Affinity Capture-Facilitated Preparation of Aequorin− Oligonucleotide

1024

Bioconjugate Chem. 2003, 14, 1024−1029

Affinity Capture-Facilitated Preparation of AequorinOligonucleotide Conjugates for Rapid Hybridization Assays Kyriaki Glynou,† Penelope C. Ioannou,*,† and Theodore K. Christopoulos*,‡,§ Department of Chemistry, University of Athens, Athens, Greece 15771, and Department of Chemistry, University of Patras, Patras, Greece 26500, and Institute of Chemical Engineering and High-Temperature Chemical Processes, P.O. Box 1414, Patras, Greece 26500. Received June 19, 2003

We report a general procedure for the preparation of biomolecular conjugates that combine the molecular recognition properties of oligonucleotides with the high detectability of the photoprotein aequorin. Central to the conjugation protocols is the use of recombinant aequorin fused to a hexahistidine tag. In one protocol, an amino-modified oligonucleotide was treated with a homobifunctional cross-linker carrying two N-hydroxysuccinimide ester groups, and the derivative was allowed to react with (His)6-aequorin. A second strategy involved the introduction of protected sulfhydryl groups into (His)6-aequorin and subsequent reaction with a heterobifunctional linker containing a Nhydroxysuccinimide and a maleimide group. The strong, but reversible, binding of (His)6-aequorin to Ni2+-nitrilotriacetic acid agarose enabled the rapid and effective removal of the unreacted oligonucleotide, which otherwise diminishes the performance of the hybridization assay by competing with the conjugate for the complementary target sequence. Aequorin-oligo conjugates prepared by affinity capture showed similar performance with those purified by anion-exchange HPLC. The conjugates were applied to the development of rapid bioluminometric hybridization assays. The analytical range extended from 2 to 2000 pmol/L of target DNA. The reproducibility was less than 10%. The conjugate obtained from a reaction of 10 nmol of (His)6-aequorin is sufficient for about 5000 hybridization assays. The proposed conjugation strategy is general because a variety of reporter proteins can be fused to hexahistidine tag by using suitable vectors that are commercially available.

INTRODUCTION

In recent years, nucleic acid analysis by hybridization has undergone a transition from radioactive labels to nonradioactive alternatives which was driven by the need to improve the detectability and facilitate automation while avoiding problems associated with the use and disposal of radioisotopes (1). (Chemi)bioluminometric hybridization assays are used widely because they are amenable to automation and offer superior detectability over conventional spectrophotometric and fluorometric ones (2). Aequorin is a photoprotein composed of a 189-amino acid polypeptide chain (apoaequorin), the imidazopyrazine chromophore coelenterazine, and oxygen that is attached to coelenterazine as a peroxide (3-5). When Ca2+ binds to aequorin, it induces a conformational change that triggers the oxidation of coelenterazine by the bound oxygen to produce coelenteramide, CO2, and light at 470 nm. Aequorin is an excellent reporter molecule since it can be detected at the attomole level in the presence of excess Ca2+ (6). The cloning of apoaequorin cDNA (7, 8) has enabled the expression of recombinant aequorin in bacteria and its subsequent use as a reporter in immunoassays (9, 10) and hybridization * Corresponding authors: P.C.I.; T.K.C.: Tel. (+30)2107274574; (+30)2610997130. FAX: (+30)2107274750; (+30)2610997118. E-mail: [email protected]; [email protected]. † University of Athens. ‡ University of Patras. § Institute of Chemical Engineering and High-Temperature Chemical Processes.

assays (9, 11-14). In these studies it was observed that although the aequorin reaction does not entail a substrate turnover, it provides a high detectability that is comparable to enzyme reporters (e.g., alkaline phosphatase) with chemiluminogenic substrates. Moreover, the detection of aequorin is complete within 3 s following the addition of Ca2+, which is a significant advantage over enzyme reactions that require much longer incubation times. Aequorin-based bioluminometric hybridization assays may be performed by two strategies. In the ‘indirect labeling’ approach, a ligand (such as biotin or the hapten digoxigenin) is attached to the DNA probe, and the hybrids are detected by using a specific binding protein conjugated or complexed to the photoprotein. Thus, the hapten digoxigenin has been attached to the probe and an aequorin-antidigoxigenin was used for detection (12). Alternatively, biotin was used as a ligand, and the hybrids were detected by an aequorin-streptavidin conjugate or a streptavidin-biotinylated aequorin complex (11, 15). The ‘direct labeling approach’ employs aequorin conjugated to the DNA probe (16). The advantage of the latter arises from the elimination of an incubation step and a washing step, thus reducing significantly the time required for assay completion. However, the preparation of aequorin-DNA conjugates requires laborious chromatographic procedures followed by concentration steps, to remove the unreacted DNA probe which otherwise competes with the conjugate for hybridization to the target sequence. Recently, we reported a simple method for expression and purification of aequorin in one step, based on

