Expression Hybridization Assays Combining cDNAs from Firefly and

Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada N9B 3P4. Two cDNAs encoding firefly luciferase (FLuc) and Re...
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Anal. Chem. 2000, 72, 4022-4028

Expression Hybridization Assays Combining cDNAs from Firefly and Renilla Luciferases as Labels for Simultaneous Determination of Two Target Sequences Eleftheria Laios, Pierre J. Obeid, Penelope C. Ioannou,† and Theodore K. Christopoulos*,‡

Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada N9B 3P4

Two cDNAs encoding firefly luciferase (FLuc) and Renilla luciferase (RLuc) were used as labels for the development of a microtiter well-based expression hybridization assay that allows simultaneous determination of two target DNA sequences. The target DNAs were denatured and hybridized with specific capture and detection probes. One detection probe was biotinylated while the other was tailed with poly(dT). The hybrids were reacted with a streptavidin-FLuc DNA complex and a poly(dA)-tailed RLuc DNA, respectively. Subsequently, the cDNA labels were expressed in vitro simultaneously and independently in the same transcription/translation reaction mixture. The activities of generated firefly and Renilla luciferases were co-determined in the same sample based on the differential requirements of their characteristic bioluminescent reactions for magnesium ions. Luciferase is a generic term for enzymes that catalyze lightproducing chemical reactions in living organisms (e.g., bacteria, fish, beetles, etc.). All known luciferases use molecular oxygen to oxidize their substrates (luciferins). Luciferases from various organisms differ greatly in their amino acid sequence and utilize substrates with quite diverse structures. Bacterial luciferases are flavin-dependent heterodimeric enzymes that catalyze the oxidation of a long-chain aldehyde to the corresponding carboxylic acid.1 Firefly (Photinus pyralis) luciferase is a monomeric protein (MW 62 000) that, in the presence of ATP and Mg2+, catalyzes the oxidative decarboxylation of beetle luciferin.2 Renilla luciferase, from the sea pansy Renilla reniformis, is also a monomeric protein (MW 36 000) which catalyzes the oxidative decarboxylation of coelenterazine.3 Luciferase activity can be measured rapidly and easily with high sensitivity and wide linear range based on the characteristic bioluminogenic reactions. However, these enzymes have found only limited use as labels in DNA hybridization assays † Present address: Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Athens, Greece, GR-15771. ‡ Present address: Department of Chemistry, University of Patras, Patras, Greece, GR-26500. (tel) (011-30-61) 997-130; (fax) (011-30-61) 997-118; (e-mail) [email protected]. (1) Meighen, E. A. FASEB J. 1993, 7, 1016-1022. (2) White, E. H.; Miano, J. D.; Umbreit, M. J. Am. Chem. Soc. 1975, 97, 198200. (3) Matthews, J. C.; Hori, K.; Cormier, M. J. Biochemistry 1977, 16, 85-91.

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and immunoassays due to the significant loss of activity upon conjugation.4 The cloning of luciferase-coding genes from different organisms5-7 has recently stimulated the development of new bioanalytical techniques that use luciferase cDNAs as reporter molecules.8 One broad category of techniques aims at monitoring gene expression and studying the strength and developmental regulation of promoters/enhancers.9,10 Recombinant constructs in which the expression of luciferase cDNA is driven by the promoter/enhancer of interest are introduced into prokaryotic or eukaryotic cells, the substrate diffuses across the cell membrane, and the enzyme activity is monitored, thus allowing the spatial and temporal visualization of gene expression. Other techniques employ the luciferase cDNA for the development of light-emitting biosensors consisting of whole cells (bacteria) as the transducer for the direct determination of, for example, pollutants in environmental samples.8,11 The luciferase cDNA is fused to an inducible promoter responsive to the analyte and introduced into bacteria. When these bacteria are exposed to the analyte, luciferase is expressed. Recently we reported that the firefly luciferase cDNA placed downstream of an RNA polymerase promoter can be used in vitro as a label of antibodies or DNA probes for immunoassays and hybridization assays, respectively.12,13 After completion of the immunocomplex or hybrid formation, the label is subjected to in vitro (cell-free) one-step transcription and translation to generate several enzyme molecules. Thus, in this technique, the gene expression provides one more amplification step in addition to substrate turnover. Moreover, the problem of luciferase inactiva(4) Kricka, L. J. Anal. Biochem. 1988, 175, 14-21. (5) de Wet, J. R.; Wood, K. V.; Helinski, D. R.; DeLuca, M. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 7870-7873. (6) de Wet, J. R.; Wood, K. V.; DeLuca, M.; Helinski, D. R.; Subramani, S. Mol. Cell. Biol. 1987, 7, 725-737. (7) Lorenz, W. W.; McCann, R. O.; Longiaru, M.; Cormier, M. J. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 4438-4442. (8) Lewis, J. C.; Feltus, A.; Ensor, C. M.; Ramanathan, S.; Daunert, S. Anal. Chem. 1998, 70, 579A-585A. (9) Bronstein, I.; Fortin, J.; Stanley, P. E.; Stewart, G. S.; Kricka, L. J. Anal. Biochem. 1994, 219, 169-181. (10) Srikantha, T.; Klapach, A.; Lorenz, W. W.; Tsai, L. K.; Laughlin, L. A.; Gorman, J. A.; Soll, D. R. J. Bacteriol. 1996, 178, 121-129. (11) Ramanathan, S.; Weiping, S.; Rosen, B. P.; Daunert, S. Anal. Chem. 1997, 69, 3380-3384. (12) Christopoulos, T. K.; Chiu, N. H. L. Anal. Chem. 1995, 67, 4290-4. (13) Chiu, N. H. L.; Christopoulos, T. K. Anal. Chem. 1996, 68, 2304-8. 10.1021/ac0004198 CCC: $19.00

