Anal. Chem. 1996, 68, 1197-1200
Comparison of an Intercalating Dye and an Intercalant-Enzyme Conjugate for DNA Detection in a Microtiter-Based Assay Beata Kolakowski, Fernando Battaglini, Yoon Suk Lee, Giannoula Klironomos, and Susan R. Mikkelsen*
Department of Chemistry and Biochemistry, Concordia University, 1455 de Maisonneuve Boulevard West, Montreal, Canada H3G 1M8
Two methods have been developed for the detection of DNA immobilized on the surface of microtiter wells. An intercalating dye, 3,6-diaminoacridine, is used in stain and rinse solutions, so that measured absorbance values (450 nm) reflect the sum of DNA-bound and free dye. With diaminoacridine, signal increases of 0.056 ( 0.010 were achieved on immobilizing double-stranded calf thymus DNA. An intercalant-enzyme conjugate, consisting of an average of four daunomycin moieties covalently bound to each glucose oxidase, was shown to provide a 10-fold signal enhancement (optimum 0.25 µM, with rinsing and peroxidase-o-dianisidine detection) compared to diaminoacridine, due to catalytic amplification; signals of 0.50 ( 0.05 were obtained. This conjugate possesses 56% of the activity of native glucose oxidase and was prepared using water-soluble carbodiimide and Nhydroxysuccinimide reagents. Single-stranded DNA was immobilized onto avidin-coated polystyrene plates and commercially available (Covalink) plates possessing secondary amine groups. Following hybridization with complementary DNA, detection was performed with the daunomycin-glucose oxidase conjugate. Both immobilization methods showed optimum DNA concentrations of 0.10 µg/mL, and maximum signal intensities were obtained when >0.5 µg/mL complementary DNA was present in the hybridization solution. Some nonspecific binding of the intercalant-enzyme conjugate was suggested by results obtained with avidin-coated polystyrene plates, but not with Covalink plates. Methods for the diagnosis of inherited disorders such as cystic fibrosis or sickle-cell anemia rely on the detection of abnormal proteins or nucleic acids.1 The genetic factors associated with many inherited disorders are understood, and the base sequences of critical regions of human DNA are now known. Diagnosis of disease at the genetic level usually relies on the selective interaction of DNA probes (labeled single-stranded oligo- or polynucleotides) with their complementary base sequences through hybridization, or double-strand formation.2 The use of a solid phase, such as microtiter plate wells, beads, or a membrane, has been described in several reports as a means by which either the analyte DNA or the probe strand can be immobilized, so that the duplex DNA formed upon hybridization can be readily separated from the test solution.3-8 We have recently focused on the use of immobilized, unlabeled DNA probes to selectively recognize a target DNA sequence (1) Skogerboe, K. J. Anal. Chem. 1993, 65, 416R. (2) Keller, G. H.; Manak, M. M. DNA Probes; Macmillan (Stockton Press): New York, 1989; pp 149-213. 0003-2700/96/0368-1197$12.00/0
© 1996 American Chemical Society