10.1021/bc0341021 CCC: $25.00 © 2003 American Chemical Society Published on Web 08/23/2003

Aequorin−Oligonucleotide Conjugates

immobilized metal-ion affinity chromatography (17). A hexahistidine-coding sequence was fused upstream of aequorin cDNA. The (His)6-apoaequorin fusion protein was purified from crude cellular extracts by using a Ni2+nitrilotriacetic acid agarose column. This procedure lasted only a few hours and gave aequorin of high purity and activity whereas previous methods required precipitation of aequorin from the culture medium at pH 4.2, resuspension, overnight dialysis, and purification by DEAE-cellulose chromatography followed by aequorin regeneration. In the present work we demonstrate that besides enabling one-step purification of the photoprotein from bacterial cultures, the hexahistidine tag greatly facilitates the preparation of conjugates of aequorin to DNA probes for the development of high-throughput hybridization assays. Conjugates are prepared by using either homobifunctional or heterobifunctional cross-linking reagents. EXPERIMENTAL SECTION

Instrumentation. Ion-exchange chromatography was performed using an HPLC system from GBC Scientific Equipment Pty (Dandenong, Victoria, Australia) consisting of the LC1150 quaternary gradient pump, the LC1205 UV/Vis Detector, and the WinChrom management System. A strong anion-exchange column Hypersil SAX (150 × 4.6 mm) was employed (GBC Scientific Equipment). Bioluminescence was measured by using the PhL microplate Luminometer/Photometer manufactured by Mediators (Vienna, Austria). Polymerase chain reaction (PCR) was carried out in a Hybaid Omn-E thermal cycler (Middlesex, UK). A digital camera, Kodak DC 120, and the Gel Analyzer software for DNA and protein documentation were purchased from Kodak (New York, NY). Microcentrifuge Mikro 20 was from Hettich Gmbh (Tuttlingen, Germany). Materials. Sulfo-succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (Sulfo-SMCC), N-succinimidyl S-acetylthioacetate (SATA), and bis(sulfosuccinimidyl) suberate (BS3) were obtained from Pierce Chemical Co (Rockford, IL). Bovine serum albumin (BSA) was from Serva (Heidelberg, Germany), blocking reagent was from Roche (Mannheim, Germany). Sephadex G-25 Spin Pure columns were purchased from CPG (Lincoln Park, NJ). Ni2+-Nitrilotriacetic acid (Ni-NTA) agarose was obtained from Qiagen (Hilden, Germany). Microcon YM-10 centrifugal filter devices were from Millipore (Bedford, MA). All oligonucleotides were synthesized by MWG Biotech (Ebersberg, Germany). The 20mer 5′-biotin-GTC CTT CCC CAG AGT TCA GT-3′ and the 31mer 5′-TTT TTC TAG AAA CAC CAT CCC TCC TCG AAC C-3′ were used as the upstream (UP) and downstream primer (DP), respectively, for the PCR of the cDNA of prostate-specific membrane antigen (PSMA). The 24mer 5′-NH2-TTT GTT TGT TTC CCA ATT TTT AGT was used as the target specific probe (p). Ultrapure 2′-deoxyribonucleoside 5′triphosphates (dNTPs) were purchased from HT Biotechnology (Cambridge, UK). Tth DNA polymerase was from Biotools (Madrid, Spain). (His)6-tagged recombinant apoaequorin was expressed in E. coli JM109 cells and purified as described previously (17). Conjugation of (His)6-aequorin to Oligonucleotides Using a Homobifunctional Cross-Linking Reagent. The 5′-amino-modified oligonucleotide probe, p, was dissolved in distilled water to a final concentration of 0.4 mmol/L. An 8.5-µL aliquot (3.4 nmol of probe) was then mixed with 4.5 µL of 0.5 mol/L sodium bicarbonate,