© 2000 American Chemical Society Published on Web 08/04/2000

tion due to coupling is avoided because the synthesized enzyme remains free in solution. In the present work, an expression hybridization assay is developed for the simultaneous determination of two target DNA sequences that combines two cDNA labels encoding firefly and Renilla luciferases. The targets are hybridized with immobilized capture probes and then linked, through detection probes, to the cDNAs. We prove that the two reporter cDNAs are expressed independently, despite the fact that they use the same promoter and share the same transcription and translation machinery. Moreover, a method is developed for co-determination of the activities of generated firefly and Renilla luciferases without splitting the sample. The method is based on the fact that, contrary to firefly luciferase, Renilla luciferase does not require the presence of divalent metal ions for full activity. EXPERIMENTAL SECTION Instrumentation. Luminescence measurements were carried out using the MLX Microtiter Plate Luminometer, with two injectors, from Dynex Technologies (Chantilly, VA).The microtiter plate washer model EAW II was from SLT-Lab Instruments (Salzburg, Austria). Hybridization assays were performed using the Amerlite shaker/incubator (Amersham, Oakville, ON, Canada). The microplate fluorometer, Fluoroskan II (Labsystems, Needham Heights, MA) was used in DNA determination with PicoGreen. Materials. Terminal deoxynucleotidyl transferase, biotin-162′, 3′-dideoxyuridine 5′-triphosphate (biotin-ddUTP), streptavidin (SA), and blocking reagent were purchased from Boehringer Mannheim Biochemica (Laval, PQ, Canada). The restriction enzymes BglI and SacII were from New England Biolabs Inc. (Mississauga, ON, Canada). Coenzyme A (CoA) and bovine serum albumin (BSA) were from Sigma (St. Louis, MO). U-bottom, transparent polystyrene microtiter wells (Nunc Maxisorp) were purchased from Life Technologies (Burlington, ON, Canada). Flatbottom, opaque polystyrene microtiter wells (Microlite 2) were from Dynatech Laboratories Inc. (Chantilly, VA). The Wizard Plus Maxipreps DNA purification system, beetle luciferin, the pRL-TK vector, and the TNT T7 wheat germ extract, used for the in vitro coupled transcription/translation reactions, were purchased from Promega Corp. (Madison, WI). Coelenterazine was from SeaLite Sciences Inc. (Norcross, GA). The PicoGreen-based DNA quantification reagent (including λ DNA standard) was from Molecular Probes (Eugene, OR). Deoxyribonucleotides (dNTPs,100 mmol/L solutions) were from Pharmacia Biotech (Montreal, PQ, Canada). The dialysis tubing used for electroelution (molecular weight cutoff of 6000-8000 and flat width of 23 mm) was obtained from Fisher Scientific (Nepean, ON, Canada; Catalog No. 21-152-3). Two DNA fragments were used as analytes in this work. Target A was a 495-bp DNA fragment synthesized by amplifying the prostate-specific antigen mRNA from LNCaP cells (a human prostatic carcinoma cell line) using reverse transcriptase polymerase chain reaction (RT-PCR), as previously described14 with the oligonucleotides 5′-CTCTCGTGGCAGGGCAGTCT-3′ and 5′GTGCTTTTGCCCCCTGTCCA-3′ as upstream and downstream primers, respectively. Target B was a 280-bp DNA fragment prepared by amplifying the BCR-ABL mRNA from K562 cells (a philadelphia chromosome positive human cell line) using RT-PCR