through hybridization, to form an immobilized duplex. The immobilized duplex is then detected following the preconcentration of a hybridization indicator, a small molecule that associates strongly but reversibly with double-stranded DNA. This concept has been tested with DNA probes immobilized on the surface of an amperometric carbon electrode, using reversibly redox-active hybridization indicators such as tris(2,2′-bipyridyl)cobalt(III) that preconcentrate at the electrode surface if double-stranded DNA is present.9,10 In this paper, we examine an equivalent DNA detection strategy based on optically detectable hybridization indicators. Single-stranded DNA, immobilized on the surface of a microtiter well, is allowed to hybridize with its complementary sequence. An intercalating dye, 3,6-diaminoacridine, preconcentrates from solution into the immobilized DNA layer; fresh intercalant solution is added to the well, and the resulting measured absorbance reflects the sum of preconcentrated (intercalated) and soluble dye. With calf thymus DNA in 0.010 M NaCl, 3,6-diaminoacridine has a reported association constant of 1.1 × 106 M-1 (calculated on the basis of the molarity of base pairs).11 To amplify the signals achieved with the simple intercalating dye, an enzyme-intercalant conjugate has been prepared by covalently binding the anthracycline antibiotic daunomycin to the enzyme glucose oxidase. In 1 M NaCl solution, daunomycin binds to a 6-base-pair oligodeoxynucleotide duplex with an association constant of 2.0 × 104 M-1.12 Intercalation of the daunomycin moieties allows preconcentration of the enzyme into the immobilized DNA layer, and the measurement of enzyme activity following rinse and substrate addition steps provides an amplified signal that allows discrimination between immobilized single- and double-stranded DNA. EXPERIMENTAL SECTION Materials and Instrumentation. Calf thymus DNA (highly polymerized), poly(dA), poly(dT), horseradish peroxidase (EC 1.11.1.7, 10 mg/mL suspension in 3.2 M ammonium sulfate, 250 units/mg), tris(hydroxymethyl)aminomethane (Tris), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), o-dianisidine hydrochloride, imidazole, and daunomycin were obtained from Sigma and were used as received. Avidin, biotin-11-dUTP, a (3) Keller, G. H.; Huang, D.-P.; Manak, M. M. Anal. Biochem. 1989, 177, 27. (4) Guesdon, J.-L. J. Immunol. Methods 1992, 150, 33. (5) Vary, C. P. H. Clin. Chem. 1992, 38, 687. (6) Forghani, B.; Yu, G.-J.; Hurst, J. W. J. Clin. Microbiol. 1991, 29, 583. (7) Radovich, P.; Bortolin, S.; Christopoulos, K. Anal. Chem. 1995, 67, 2644. (8) Mazza, C.; Mantero, G.; Primi, D. Mol. Cell. Probes 1991, 5, 459. (9) Millan, K. M.; Mikkelsen, S. R. Anal. Chem. 1993, 65, 2317. (10) Millan, K. M.; Saraullo, A.; Mikkelsen, S. R. Anal. Chem. 1994, 66, 2943. (11) Gaugain, B.; Barbet, J.; Capelle, N.; Roques, B. P.; LePecq, J. B. Biochemistry 1978, 17, 5078. (12) Ragg, E.; Mondelli, R.; Battistini, C.; Garbesi, A.; Colonna, F. P. FEBS Lett. 1988, 236, 231.
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terminal deoxynucleotide transferase kit (EC 2.7.7.31), and glucose oxidase (EC 1.1.3.4 from Aspergillus niger, grade II) were obtained from Boehringer-Mannheim. Coomassie brilliant blue G-250 protein assay dye reagent, acrylamide, N,N′-methylenebis(acrylamide) and silver stain kits were purchased from Bio-Rad. Aldrich supplied R-D-glucose, 3,6-diaminoacridine, and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC). N-Hydroxysulfosuccinimide (NHS) was obtained from Pierce. Sephadex G-15 and G-25 fine gel filtration resins were from Pharmacia. Amicon cells and YM 30 membranes (30 000 MW cutoff) were used for ultrafiltration. Mono- and dibasic potassium phosphate, boric acid, disodium ethylenediaminetetraacetic acid (EDTA), and sodium chloride were obtained from Fisher. Flat-bottom, 96-well polystyrene microtiter plates were obtained from Sigma, and eightwell microtiter strips modified to possess surface secondary amine groups (Covalink, NUNC) were obtained from Gibco-BRL. Electrophoresis was performed in a