Use of Maleimide-Thiol Coupling Chemistry for Efficient Syntheses Of

Bioconjugate Chem. 1990, 1, 71-76 71

radiopharmaceuticals. J . Nucl. Med. 28, 83. (e) Desh- pande, S. V., DeNardo, S. J., Meares, C. F., McCall, M. J., Adams, G. P., Moi, M. K., and DeNardo, G. L. (1988) 67Cu- labeled monoclonal antibody Lym-1, a potential radiophar- maceutical for cancer therapy: Labeling and biodistribution in RAJI tumored mice. J . Nucl. Med. 29, 217. (f) Moi, M. K., Meares, C. F., and DeNardo, S. J. (1988) The peptide way to macrocyclic bifunctional chelating agents: Synthesis of 2-@)nitro-benzylDOTA, and study of its yttrium (111) complex. J. Amer. Chem. SOC. 110, 6266.

(14) Desreux, J. F. (1980) Nuclear magnetic resonance spectroscopy of lanthanide complexes with a tetraacetic tetraaza macrocycle. Unusual conformation properties. Znorg. Chem. 19, 1319.

(15) Rocklage, S. M., Cacheris, W. P., Quay, S. C., Hahn, F. E., and Raymond, K. N. (1989) Manganese(I1) N,N‘-dipyridox- ylethylenediamine-N,N‘-diacetate 5,5’-Bis(phosphate). Syn- thesis and characterization of a paramagnetic chelate for magnetic resonance imaging enhancement. Znorg. Chem. 28,477.

(16) Ellman, G. L. (1958) A colorimetric method for determin- ing low concentrations of mercaptans. Arch. Biochem. Bio- piys . 74, 443.

(17) Magerstadt, M., Gansow, 0. A., Brechbiel, M. W., Colcher, D., Baltzer, L., Knop, R. H., Girton, M. E., and Naegele, M. (1986) Gd(D0TA): An alternative to Gd(DTPA) as a TI,* relaxation agent for NMR imaging or spectroscopy. Magn. Reson. Med. 3, 808.

(18) Tweedle, M. F., Eaton, S. M., Eckelman, W. C., Gaughan, G. T., Hagan, J. J., Wedeking, P. W., and Yost, F. J. (1988) Comparative chemical structure and pharmacokinetics of MRI contrast agents. Invest. Radiol. 23, S236.

(19) Sherry, A. D., Brown, R. D., Geraldes, C. F. G. C., Koenig, S. H., Kuan, K.-T., and Spiller, M. (1989) Synthesis and characterization of the gadolinium(3+) complex of DOTA-pro-

pylamide: A model DOTA-protein conjugate. Znorg. Chem. 28, 620.

(20) Lauffer, R. B. (1987) Paramagnetic metal complexes as water proton relaxation agents for NMR imaging: Theory and design. Chem. Rev. 87, 901.

(21) Cassidy, R. M., Elchuk, S., and Dasgupta, P. K. (1987) Performance of annular membrane and screen-tee reactors for postcolumn-reaction detection of metal ions separated by liquid chromatography. Anal. Chem. 59, 85.

(22) Hughes, W. L. (1947) An albumin fraction isolated from human plasma as a crystalline mercuric salt. J. Am. Chem. SOC. 69, 1836.

(23) May, S. W., Lee, L. G., Katopodis, A. G., Kuo, J.-Y., Wimalasena, K., and Thowsen, J. R. (1984) Rubredoxin from Pseudomonas oleovorans: Effects of chemical modification and metal substitution. Biochemistry 23, 2187.

(24) Jue, R., Lambert, J. M., Pierce, L. R., and Traut, R. R. (1978) Addition of sulfhydryl groups to Escheria coli ribo- somes by protein modification with 2-iminothiolane (methyl- 4-mercapto-butyrimidate). Biochemistry 17(25), 5399.

(25) Yoshitake, S., Imagawa, M., Ishikawa, E., Niitsu, Y., Urush- izaki, I., Nishiura, M., Kanazawa, R., Kurosaki, H., Tachibana, S., Nakazawa, N., and Ogawa, H. (1982) Mild and efficient conjugation of rabbit Fab’ and horseradish peroxidase using a maleimide compound and its use for enzyme immunoassay. J. Biochem. 92, 1413.

(26) Barbeau, A. (1984) Manganese and extrapyramidal disorders. Neurotoxicology 5 (l), 13.