as described in ref 15, with the oligonucleotides 5′- GGAGCTGCAGATGCTGACCAAC-3′ and 5′-CAGTGCAACGAAAAGGTTGGG GTC-3′ as upstream and downstream primers, respectively. The concentrations of stock target DNA solutions were determined fluorometrically using PicoGreen. Oligonucleotides 1 and 3, used as capture and detection probes for target A, were complementary to target regions 67-90 and 214-233, respectively. The capture and detection probes for target B (oligonucleotides 2 and 4) were complementary to regions 139-164 and 179-200, respectively. The blocking solution contained 10 g/L blocking reagent in 0.1 mol/L maleate and 0.15 mol/L NaCl, pH 7.5. Solutions with various target DNA concentrations were prepared by diluting the DNA in 10 g/L blocking reagent in water. The hybridization buffer contained 60 mmol/L citrate, 0.6 mol/L NaCl, and 10 g/L blocking reagent, pH 7.5. The wash solution consisted of 50 mmol/L Tris, pH 7.4, 0.15 mol/L NaCl, and 1 mL/L Tween-20. The phosphatebuffered saline (PBS) was a 10 mmol/L sodium phosphate, 1.76 mmol/L potassium phosphate, 0.14 mol/L NaCl, and 2.7 mmol/L KCl solution, pH 7.4. The Tris-EDTA (TE) buffer consisted of 10 mmol/L Tris and 1 mmol/L EDTA, pH 8.0. The Tris-acetateEDTA (TAE) buffer contained 40 mmol/L Tris-acetate (pH 7.6) and 1 mmol/L EDTA. The Tris-borate-EDTA (TBE) buffer contained 18 mmol/L Tris-borate (pH 7.6) and 0.4 mmol/L EDTA. Labeling of Detection Probes. The oligonucleotide used as a detection probe for target A was labeled at the 3′ end with a single biotin. The reaction was performed in 20 µL containing 0.2 mol/L potassium cacodylate, 25 mmol/L Tris-HCl (pH 6.6), 0.25 g/L BSA, 5 mmol/L CoCl2, 0.05 mmol/L biotin-ddUTP, 25 units of terminal deoxynucleotidyl transferase, and 100 pmol of probe. The reaction was carried out at 37 °C for 1 h and was terminated by the addition of 2 µL of 0.2 mol/L EDTA. Purification of the biotinylated probe was not necessary. The oligonucleotide used as a detection probe for target B was enzymically tailed with dTTP as described above except that 0.05 mmol/L dTTP was included in the reaction mixture instead of biotin-ddUTP. Purification of the dTTP-tailed probe was not required. Preparation of Firefly Luciferase-Coding DNA Fragment (FLuc). A 4.3-kbp plasmid, containing the firefly luciferase coding sequence downstream of the T7 RNA polymerase promoter, was used as a label for the determination of target A. The plasmid (50 µg) was digested with 100 units of Bgl I (2 h), in a total volume of 100 µL, to give a linear fragment with protruding 3′ ends. After ethanol precipitation, 30 µg (10 pmol) of FLuc DNA was enzymically labeled at the 3′ end with biotin-ddUTP as described above (under Labeling of Detection Probes). After ethanol precipitation, the biotinylated DNA was quantitated with PicoGreen. Preparation and Purification of the Streptavidin-FLuc DNA Complex. The streptavidin-biotinylated FLuc DNA complex was prepared in a final volume of 50 µL containing 1 mol/L NaCl, 21.8 µg of biotinylated DNA (7.6 pmol), and 23.9 µg (458 pmol) of streptavidin in TE buffer. After being incubated for 30 min at room temperature, the complex was purified from the excess streptavidin by electroelution. The mixture was first subjected to agarose (0.7%) gel electrophoresis at 4 °C in TAE

(14) Galvan, B.; Christopoulos, T. K. Clin. Biochem. 1997, 30, 391-397.

(15) Bortolin, S.; Christopoulos, T. K. Anal. Chem. 1994, 66, 4302-4307.