Mini-Protean II cell (Bio-Rad) using 8.3%T, 3.3%C polyacrylamide gels cast and run in TBE buffer (0.134 M Tris, 0.08 M boric acid, 0.0026 M EDTA, pH 8.8) at 100 V for 3 h, followed by silver staining according to the procedure recommended by Bio-Rad. Absorbance measurements in the microtiter plates or strips were made with Dynatech MR300 or Bio-Tek EL307C microtiter plate readers, using 450 nm interference filters. A Varian Cary 1 double-beam UV-visible spectrophotometer was used for all other absorbance measurements. Preparation and Characterization of Daunomycin-Modified Glucose Oxidase. (A). EDC/NHS Method. To a solution of 47 mg (0.3 µmol) of glucose oxidase in 20 mL of 0.1 M HEPES buffer, pH 7.0, were added a 7000-fold molar excess of EDC and a 400-fold molar excess of NHS. This solution was incubated in an ice bath for 30 min, and 4 mg (7 µmol) of daunomycin was then added. The reaction continued overnight at 4 °C. To test for adsorption, rather than covalent binding of daunomycin to glucose oxidase, a control reaction was performed under identical conditions, but in the absence of the coupling reagents EDC and NHS. The resulting solutions were concentrated to 4 mL by ultrafiltration and applied to a 1.5- × 40-cm G-15 gel filtration column. The modified enzyme eluted in the first, orange fraction. Protein concentrations were determined by the Coomassie blue protein-dye binding method,13 using native glucose oxidase for calibration. Activities were measured at 436 nm in air-saturated 0.1 M phosphate buffer containing 0.1 M mutarotated glucose, using the peroxidase-o-dianisidine assay.14 The extent of modification of the enzyme with daunomycin was determined spectrophotometrically, using molar absorptivities for daunomycin of 9620 and 5560 M-1 cm-1 at 476 and 550 nm, respectively. For these measurements, N2-purged solutions of the modified and native enzyme containing 0.1 M glucose in 0.1 M phosphate buffer, pH 7.0, were used to eliminate absorbance due to flavin adenine dinucleotide, which absorbs over this wavelength range in its oxidized form and is present at each of the two active sites of the 160-kDa homodimeric enzyme.15 (B) Periodate Method. Glucose oxidase (50 mg) was dissolved in 5.0 mL of 0.1 M sodium periodate and incubated at 4 °C for 5 h to partially deglycosylate the enzyme and to generate aldehyde groups. The reaction was quenched by the addition of 1.0 mL of ethylene glycol, and the mixture was allowed to stand 30 min at ambient temperature. The sample was applied to a (13) Bradford, M. M. Anal. Biochem. 1976, 72, 248. (14) Worthington Enzyme Manual; Worthington Biochemical Corp.: Freehold, NJ, 1972; p 19. (15) Hecht, H.-J.; Kalisz, H. M.; Hendle, J.; Schmid, R. D.; Schomburg, D. J. Mol. Biol. 1993, 229, 153.
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Sephadex G-25 column (1.5 cm × 40 cm) and eluted with 0.050 M phosphate, pH 7.5, and the first of the three bands was collected. Daunomycin (1.4 mg) was added to this fraction along with 25 mg of sodium cyanoborohydride, and the mixture was incubated overnight at 4 °C. The sample volume was reduced to 3 mL by ultrafiltration, and gel filtration (G-25) in phosphate buffer (0.050 M, pH 7.5) was used to purify the product, which eluted as the first band. The product was characterized as described above for the EDC/NHS method. Catalytic Elongation of Poly(dA) with Biotin-11-dUTP. Poly(dA) (average length 4 kilobases) was elongated enzymatically with biotin-11-dUTP, using a terminal deoxynucleotidyl transferase kit. To five A260 units (1.0 mg) of poly(dA) in 80 µL