Bioconjugate Chem., Vol. 14, No. 5, 2003 1025

pH 8, and 9.7 µL of 20 g/L BS3 (dissolved in DMSO). The mixture was incubated for 15 min at ambient temperature in the dark, and the unreacted BS3 was removed by gel filtration using spin-pure G-25 column prehydrated with distilled water. The derivatized oligonucleotide (∼32 µL) was mixed with 9.3 µL of 0.2 mol/L sodium phosphate buffer, pH 8.6, containing 5 mmol/L EDTA and 61 µL of 165 µmol/L aequorin solution (10 nmol) in 50 mmol/L N-(3-sulfopropyl)morpholine (MOPS), pH 7.2, 1 mol/L KCl, 5 mmol/L EGTA and 0.3 mol/L glucose. KCl was added to give a final concentration of 2 mol/L, and the mixture was incubated for 12-16 h at 4 °C in the dark. Conjugation of (His)6-aequorin to Oligonucleotides Using a Heterobifunctional Cross-Linking Reagent. Sulfhydryl groups were first introduced into aequorin by using SATA. A 61-µL aliquot of 165 µmol/L aequorin (10 nmol), in 50 mmol/L MOPS, pH 7.2, 1 mol/L KCl, 5 mmol/L EGTA and 0.3 mol/L glucose, was mixed with 1.8 µL 1.25 g/L SATA solution (10 nmol) in DMSO and 6.9 µL of 0.2 mol/L sodium phosphate buffer, pH 8.0, 5 mmol/L EDTA and 2 mol/L KCl. The reaction mixture was incubated for 90 min at ambient temperature in the dark. Excess SATA was removed by ultrafiltration using the Microcon YM-10 centrifugal filter device. Prior to ultrafiltration, the reaction mixture was diluted with 0.4 mL of a buffer solution (20 mmol/L phosphate buffer, pH 7.5, containing EDTA 0.5 mmol/L and KCl 0.2 mol/L) and it was centrifuged at 10000 rpm, in the microcentrifuge, for 50 min at 4 °C. This step was repeated, and then the retentate was collected by centrifugation at 3000 rpm for 3 min (retentate volume about 8 µL). The 5′-amino-modified oligonucleotide probe was activated by reacting with the heterobifunctional crosslinking reagent sulfo-SMCC. An 8.5-µL aliquot of oligonucleotide, p, solution (3.4 nmol) was mixed with 3.9 µL of 0.5 mol/L sodium bicarbonate, pH 8, and 7.4 µL of 20 g/L sulfo-SMCC (340 nmol) solution in DMSO. Following 30 min incubation at ambient temperature in the dark, the excess of sulfo-SMCC was removed by gel filtration using a G-25 Spin Pure column prehydrated with water. The reaction between SATA-derivatized aequorin and maleimide-activated oligonucleotide was initiated by the addition of hydroxylamine. A 30-µL aliquot of maleimideactivated oligo was mixed with 8 µL of SATA-aequorin, 10.5 µL of 0.5 mol/L NH2OH, and 5.2 µL of 0.2 mol/L phosphate buffer, pH 7.5, containing 5 mmol/L EDTA. Then, KCl was added to a final concentration of 2 mol/L, and the mixture was incubated for 12-16h at 4 °C. Purification of Oligonucleotide-Conjugated (His)6aequorin Using Ni-NTA Agarose. A 35-µL aliquot of Ni-NTA agarose slurry was placed to a disposable spin column and centrifuged at 3000 rpm (in the microcentrifuge) for 3 min at ambient temperature, and the flowthrough was discarded. Ni-NTA agarose was shaked gently in the column for 25 min with 0.2 mL of blocking solution (20 mmol/L NaH2PO4, 0.5 mmol/L EDTA, 0.2 mol/L KCl, pH 7.5, 10 g/L BSA, and 1.5 mL/L Tween20). The mixture was then centrifuged at 3000 rpm for 3 min, and the flow-through was discarded. The conjugation reaction mixture was diluted about 10 times with water so that the final concentration of EDTA did not exceed 0.5 mmol/L, and then it was loaded onto the spin column. Following a 20 min incubation period, under gentle agitation, the mixture was centrifuged, and the flow-through was discarded. Ni-NTA agarose was washed with 0.1 mL of 20 mmol/L NaH2PO4, 0.5 mmol/L EDTA, 0.2 mol/L KCl, pH 7.5. The bound conjugate was eluted by applying 3 × 0.1 mL of a solution containing 20 mmol/L NaH2PO4, 0.1 mol/L EDTA, 0.2 mol/L KCl, pH