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buffer containing 0.125 mg/L ethidium bromide. The band containing the SA-FLuc complex was excised and placed in short lengths of dialysis tubing containing TBE buffer. The dialysis tubing had been previously soaked in distilled water for 15 min, heated with stirring at 80 °C for 30 min in 10 mmol/L sodium bicarbonate, soaked in 10 mmol/L EDTA for 30 min, and heated again at 80 °C for 30 min in water. The tube containing the gel slice was closed by plastic clips, placed in an electrophoresis tank filled with TBE buffer, and run at 60 V at 4 °C. The tube was periodically checked with a hand-held UV lamp to determine whether the DNA had been completely eluted from the gel slice. The buffer containing the complex was then removed from the tubing and concentrated to about 150-250 µL, in the presence of 0.2 g/L blocking reagent, by using Centricon-30 concentrators. The complex was stored at 4 °C in a solution containing 1 mol/L NaCl. Preparation of Renilla Luciferase-Coding DNA Fragment (RLuc DNA). A 4.0-kbp plasmid (pRL-TK) containing the Renilla luciferase coding sequence downstream of the T7 RNA polymerase promoter was used as a label for determination of target B. For growing E. coli JM109 bacteria, preparation of competent cells, and transformation with plasmid DNA, standard procedures were followed.16 Transformed bacteria were grown overnight in LB broth (10 g/L tryptone, 5 g/L yeast extract, 0.17 mol/L NaCl, and 2 mmol/L NaOH) containing 0.1 g/L ampicillin. The plasmid DNA was purified from the bacterial culture using the Wizard Plus Maxipreps DNA purification system according to the manufacturer’s instructions except that we used phenol/chloroform extraction and ethanol precipitation in the last step. The size of the plasmid was confirmed by agarose (0.7%) gel electrophoresis and ethidium bromide staining. The plasmid concentration was determined fluorometrically. For preparation of the DNA template, 50 µg of plasmid was digested with 100 units of SacII (overnight), in a total volume of 100 µL, to give a linear fragment with protruding 3′ ends. After ethanol precipitation and quantification, 9.3 µg (3.5 pmol) of RLuc DNA was enzymically tailed with dATP as described above (under Labeling of Detection Probes) by using 0.038 mmol/L dATP. The tailed DNA was used directly without purification. Assay of Firefly Luciferase Activity. The substrate solution for firefly luciferase contained 20 mmol/L tricine, 1.1 mmol/L magnesium carbonate pentahydrate, 2.7 mmol/L MgSO4, 0.1 mmol/L EDTA, 270 µmol/L CoA, 530 µmol/L ATP, and 470 µmol/L luciferin, pH 7.8, 17 and 3.3 mmol/L dithiothreitol (DTT). For the firefly luciferase assay, 50 µL of substrate solution was added with automated injection to aliquots of transcription/ translation reactions in flat-bottom, opaque microtiter wells. The luminescence was measured for 30 s (2-s delay time and 28-s integration time). Assay of Renilla Luciferase Activity. The substrate solution contained 10 mmol/L KH2PO4, 0.5 mol/L NaCl, 1 mmol/L EDTA, pH 7.5, and 1 µmol/L coelenterazine. For the Renilla luciferase assay, 50 µL of substrate solution was added with automated injection to aliquots of transcription/translation reactions in flat(16) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning. A Laboratory Manual, 2nd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor. NY, 1989. (17) Titus, D. E. Promega Protocols and Applications Guide, 2nd ed., Promega Corp., Madison, WI, 1991.

