmetric cyanine dyes into double-stranded DNA. The reaction times were also varied to optimize the conjugation efficiency. The majority of the conjugation occurred during the first hour, but after 3 h, the conjugation of the dyes to the primer was essentially complete (>70%yield with most of the dyes). The conjugation efficiency of dye 3 (