1026 Bioconjugate Chem., Vol. 14, No. 5, 2003

Glynou et al.

Figure 1. Outline of the strategies used for preparation and purification of conjugates of hexahistidine-tagged photoprotein aequorin to oligonucleotide probes. (A) Conjugation through a homobifunctional cross-linking reagent. (B) Conjugation through a heterobifunctional cross-linker. (C) Affinity capture-facilitated purification of the conjugates. (D) Hybridization assay configuration. SA ) Streptavidin; B ) Biotin.

7.5. The eluate was concentrated to a volume of 20 to 40 µL using a Microcon filter device. Purification of Oligonucleotide-Conjugated (His)6aequorin by HPLC. A 20-µL aliquot of the concentrated eluate from the previous step was injected onto a Hypersil SAX anion-exchange column. Initially the mobile phase contained 55% buffer A (10 mmol/L Tris-HCl, 2 mmol/L EDTA pH 7.5) and 45% buffer B (10 mmol/L Tris-HCl, 2 mmol/L EDTA and 1 mol/L NaCl pH 7.5). After 10 min, a linear gradient of 3.25% buffer B/min was applied until buffer B reached to 58%. The composition of the mobile phase was kept constant for 10 min, and then a linear gradient of 3.8% buffer B/min was applied until buffer B reached 100%. The flow rate was 1 mL/min. During separation, 1-mL fractions were collected, and the activity of aequorin was measured. Polymerase Chain Reaction. The target DNA was a 263-bp fragment synthesized by amplifying the prostatespecific membrane antigen (PSMA) cDNA. PCR was performed in a total volume of 50 µL consisting of 75 mmol/L Tris-HCl (pH 9.0), 20 mmol/L (NH4)2SO4, 50 mmol/L KCl, 2 mmol/L MgCl2, 0.2 mmol/L dNTPs, 25 pmol each of the upstream (biotinylated) and downstream primer, and 1.5 units of Tth DNA polymerase. Amplification was carried out for 35 cycles using the following program: 95 °C, 15 s (1 min for the first cycle), 60 °C, 15 s, 72 °C, 30 s (10 min for the last cycle). The PCR products were stored at 4 °C until hybridization. The concentration of the stock solutions of target DNA was determined by densitometry from pictures of ethidium bromide-stained agarose gels using the φΧ174 DNA fragments as calibrators. Hybridization Assays Using Oligonucleotide Probe-Conjugated Aequorin. Opaque polystyrene wells were coated overnight at ambient temperature with 50 µL of 1.4 mg/L streptavidin diluted in phosphate-buffered saline (PBS). Prior to use, the wells were washed three times with wash solution (50 mmol/L Tris-HCl, 0.15

mol/L NaCl, 2 mmol/L EGTA, pH 7.5, and 1 mL/L Tween20). Biotinylated PCR products were diluted in blocking solution (10 g/L blocking reagent in 0.1 mol/L maleic acid, 0.15 mol/L NaCl, 2 mmol/L EGTA, and 1.5 mL/L Tween20), and then 50 µL was pipetted into the wells. The DNA fragments were allowed to bind to streptavidin for 30 min with shaking. The wells were washed three times, and then the nonbiotinylated strand was dissociated by incubating for 20 min with 50 µL of 0.2 mol/L NaOH. The wells were washed three times, and 50 µL of oligonucleotide probe-conjugated aequorin (22 nmol/L with respect to aequorin), diluted in blocking solution containing 10% DMSO, was added. Following 30 min incubation at 37 °C, the wells were washed three times and placed in the luminometer. The activity of aequorin was measured by injecting 50 µL of triggering solution (20 mmol/L Tris-HCl, 25 mmol/L CaCl2, pH 7.5) into each well and integrating the signal for 3 s. RESULTS AND DISCUSSION