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bottom, opaque microtiter wells. The luminescence was measured for 10 s (2-s delay time and 8-s integration time). Co-Determination of Firefly Luciferase and Renilla Luciferase Activities in the Same Sample. Samples (e.g., transcription/translation reaction mixtures) containing both firefly and Renilla luciferases were analyzed as follows. The firefly luciferase substrate solution (50 µL) was added with automated injection to aliquots of transcription/translation reactions in flat-bottom, opaque microtiter wells. The activity of firefly luciferase was measured for 30 s (2-s delay and 28-s integration). Subsequently, the firefly luciferase reaction was terminated by the manual addition of 25 µL of 300 mmol/L EDTA and shaking for 1 min. The activity of Renilla luciferase was then measured by injecting 50 µL of Renilla luciferase substrate solution (containing 2.5 µmol/L coelenterazine). The luminescence was measured for 10 s (2-s delay and 8-s integration). Expression Hybridization Assays Combining Firefly Luciferase- and Renilla Luciferase-Coding DNA Fragments as Labels. U-Bottom polystyrene microtiter wells were coated overnight at 4 °C, by physical adsorption, with 25 µL of a solution containing 5 mg/L each of BSA-oligo 1 conjugate and BSAoligo 2 conjugate diluted in PBS containing 5 mmol/L EDTA. The wells were washed three times with wash solution, and the remaining binding sites were blocked with 100 µL of blocking solution for 60 min at room temperature. The wells were washed three times as above followed by the addition of 15 µL/well of a solution containing 6.7 nmol/L each of oligo 3 and oligo 4 diluted in hybridization buffer and preheated at 42 °C. Samples containing both DNA targets (A and B), diluted in 10 g/L blocking reagent in water, were denatured by heating at 95 °C for 10 min and then 10 µL was added into each well and allowed to hybridize simultaneously with the capture probes (oligos 1 and 2) and the detection probes (oligos 3 and 4) for 60 min at 42 °C. After washing the wells, 25 µL of a solution containing the streptavidinFLuc DNA complex (0.7 nmol/L with respect to DNA) and the dATP-tailed RLuc DNA (1.6 nmol/L), diluted in hybridization buffer and 2 mmol/L EDTA, was added to each well and allowed to anneal with the detection probes for 20 min at room temperature. After the wells were washed five times with wash solution and twice with 50 mmol/L potassium acetate, the bound FLuc DNA and RLuc DNA were subjected to a coupled (one-step) transcription/translation by adding 25 µL of transcription/translation (TNT) mixture and incubating for 90 min at 30 °C. At the end of this period, the activities of the generated firefly luciferase and Renilla luciferase were determined in a 10-µL aliquot of the expression mixture using the protocol described above. RESULTS AND DISCUSSION The principle of the proposed hybridization assay is illustrated in Figure 1. The following requirements must be fulfilled for the simultaneous determinations of two target DNA sequences by an expression hybridization assay: (a) The reporter genes (labels) are expressed independently in the same reaction mixture; namely, the transcription and translation of one gene does not affect the expression of the other. (b) The gene products (enzymes in this work) are co-determined in the same sample, i.e., the presence of one enzyme and its substrate does not interfere with

Figure 1. Principle of expression hybridization assay that combines firefly luciferase (FLuc) DNA and Renilla luciferase (RLuc) DNA as labels for the simultaneous determination of two target DNA sequences. The target DNA fragments are denatured and hybridized simultaneously on immobilized specific capture probes and detection probes. One detection probe is labeled with biotin (B) and the other carries a poly(dT) tail. The hybrids are reacted with streptavidin (SA)FLuc complex and poly(dA)-tailed Renilla luciferase DNA. The FLuc and RLuc DNA bound to the solid phase were measured by coupled (one-step) in vitro transcription/translation.

the assay of the other enzyme. (c) There is no cross-reactivity between probes and target DNA sequences during hybridization. We first studied the relation between input RLuc DNA in the cell-free transcription/translation reaction and the activity of synthesized Renilla luciferase. Various amounts of RLuc plasmid DNA were subjected to coupled transcription/translation in a total volume of 12.5 µL. After completion of the reaction, 50 µL of the RLuc substrate solution (containing 1 µmoL/L coelenterazine) was added to 5-µL aliquots of the mixtures and the luminescence was measured as above (see Assay of Renilla Luciferase Activity). Figure 2 shows that the luminescence is a linear function of the number of RLuc DNA molecules in the sample, in the range of 105-6 × 107 molecules (0.17-99.6 amol). The signal-to-background ratio for 105 RLuc DNA molecules was 2.6. A similar experiment was performed to assess the relation between input Fluc DNA in the transcription/translation reaction and the luminescence signal. The linear range extends from 2 × 104 to 107 FLuc DNA molecules (33.2 zmol-16.6 amol) (Figure 2). The signal-to-background ratio for 2 × 104 FLuc DNA molecules was 8.2. Coexpression of RLuc and FLuc DNA. To investigate if the RLuc and FLuc genes can be expressed simultaneously in a cellfree system, we performed transcription/translation reactions in which each gene was expressed either alone or in the presence of a large excess of the other gene. The activity of the corresponding luciferase was then measured and the luminescence values were compared. In a typical experiment, the luminescence (relative light units) of a transcription/translation reaction mixture containing 9 × 104 molecules (0.15 amol) of FLuc DNA was 1739 ( 90 (n ) 4), as measured with the firefly luciferase assay. When 9 × 104 FLuc DNA molecules (0.15 amol) were coexpressed with 107 molecules (16.6 amol) of RLuc DNA (a 100-fold excess), the luminescence obtained for firefly luciferase was 1951 ( 90 (n ) 4). Similarly, the luminescence obtained from the expression of 1.8 × 105 molecules (0.3 amol) of RLuc DNA was 26.2 ( 1.5 (n ) 4), as measured with the Renilla luciferase assay. When 1.8 × 105 molecules of RLuc DNA were coexpressed with 107 FLuc DNA molecules (a 50-fold excess), the luminescence obtained for