of H2O were added 10 µL (0.25 mg) of biotin-16-dUTP, 35 µL of H2O, 60 µL of terminal transferase buffer, 10 µL of CoCl2 solution, and 4 µL of terminal transferase solution (50 IU). Incubation proceeded at 37 °C, and three additions of enzyme (4 µL) were made at 4-h intervals. Addition of phenol-chloroform-isoamyl alcohol and centrifugation at 12000g for 10 min produced a pellet that was rinsed with cold ethanol and stored at -20 °C. The singlestranded DNA concentration in the product was estimated from absorbance values measured at 260 and 280 nm and was reconstituted to a stock solution of 1 mg/mL in distilled, deionized water. Covalent Immobilization of DNA. (A) Carbodiimide Method. Double-stranded calf thymus DNA or poly(dA) (average length 4 kilobases) in varying concentrations was incubated in 300 µL of 0.2 M EDC in 0.1 M imidazole buffer, pH 7.0, in covered wells of the Covalink eight-well microtiter strips for 5 h at 50 °C, as recommended by Rasmussen et al.16 These conditions allow immobilization through the 5′-phosphate groups of DNA by the formation of a phosphoramidate bond with the surface-bound amine groups. Excess reagents were removed by rinsing with distilled water, and 300 µL of 1 mg/mL bovine serum albumin in distilled water was then added to each well and incubated overnight at 4 °C. For poly(dA) immobilizations, an overnight hybridization at 42 °C was performed with 300 µL of 1 µg/mL poly(dT) in 0.1 M phosphate buffer. (B) Biotin-Avidin Method. Polystyrene microtiter plates were rinsed with distilled water, and the wells were filled with 300 µL of 12.5 µg/mL avidin in 0.1 M phosphate buffer containing 1 M NaCl and covered to prevent evaporation. Following a 2-h incubation at 4 °C, the wells were rinsed with phosphate buffer and filled with 300 µL of 1 mg/mL bovine serum albumin in distilled water. Following overnight incubation at 4 °C, the plates were rinsed with phosphate buffer, and the wells were filled with 300 µL of biotinylated poly(dA) solutions in 0.1 M phosphate buffer, pH 7.0, of various concentrations. Under these conditions, surface-adsorbed avidin captures biotinylated DNA to bind it to the surface of the microtiter wells. After incubation at 22 °C, the wells were rinsed three times with phosphate buffer, and 300 µL of a 1 µg/mL poly(dT) solution in phosphate buffer was added to each well. Following hybridization, the wells were again rinsed three times with 0.1 M phosphate buffer, pH 7.0. Detection of Immobilized DNA. (A) 3,6-Diaminoacridine. The prepared microtiter strips and wells were stained with the intercalating dye 3,6-diaminoacridine in two steps. In the first step, 300 µL of 1.5 mM 3,6-diaminoacridine in 0.1 M phosphate buffer, pH 7.0, was added to each well and incubated for 30 min at ambient temperature. The wells were then rinsed three times with a more dilute solution of 3,6-diaminoacridine in the same (16) Rasmussen, S.; Larsen, M. R.; Rasmussen, S. E. Anal. Biochem. 1991, 198, 138.
Figure 2. Change in absorbance upon calf thymus DNA immobilization (carbodiimide method) versus concentration of 3,6diaminoacridine used in rinse and measurement steps. Each point is the average of eight measurements ( SD.
Figure 1. (A) UV-visible absorption spectra of 2.5 × 10-5 M 3,6diaminoacridine in the absence (a) and presence (b) of calf thymus DNA, 2.6 mM base pairs, in 0.1 M phosphate buffer, pH 7.0. (B) Scatchard plot for binding of diaminoacridine to DNA, where r ) [bound dye]/[total DNA]; line is predicted by the McGhee-von Hippel equation,17 where K ) (2.8 ( 0.1) × 104 M-1 and n ) 8.4 base pairs.