The principle of affinity capture-facilitated preparation of aequorin-oligonucleotide conjugates is illustrated diagrammatically in Figure 1. The vector pHISAEQ (17) was used for expression of recombinant (His)6-aequorin in E. coli and subsequent purification of the photoprotein. Two conjugation strategies were studied in the course of the present work using an oligonucleotide probe modified with a primary amino group at the 5′ end. The first approach involves reaction of the probe with an excess (100-fold) of the homobifunctional cross-linking reagent BS3 that contains two N-hydroxysuccinimide groups. By adding an excess of BS3, the bridging of two oligonucleotides by the cross-linker was avoided and one succinimide group remained free on the derivatized oligo for subsequent reaction with primary amino groups of aequorin (-amino groups of lysine residues). Following synthesis of the conjugate, the effective removal of the unreacted oligonucleotide is crucial because it competes

Aequorin−Oligonucleotide Conjugates

Figure 2. Chromatogram of aequorin-conjugated oligonucleotide synthesized using homobifunctional linker (BS3). The conjugation reaction was performed with 5 nmol of (His)6aequorin and 5 nmol of oligonucleotide modified with BS3 (1: 100 molar ratio). The luminescence of the eluted fractions are plotted versus the fraction number.

with the aequorin-oligo conjugate for hybridization to the target DNA sequence. The hexahistidine tag of aequorin enables the affinity capture of the conjugate to Ni-NTA agarose and removal of the free oligo by washing. The conjugate is then eluted with EDTA and used directly in hybridization assays. The second approach involves chemical modification of (His)6-aequorin with SATA to introduce protected thiol groups. The amino group of the probe was converted to a maleimide group by reacting with the heterobifunctional cross-linker sulfo-SMCC. The SATA-modified aequorin was then mixed with the SMCC-oligo, and the reaction was initiated by deprotecting the sulfhydryl group with hydroxylamine. Again, the conjugate was captured on Ni-NTA agarose for effective removal of the free probe. Conjugation of (His)6-aequorin to DNA Using a Homobifunctional Cross-Linker. The conjugation reaction products were first separated by HPLC using an anion-exchange column. Elution was accomplished by applying a NaCl gradient. Five nanomoles of (His)6aequorin were conjugated to BS3-derivatized oligo at a 1:1 molar ratio. Fractions (1 mL) were collected, and the luminescence of aequorin was measured by pipetting 50 µL from a 100-fold dilution of each fraction into a microtiter well, adding 50 µL of triggering solution and integrating the light emission for 3 s. The chromatographic profile of the conjugation reaction mixture, presented as luminescence versus the fraction number, is shown in Figure 2. Unreacted (His)6-aequorin is eluted first followed by the aequorin-DNA conjugate (fractions 15-23). A small peak also appears at fractions 28-33 that corresponds to higher oligo-aequorin molar ratios. According to absorbance data (260 nm), the free oligonucleotide was eluted at fractions 25 to 31. Fractions from the first conjugate peak were pooled, supplemented with 1 g/L BSA and 200 g/L glycerol, and stored at -20 °C. Pooled fractions from the first conjugate peak were tested further as a detection reagent in hybridization assays. Biotinylated (through PCR) target DNA (263 bp) was captured on the surface of polystyrene microtiter wells coated with streptavidin. One strand was removed by NaOH treatment followed by hybridization with the

Bioconjugate Chem., Vol. 14, No. 5, 2003 1027

Figure 3. Luminescence as a function of the target DNA concentration. The hybridization assay was performed as described under Materials and Methods using aequorin-oligonucleotide conjugates prepared with a homobifunctional linker and purified: (A) by anion-exchange HPLC and (B) by affinity capture on Ni-NTA agarose and anion-exchange chromatography.