Figure 2. Luminescence as a function of the number of Renilla luciferase (RLuc)-coding DNA molecules (circles) and firefly luciferase (FLuc)-coding DNA molecules (squares). In a typical experiment, various amounts of the enzyme-coding DNA fragment were subjected to in vitro one-step transcription/translation. After completion of the reaction, 50 µL of RLuc or FLuc substrate solution was added to 5 µL of the expression mixture and the luminescence was measured as described in the Experimental Section. The triangles correspond to the signal obtained when Rluc was measured with the luciferase co-determination method, which involves injection of the firefly luciferase assay reagent, addition of EDTA, and then injection of RLuc substrate solution (see Experimental Section).

Renilla luciferase was 28.8 ( 2.1 (n ) 4). Consequently, the RLuc and FLuc genes are coexpressed in vitro independently and in a wide range of concentrations despite the fact that they contain the same promoter sequence (T7) and share the same transcription and translation machinery. These experiments also demonstrate that the presence of excess firefly luciferase enzyme does not interfere with the activity of Renilla luciferase and vice versa (specificity of the enzymes for their substrates). Co-Determination of Firefly and Renilla Luciferases. The method is based on the fact that the firefly luciferase-catalyzed reaction requires the presence of Mg2+, whereas the reaction catalyzed by Renilla luciferase takes place in the absence of divalent cations. First, the substrate for firefly luciferase is added and the luminescence is measured. Mg2+ is then chelated with EDTA, causing termination of the firefly luciferase reaction. Afterward, the substrate for Renilla luciferase is added followed by luminescence measurement. The entire protocol is complete in 2 min. To test whether the presence of FLuc assay reagents interferes with the RLuc assay, we measured the activity of synthesized RLuc either by using the single assay (direct addition of RLuc substrate solution) or by the co-determination method (FLuc reagents, EDTA, and then RLuc substrate). The data presented in Figure 2 suggest that there is no effect of FLuc assay reagents on the activity of RLuc. The effect of EDTA concentration on the firefly luciferase reaction was studied in the range of 2.5-100 mmol/L. We observed that a final concentration of 100 mmol/L was required Analytical Chemistry, Vol. 72, No. 17, September 1, 2000

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Figure 3. Time course of light emission during co-determination of firefly luciferase and Renilla luciferase. The substrate solution of firefly luciferase is injected into the well and the luminescence is measured (delay time 2 s, integration time 28 s). The reaction is stopped by the manual addition of EDTA. Subsequently, the substrate solution for Renilla luciferase is injected and the luminescence is measured (delay time 2 s, integration time 8 s). The reactions catalyzed by firefly and Renilla luciferases are also shown.

for effective inhibition. The luminescence of a sample containing a high concentration of firefly luciferase was 11 947. Following the addition of 100 mmol/L EDTA, the remaining luminescence signal measured with the instrument settings of Renilla luciferase (2-s delay, 8-s integration) was found to be 1.55. Therefore, only 0.013% of the firefly luciferase signal was carried over to the Renilla luciferase assay. Moreover, we observed that EDTA did not affect the Renilla luciferase reaction. The EDTA was not included in the RLuc reagent in order to ensure complete quenching of the FLuc signal prior to the initiation of the RLuc reaction. An experiment was carried out in which a sample containing only RLuc was mixed with the FLuc substrate solution. The luminescence was found to be indistinguishable from the background (the background was the luminescence obtained from a sample containing neither enzyme). In another experiment, a sample containing only FLuc was measured with its own substrate solution, quenched with EDTA, and then mixed with the RLuc substrate solution (according to the co-determination assay protocol). The luminescence, after addition of coelenterazine, was also indistinguishable from the background. These experiments prove that the FLuc substrate solution does not trigger the RLuc reaction and vice versa. The kinetics of light emission during co-determination of firefly and Renilla luciferases is shown in Figure 3. The chemiluminescence quantum yields for the firefly and Renilla luciferase reaction 4026 Analytical Chemistry, Vol. 72, No. 17, September 1, 2000