buffer (the concentration was varied), and 300 µL of this solution was then added to each well. Absorbance was measured at 450 nm. (B) Daunomycin-Glucose Oxidase Conjugate. A solution of the enzyme-intercalant conjugate (300 µL, varying concentration) in 0.1 M phosphate buffer, pH 7.0, was added to each well and incubated 2 h at 4 °C. The wells were rinsed three times with phosphate buffer, and 300 µL of an air-saturated substrate solution (0.1 M mutarotated glucose, 5 mM o-dianisidine, and 6.5 µg/mL horseradish peroxidase in 0.1 M phosphate buffer containing 10 mM ammonium sulfate) was added to each well. After 2 h at 37 °C, absorbance was measured at 450 nm. Safety Considerations. Daunomycin and 3,6-diaminoacridine are toxic and must be treated with caution. Disposable latex gloves and eye protection were used during the preparation and use of solutions. Care was taken to dispose of waste solutions properly. RESULTS AND DISCUSSION Characterization of Hybridization Indicators. Absorption spectra of 3,6-diaminoacridine in the presence and absence of calf thymus DNA are shown in Figure 1A. At 450 nm, the free and bound forms of the dye absorb with similar molar absorptivities of 3.5 × 104 and 3.2 × 104 M-1 cm-1, respectively. The absorbance maxima of the free and bound forms, 444 and 460 nm, were used to quantitate the binding of 3,6-diaminoacridine with calf thymus DNA in 0.1 M phosphate buffer, pH 7.0. The results, shown in Figure 1B as a Scatchard plot fitted to the McGhee-von Hippel equation17 by nonlinear regression, yield an association constant of (2.8 ( 0.1) × 104 M-1 and a binding site size of 8.4 base pairs. All measurements of dye absorbance in the untreated and DNAmodified microtiter wells were made at 450 nm. The second hybridization indicator tested in the DNA assay, the daunomycin-glucose oxidase conjugate, was prepared using two different methods that yielded different daunomycin-enzyme stoichiometries. Absorbance measurements at 476 and 550 nm (17) McGhee, J. D.; von Hippel, P. H. J. Mol. Biol. 1974, 86, 469.
for identical concentrations of modified and native glucose oxidase indicate a 4:1 daunomycin-enzyme stoichiometry for the species prepared by the carbodiimide/N-hydroxysuccinimide method and a 25:1 stoichiometry for the species prepared by the periodate method. Attempts to compare the UV-visible absorbance spectra of the two conjugates in the presence of DNA were unsuccessful, because solutions containing the 25:1 conjugate and calf thymus DNA formed a precipitate. No daunomycin was found in the purified product of the control reaction, in which coupling reagents were absent. The activities of the conjugates were 56% (carbodiimide/NHS) and 60% (periodate) relative to the native enzyme. Detection of Immobilized Calf Thymus DNA. Calf thymus DNA (20 µg/mL) was bound to the surface of the Covalink microtiter strips, and detection with 3,6-diaminoacridine was performed using unmodified microtiter strips as controls. Following a 30-min incubation with 1.5 mM 3,6-diaminoacridine, the concentration of dye used for the rinse steps and as the measurement solution was varied from 5 to 40 µM. Figure 2 shows the increase in absorbance that occurs upon hybridization as a function of 3,6-diaminoacridine concentration. The optimum dye concentration for the detection of immobilized, double-stranded DNA is ∼15 µM 3,6-diaminoacridine. At this concentration, the absorbance difference between wells modified with doublestranded DNA and unmodified wells is 0.056 ( 0.010 AU. Microtiter strips (Covalink) freshly modified with doublestranded calf thymus DNA were exposed to the daunomycinglucose oxidase conjugates for a single 2-h incubation. Following a buffer rinse, substrate incubation was allowed to proceed at 37 °C for 2 h. Figure 3 shows a plot of absorbance versus enzyme concentration over the 10-16-10-4 M range, obtained with DNAmodified and unmodified microtiter strips. The maxima obtained with the 4:1 and 25:1 daunomycin-glucose oxidase conjugates occur at different concentrations and yield different signal magnitudes. Interestingly, the 4:1 conjugate shows a maximum signal at a lower concentration (0.25 µM) than the 25:1 conjugate (2.5 µM), which is not expected, since the 25:1 conjugate should bind DNA much more strongly. It is possible that the bound 25:1 conjugate possesses lower activity than the bound 4:1 conjugate due to conformational or steric factors; if this were the case, then the 25:1 conjugate could bind to immobilized DNA to a greater extent than the 4:1 conjugate, yet produce smaller signals due to lower activity. Additionally, the signal obtained with the 4:1 conjugate is approximately twice that obtained with the 25:1 conjugate. Clearly, the abilities of these conjugates to bind DNA and retain catalytic activity once bound are quite different. Repetitions of these experiments showed that the maximum signal intensity obtained with the 4:1 conjugate was 0.50 ( 0.05 AU, Analytical Chemistry, Vol. 68, No. 7, April 1, 1996
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Figure 3. Absorbance change upon calf thymus DNA immobilization (carbodiimide method) as a function of daunomycin-glucose oxidase concentration used prior to rinsing and substrate addition: (a) 4:1 daunomycin-glucose oxidase conjugate, prepared by the EDC/NHS method, and (b) 25:1 daunomycin-glucose oxidase conjugate, prepared by the periodate method. Each point is the average of eight measurements ( SD.