aequorin-oligonucleotide conjugate. The excess of conjugate was removed by washing and the bioluminescent reaction of bound aequorin was triggered by the addition of Ca2+. The effect of the conjugate concentration on the signal of the assay was studied in the range of 0.08 to 0.64 nmol/L at 0.2 nmol/L target DNA. The optimized conjugate concentration was found to be 0.45 nmol/L (calculated on the basis of aequorin activity). Subsequently, the luminescence was studied as a function of the concentration of target DNA, and the results are presented in Figure 3. Signal-to-background (S/B) ratios of 1.2 and 2.8 were obtained for 8 and 32 pmol/L of target DNA, respectively. The background is defined as the luminescence signal in the absence of target DNA, and it is due to the nonspecific binding of the detection reagent to the surface of the microtiter well. Subsequently, we investigated whether affinity capture of the aequorin-DNA conjugate on Ni-NTA agarose, through the hexahistidine tag, could facilitate its purification by replacing the anion-exchange HPLC step. Initial affinity capture experiments were carried out by using (His)6-aequorin. A solution containing 10 nmol of (His)6-aequorin (the amount typically used in conjugation reactions) was incubated, under gentle agitation, with 10-50 µL of Ni-NTA agarose slurry for various time intervals between 20 and 75 min. The aequorin was eluted with EDTA. These experiments showed that the capture of aequorin to agarose matrix was efficient, but the yield of the elution of bound aequorin was relatively low (40-60%). This was attributed to the nonspecific binding (adsorption) of the photoprotein directly to agarose. To decrease the adsorption of aequorin, blocking of Ni-NTA agarose (prior to the affinity capture) was investigated by applying various concentrations of bovine serum albumin. The most efficient blocking of agarose was achieved by using a 10 g/L BSA solution containing 1.5 mL/L Tween-20. Under these conditions, the recovery of bound (His)6-aequorin was about 90%. The optimized affinity capture protocol was applied to the purification of aequorin-oligo conjugates from the free oligo. The performance of aequorin-oligo conjugates purified both by affinity capture and anion-exchange HPLC, was

1028 Bioconjugate Chem., Vol. 14, No. 5, 2003

Glynou et al.

Figure 4. Effect of SATA on the activity of aequorin. A constant amount of (His)6-aequorin (0.5 nmol) was treated with various amounts of SATA for 90 min, and the luminescence was measured as described in the Experimental Section. The percent luminescence is plotted versus the molar ratio SATA:aequorin. The value 100% is defined as the signal obtained from nonderivatized (with SATA) aequorin.

Figure 5. Chromatogram of aequorin-conjugated oligonucleotide synthesized using a heterobifunctional linker. The conjugation reaction was performed with 5 nmol of (His)6-aequorin modified with SATA (1:1 molar ratio) and 1.7 nmol of oligonucleotide modified with sulfo-SMCC (1:100 molar ratio). The reaction product was purified from unreacted oligonucleotide by Ni-NTA agarose and by anion-exchange HPLC. The luminescence of the eluted fractions are plotted versus the fraction number.

tested in hybridization assays. The results are also presented in Figure 3. It is observed that the signals were four times higher with the affinity-purified conjugate compared to the one purified only by HPLC. This was attributed to the more effective removal of unreacted probe by affinity capture. As low as 2 pmol/L target DNA was detected using this conjugate with a signal-tobackground ratio of 1.8. Moreover, we found that conjugates purified by a single rapid affinity capture step offer the same detectability and analytical range as those purified both by affinity capture and HPLC. The reproducibility of the hybridization assay was tested by analyzing samples containing 3.2, 12.4, 50, and 400 pmol/L of target DNA. The CVs were 2, 9, 2, and 2%, respectively (n ) 3). Conjugation of (His)6-aequorin to DNA Using a Heterobifunctional Cross-Linker. It is known that treatment of aequorin with cross-linkers containing a maleimide group results in a rapid inactivation of the photoprotein due to derivatization of cysteine groups that appear to play an important role in the Ca2+-dependent bioluminescent reaction (16, 18). This problem was circumvented either by introducing extra thiol groups to aequorin (16) or by substituting the cysteines with serines (through site-directed mutagenesis) and simultaneous introduction of a single cysteine at a position that has no effect on the activity (19, 20). The latter approach has only been applied to immunoassay development. We have converted the amino group of the probe to a maleimide group by treating with sulfo-SMCC. Protected thiol groups were introduced to (His)6-aequorin by reacting with SATA. The effect of SATA on the activity of (His)6-aequorin was studied at molar ratios (Aeq:SATA) ranging from 1:1 to 1:20, and the results are presented in Figure 4. It is observed that at 1:1 and 1:5 Aeq:SATA ratios 100% and 85% of the activity of aequorin was preserved after derivatization. At a higher excess of SATA, aequorin is inactivated as it is concluded by the rapid drop of luminescence. The SATA-derivatized aequorin was mixed with SMCC-modified oligo and hydroxylamine was added to expose the thiol groups. Figure 5 presents the chromatographic analysis of the conjuga-