are 0.88 and 0.055, respectively.3,18 This explains the higher luminescence intensity observed with the firefly reaction. The integration times for both FLuc and RLuc assays were optimized to achieve the maximum signal-to-background ratios. A longer than 8-s integration time for RLuc leads to a disproportionately higher background with respect to the signal, hence a decrease in the signal-to-background ratio. To assess the reproducibility of the proposed method for codetermination of firefly and Renilla luciferases, we carried out transcription/translation reactions of three samples containing both FLuc and RLuc DNA at various ratios (low, medium, and high). The synthesized FLuc and RLuc were then measured four times by the co-determination method. Samples 1, 2, and 3 contained 9 × 104/107, 1.4 × 106/2.3 × 106, and 107/1.8 × 105 FLuc/Rluc DNA molecules, respectively. The corresponding CVs were 4.6/1.9, 1.6/3.4, and 6.8/1.8%, respectively (the first value of each pair refers to FLuc). Simultaneous Determination of Two Target DNA Sequences by Expression Hybridization Assay Combining Firefly and Renilla Luciferase DNAs as Labels. The ability of the proposed hybridization assay (Figure 1) to quantify both target DNA sequences (A and B) in a mixture was assessed. We analyzed samples containing various amounts of each target (18) Seliger, H. H.; McElroy, W. D. Arch. Biochem. Biophys. 1960, 88, 136141.

Figure 4. Luminescence as a function of the amount of target A DNA (a) and target B DNA (b). The signals were obtained from samples containing various amounts of each target DNA either alone (solid squares) or in the presence of 10 fmol of the other target (open squares).

sequence either alone or in the presence of 10 fmol of the other target (this amount corresponds to the upper point of the calibration curve of either target DNA). The results are presented in Figure 4. It is observed that the luminescence obtained from synthesized firefly luciferase is linearly related to the amount of DNA target A in the sample (Figure 4a). The signal-to-background ratio obtained for 66 amol of target A in the presence of 10 fmol of target B (a 150-fold excess) was 2.7. The background was defined as the luminescence corresponding to a sample without target A but with 10 fmol of target B. Similarly, the luminescence obtained from synthesized Renilla luciferase is a linear function of the amount of DNA target B (Figure 4b). The signal-tobackground ratio obtained for 66 amol of target B (analyzed in the presence of 10 fmol of target A) was 6. The coincidence of the two lines (single target vs both targets) in Figure 4a and b shows that there is no cross-reactivity between probes and target DNA sequences in the assay. To test the reproducibility of the hybridization assay, we analyzed three samples, each containing both targets A and B at the following quantities (respectively): sample 1, 0.5 and 10 fmol; sample 2, 2 fmol of each; sample 3, 10 and 0.07 fmol. The samples were analyzed six times. The CVs obtained for targets A and B were 18 and 7% (sample 1), 7.3 and 8.9% (sample 2), and 4.8 and 14% (sample 3), respectively. In other reports, the complex of streptavidin with biotinylated firefly luciferase-coding DNA fragment was used as a label in a model immunoassay and a hybridization assay.12,13 The preparation of the complex involved digestion of the plasmid to produce three fragments with recessed 3′ ends, a fill-in reaction with Klenow DNA polymerase in the presence of biotin-dATP to create fragments biotinylated at both ends, ethanol precipitation, and a digestion leaving a 2.1-kbp fragment labeled with biotin only at one terminus. After electrophoretic separation, the 2.1-kbp fragment was excised, purified, and complexed with streptavidin. The complex was purified by size exclusion HPLC. The yield was between 10 and 20%. In this work, the entire FLuc 4.3-kbp plasmid was used as a label. The procedure involved a single digestion to linearize the plasmid, ethanol precipitation, enzymic labeling of