almost an order of magnitude larger than that obtained with 3,6diaminoacridine, illustrating the effect of catalytic signal amplification, since the daunomycin-enzyme conjugate produces multiple copies of the detected o-dianisidine oxidation product. The magnitudes of the observed absorbances are expected to be even greater if measurements are made at 436 nm, where the odianisidine oxidation product absorbs maximally. It should be noted that this product irreversibly stains the microtiter wells, so that measurements can be made only once on a particular well. Capture and Detection of Single-Stranded DNA. Singlestranded poly(dA) and poly(dA) modified by biotinylation were immobilized onto Covalink strips and avidin-coated microtiter plates, respectively, varying the poly(dA) concentration in the immobilization solutions. The resulting DNA-modified wells were hybridized with 1 µg/mL poly(dT), and detection proceeded with the 4:1 daunomycin-glucose oxidase conjugate. The results, shown in Figure 4, indicate that the optimum concentrations of poly(dA) for immobilization are similar for the two methods at 0.1 µg/mL. The larger absolute signal magnitudes and uncertainties observed in the case of biotin-avidin immobilization suggest nonspecific interactions of the conjugate with the DNA/avidin layer on the well surfaces. Dye detection experiments performed before and after hybridization with varying concentrations of complementary DNA (0.1-2 µg/mL) show that maximum signals are obtained for complementary DNA concentrations in excess of 0.5 µg/mL; values obtained for 0.5, 1, and 2 µg/mL were identical, with a 90% increase upon hybridization (data not shown). Of the methods studied, optimum conditions are thus 0.1 µg/ mL DNA immobilized by the carbodiimide method, hybridized with >0.5 µg/mL complementary DNA, and detected with 0.25 µM of the 4:1 daunomycin-glucose oxidase conjugate. It should be noted, however, that the carbodiimide reagent can cause polymerization of DNA, as shown by polyacrylamide gel electrophoresis experiments. These experiments showed greater molecular weight for poly(dT) incubated with EDC overnight at 4 °C, compared to a control sample with no EDC. (18) Dianzani, I.; Camaschella, C.; Saglio, G.; Forrest, S. M.; Ramus, S.; Cotton, R. G. H. Genomics 1991, 11, 48.
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Figure 4. Optimization of DNA concentration for immobilization. Absorbance values are corrected for unmodified microtiter wells and are shown as a function of the concentration of poly(dA) used during immobilization. Hybridization of 1.0 µg/mL poly(dT) preceded detection with 0.25 µM 4:1 daunomycin-glucose oxidase conjugate. (a) Biotin-avidin immobilization method (n ) 6). (b) Carbodiimide immobilization method (n ) 8).
This work demonstrates the feasibility of discriminating between double- and single-stranded DNA using simple intercalating dyes and intercalant-enzyme conjugates. These species could be applied to the detection of point mutations, using the chemical mismatch cleavage method, whereby hybridization of long, immobilized probe-soluble target species is allowed to occur, and reagents that cleave DNA only at mismatched bases are used to cleave the immobilized duplex DNA if a single- or multiple-base mismatch is present.18 The final absorbance signals would be smaller and larger for microtiter wells where the mismatch cleavage step is included and omitted, respectively, if a probetarget mismatch is present. Further work with short oligonucleotides, 20 or 30 bases in length, should also be undertaken to determine the applicability of intercalant-enzyme species to assays involving the formation of short duplex DNA products. ACKNOWLEDGMENT Financial support from the Natural Sciences and Engineering Research Council of Canada is gratefully acknowledged. B.K. acknowledges helpful discussions with J. B. Giorgi.
Received for review August 29, 1995. Accepted December 18, 1995.X AC950878M X
Abstract published in Advance ACS Abstracts, February 1, 1996.