tion reaction mixture by anion-exchange HPLC. Fractions 15-23 contain the conjugate. The small conjugate peak collected in fractions 26-31 corresponds to higher oligo:aequorin molar ratios. By comparing the chromatographic data from Figures 2 and 5, it is deduced that the elution volumes of aequorin-oligo conjugates prepared by using either homobifunctional or heterobifunctional cross-linkers were identical. We assessed the performance of conjugates purified (a) by affinity capture and anion-exchange HPLC and (b) by a single affinity capture step. The effect of the concentration of the affinity purified conjugate was studied in the range of 4.4-35 nmol/L and the optimum concentration was 11 nmol/L. Then, a series of hybridization assays was performed at various concentrations of target DNA and aequorin-oligo conjugates (a and b) were employed as detection reagents. In Figure 6, the luminescence is plotted as a function of target DNA concentration. A linear response was obtained for both conjugates in the range of 2 to 2000 pmol/L target DNA. The conjugate that was purified only by affinity capture gave higher signals. However, the S/B ratios at 2 pmol/L target DNA were 3.9, for the conjugate that was purified by affinity capture and HPLC, and 2.9 for the conjugate that was only subjected to affinity capture. The slight decrease in the S/B ratio is due to the higher background signal caused by the presence of unreacted aequorin. We have also tested conjugates prepared with (His)6-aequorin derivatized with 1-, 2.5-, and 5-fold molar excess of SATA. It was found that the 1:1 molar ratio gave the highest signal and lowest background. To summarize, (His)6-aequorin-oligo conjugates prepared by affinity capture followed by HPLC and conjugates purified simply by affinity capture on the Ni-NTA agarose slurry showed similar performance. This fact greatly simplifies the preparation of oligonucleotide conjugated aequorin because it eliminates the necessity for HPLC purification. HPLC requires costly instrumentation and long time to setup, to carry out the separation and to concentrate the pooled fractions. By comparing the two conjugation chemistries studied, we found that

Aequorin−Oligonucleotide Conjugates

Figure 6. Calibration graph for the hybridization assay of amplification product from the PSMA cDNA, using aequorinoligonucleotide conjugate prepared with SATA-sulfo-SMCC chemistry and purified: (A) by Ni-NTA agarose and anionexchange chromatography and (B) by Ni-NTA agarose.

conjugates prepared with the heterobifunctional reagent protocol showed about 2 times higher S/B ratios than those prepared using the homobifunctional cross-linker. However, conjugation using the homobifunctional linker is simpler because it requires fewer reaction steps. Assessment of the performance of the conjugates showed that the detectability and analytical range are similar to those obtained previously (12) by using the ‘indirect labeling’ approach in which a digoxigenin-labeled probe hybridized to the target and the hybrids were reacted with antidigoxigenin-aequorin conjugate. The hybridization assay, however, is much faster and less costly when using the aequorin-DNA conjugate because the antibody incubation and washing steps are eliminated. The conjugate obtained from a reaction that uses 10 nmol of (His)6-aequorin is sufficient for about 5000 hybridization assays. The proposed method for preparing conjugates is general because a variety of reporter proteins can be fused to a hexahistidine tag at their N- or C-terminus without significant loss of activity, by using suitable vectors that are available commercially. ACKNOWLEDGMENT

This work was supported by a research grant from the General Secretariat of Research and Technology (GSRT) of Greece and Medicon Hellas, SA. We also acknowledge the financial support from the Secretariat of the Research Committee of the University of Athens and a Karatheodory research grant from the University of Patras. LITERATURE CITED (1) Christopoulos, T. K. (1999) Nucleic acid analysis. Anal. Chem. 71, 425R. (2) Lewis, L. C., and Daunert, S. (2000) Photoproteins as luminescent labels in binding assays. Fresenious J. Anal. Chem. 366, 760.