the DNA with biotin-ddUTP, ethanol precipitation, and complexation with streptavidin. The complex was purified by electroelution. The yield was between 40 and 60%, and the overall procedure was much simpler and faster. Electroelution is usually applied for the purification of naked DNA fragments from a gel following electrophoretic separation. However, in this work, electroelution is used as a tool for purification of protein-DNA complexes while maintaining their activities. The Renilla luciferase-coding DNA was chosen in this study because Renilla luciferase, like firefly luciferase, is a monomeric protein that does not require posttranslational modification and its activity can be readily measured in the transcription/translation reaction without prior purification. To our knowledge, this is the first time that Renilla luciferase has been used as a reporter molecule in hybridization assays. The 90-min incubation required for the transcription/translation reaction is a lengthy step, but it is at least compensated partially by the short (less than 2 min) assay of both FLuc and RLuc activities. Previous work by our group12 have shown that the expression of an immobilized FLuc DNA template, monitored by the activity of synthesized luciferase, reaches a plateau at 90 min. At 60 min, the luminescence is ∼80% of the maximum. Therefore, a 60-min transcription/translation step would compromise the sensitivity of the assay accordingly. Incubation times shorter than 60 min lead to a steep decrease in the luciferase activity.12 In the process of this work, we prepared complexes or conjugates of each luciferase-coding DNA with the respective detection probe, thus creating a new reagent with dual function, i.e., possessing both a molecular recognition part (probe) and a signal-generating part (reporter). The reagent could then be used in a single step during hybridization. Initially, we assembled these bifunctional probe/reporter molecules by incubating poly(dT)tailed oligo 3 (detection probe for target A) with poly(dA)-tailed FLuc DNA and poly(dT)-oligo 4 (specific for target B) with poly(dA)-tailed RLuc DNA. The complexes performed well in a hybridization assay of a single target. However, when they were applied to the simultaneous hybridization assay of both targets, Analytical Chemistry, Vol. 72, No. 17, September 1, 2000

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we observed an exchange of reporter cDNAs between the probes, thus introducing errors in the analysis. The interaction between poly(dG) and poly(dC) tails was also used in order to preform bifunctional probe/reporter DNA complexes but the signal-tobackground ratio was decreased 4 times. Another strategy involved the covalent attachment of an adapter to FLuc DNA. The adapter consisted of a single-stranded segment complementary to target A (probe sequence) and a double-stranded segment with cohesive ends to digested FLuc DNA. Ligation of the adapter with FLuc DNA gave a conjugate that could both recognize the target sequence and serve as a template for synthesis of firefly luciferase. However, this approach also resulted in lower assay sensitivity. The decrease in sensitivity was attributed to interactions between the single-stranded probe sequence and the reporter cDNA that interfere with the subsequent hybridization of the probe to the corresponding target. Consequently, the maximum sensitivity was achieved when each probe first hybridized to the corresponding target prior to the addition of reporter cDNA. The utilization of a protein as a reporter molecule presupposes that the protein maintains its activity during (a) isolation from the corresponding cells/organism, (b) linking to molecules that are responsible for binding to the analyte, and (c) storage. On the contrary, DNAs are much more stable than enzymes. To avoid (19) Hastings, J. W. Gene 1996, 173, 5-11. (20) Wood, K. V.; Lam, Y. A.; Seliger, H. H.; McElroy, W. D. Science 1989, 244, 700-702. (21) Branchini, B. R.; Magyar, R. A.; Murtiashaw, M. H.; Anderson, S. M.; Helgerson, L. C.; Zimmer, M. Biochemistry 1999, 38, 13223-13230.

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steric hindrance during transcription, the reporter luciferase cDNAs were attached to the specific probes through their 3′ termini. In this work, it was proved that firefly and Renilla luciferase cDNAs can be transcribed and translated simultaneously and independently in the same in vitro expression mixture and in a broad range of concentrations. This fact provides a perspective for the application of multiple cDNAs as labels for simultaneous determination of many target DNA sequences by expression hybridization assay. In this context, the color of emitted light can serve as an additional parameter to distinguish between synthesized luciferases in a multianalyte system.19 Both the substrate structure and the amino acid sequence at the active site of a luciferase determine the emission wavelength. Different beetle luciferase genes have been cloned which express luciferases that catalyze the oxidation of a common substrate to produce various wavelengths.20 In vitro mutagenesis of the luciferase-coding DNA has also been used to alter the emission spectra.21 ACKNOWLEDGMENT This work was supported by grants to T.K.C. from the National Science and Engineering Research Council of Canada (NSERC). P.C.I. acknowledges financial support from the University of Athens, Greece, for her sabbatical leave. Received for review April 10, 2000. Accepted June 19, 2000. AC0004198