Bioconjugate Chem., Vol. 14, No. 5, 2003 1029 (3) Jones, K., Keenan, M., and Keenan, M. (1999) Glowing jellyfish, luminescence and a molecule called coelenterazine. Trends Biotechnol. 17, 477. (4) Shimomura, O., and Johnson, F. H. (1978) Peroxidized coelenterazine, the active group in the photoprotein aequorin. Proc. Natl. Acad. Sci. U.S.A. 75, 2611. (5) Head, J. F., Inouye, S., Teranishi, K., and Shimomura, O. (2000) The crystal structure of the photoprotein aequorin at 2.3 A resolution. Nature 405, 372. (6) Kendall, J. M., and Badminton, M. N. (1998) Aequorea Victoria bioluminescence moves into an exciting new era. Trends Biotechnol. 16, 216. (7) Inouye, S., Noguchi, M., Sakaki, Y., Takagi, Y., Miyata, T., Iwanaga, S., Miyata, T., and Tsuji, F. I. (1985) Cloning and sequencing analysis of cDNA for the luminescent protein aequorin. Proc. Natl. Acad. Sci. U.S.A. 82, 3154. (8) Prasher, D., McCann, R. O., and Cormier, M. J. (1985) Cloning and expression of the cDNA coding for aequorin, a bioluminescent calcium-binding protein. Biochem. Biophys. Res. Commun. 126, 1259. (9) Stults, N. L., Stocks, N. F., Rivera, H., Gray, J., McCann, R. O., O′Kane, D., Cummings, R. D., Cormier, M. J., and Smith, D. F. (1992) Use of recombinant biotinylated aequorin in microtiter and membrane-based assays: purification of recombinant apoaequorin from Escherichia coli. Biochemistry 31, 1433. (10) Sgoutas, D. S., Tuten, T. E., Verras, A. A., Love, A., and Barton, E. G. (1995) AquaLite bioluminescence assay of thyrotropin in serum evaluated. Clin. Chem. 41, 1637. (11) Galvan, B., and Christopoulos, T. K. (1996) Bioluminescence hybridization assays using recombinant aequorin. Application to the detection of prostate-specific antigen mRNA. Anal. Chem. 68, 3545. (12) Verhaegen, M., and Christopoulos, T. K. (1998) Quantitative polymerase chain reaction based on a dual-analyte chemiluminescence hybridization assay for target DNA and internal standard. Anal. Chem. 70, 4120. (13) Laios, E., Ioannou, P. C., and Christopoulos, T. K. (2001) Enzyme-amplified aequorin-based bioluminometric hybridization assays. Anal. Chem. 73, 689. (14) Actor, J. K. (2000) Bioluminescent quantitation and detection of gene expression during infectious disease. Comb. Chem. High Throughput Screen. 3, 273. (15) Verhaegen, M., and Christopoulos, T. K. (2002) Bacterial expression of in vivo-biotinylated aequorin for direct application to bioluminometric hybridization assays. Anal. Biochem. 306, 314. (16) Stults, N. L., Rivera, H. N., Burke-Payne, J., Ball, R. T., and Smith, D. F. (1997) Preparation of stable conjugates of recombinant aequorin with proteins and nucleic acids, in Bioluminescence and Chemiluminescence: Molecular reporting with photons (Hastings, J. W., Kricka, L. J., Stanley, P. E., Eds), p 423, John Wiley and Sons, Chichester, UK. (17) Glynou, K., Ioannou, P. C., and Christopoulos, T. K. (2003) One-step purification and refolding of recombinant photoprotein aequorin by immobilized metal-ion affinity chromatography. Protein Expr. Purif. 27, 384. (18) Erikaku, T., Zenno, S., and Inouye, S. (1991) Bioluminescent immunoassay using a monomeric Fab′-photoprotein aequorin conjugate. Biochem. Biophys. Res. Commun. 174, 1331. (19) Lewis, J. C., Cullen, L. C., and Daunert, S. (2000) Sitespecifically labeled photoprotein-thyroxin conjugates using aequorin mutants containing unique cysteine residues: applications for binding assays. Bioconjugate Chem. 11, 140. (20) Shrestha, S., Paeng, I. R., and Daunert, S. (2002) Cysteinefree mutant of aequorin as a photolabel in immunoassay development. Bioconjugate Chem. 13, 269.

BC0341021