Registry No. IBCF, 543-27-1; DOTA, 60239-18-1; TMG, 80- 70-6; SMCC, 85060-00-0; DTPA, 67-43-6; DTPA mixed anhydride, 124098-80-2; DOTA mixed anhydride, 124098-82-4; Et,N, 121-44-8; poly(L-lysine), 25104-18-1; poly(t1ysine) SRU, 38000-06-5.

Use of Maleimide-Thiol Coupling Chemistry for Efficient Syntheses of Oligonucleotide-Enzyme Conjugate Hybridization Probes

Soumitra S. Ghosh,’ Philip M. Kao, Ann W. McCue, and H u g h L. Chappelle

SISKA Diagnostics, Inc., and Salk Institute Biotechnology/Industrial Associates, Inc., La Jolla, California 92037. Received August 10, 1989

Two general methods which exploit t he reactivity of sulfhydryl groups toward maleimides are described for t he synthesis of oligonucleotide-enzyme conjugates for use as nonradioisotopic hybridization probes. I n the first approach, 6-maleimidohexanoic acid succinimido ester was used t o couple 5’-thiolated oligonucleotide t o calf intestine alkaline phosphatase to provide a 1:l conjugate in 80445% yield. T h e second strategy employed N,Nf-1,2-phenylenedimaleimide t o cross-link thiolated horseradish peroxidase or @-galactosidase with a 5’-thiolated oligonucleotide in 58% and 65% yields, respectively. T h e oligonucleotide-alkaline phosphatase conjugate was able t o detect 6 amol of target DNA in 4 h, while the horseradish peroxidase conjugate was found t o be 40-fold lower in its sensitivity of detection by using dye precipitation assays.

In recent years, considerable interest has focused on exploiting the specificity of nucleic acid hybridization reactions for t he diagnosis of genetic disorders and infec- tious diseases. T h e sensitivity of t h e technology has ben-

efited in great measure from the use of in vitro nucleic acid amplification procedures (1, 2) before detection by hybridization. Such target-directed amplification strategies are particularly essential for t he detection of patho- gens such as the HIV-1 virus, which are often present in immeasurably low titers in blood samples.

Traditionally, radioisotopes, such as 32P, have been utilized as detection labels for nucleic acid probes in nucleic

* To whom correspondence should be addressed at SIBIA, P.O. Box 85200, San Diego, CA 92138-9216.

1043-1802/90/2901-0071$02.50/0 0 1990 American Chemical Society

72 Bioconjugate Chem., Vol. 1, No. 1, 1990

acid hybridization reactions. However, concerns about safety, short lifetimes, and cost have prompted the inves- tigation of nonisotopic detection alternatives (3). Recently, we ( 4 ) and others (5-7) have demonstrated the efficacy of using enzymes as reporter groups in oligonucleotide probes. The signal amplification afforded by the enzyme component in these covalently linked conjugates results in sensitivities of detection equivalent to those of ,'P-labeled probes.

We have been interested in using the unique chemistry of the sulfhydryl group to develop a general method- ology for covalently coupling signal-generating enzymes to oligonucleotides. Prior to our work, a ligation method was described for attaching oligonucleotides to nucleic acids, proteins, and peptides (8) based on the propensity of sulfhydryl groups to form disulfides. A drawback of this approach is the susceptibility of the disulfide bond to cleavage by thiols. This problem can be circum- vented by using a stable thioether linkage, as exempli- fied by the synthesis of oligonucleotide-alkaline phosphatase conjugates using bromoacetyl-sulfhydryl coupling chemistry (6). This report presents alternate and extremely efficient conjugation approaches for the preparation of oligonucleotide-enzyme hybridization probes which utilize the high and rather specific reactivity of sulfhydryl groups for maleimides. The oligonucleotide- enzyme conjugates are capable of detecting complementary DNA sequences with high sensitivities with dye precipitation assays.

Ghosh et al.

EXPERIMENTAL PROCEDURES

T4 polynucleotide kinase (EC 2.7.1.78) was obtained from New England Biolabs. Calf intestine alkaline phosphatase (EC 3.1.3.1., enzyme immunoassay grade) and P-galactosidase (EC 3.2.1.23., enzyme immunoassay grade) were purchased from Boehringer Mannheim, and [y- 32P]ATP was from ICN. Bovine serum albumin fraction V (BSA),' horseradish peroxidase (Type VII, EC 1.11.1.7), sodium dodecyl sulfate (SDS), and polyvinylpyr- rolidone (PVP) were from Sigma. Bio-Gel P-100 fine and acrylamide were obtained from Bio-Rad, and DEAE- cellulose (DE-52) came from Whatman. N-(2-hydroxy- ethy1)piperazine-N'-ethanesulfonic acid (HEPES), 3-N- morpholinopropanesulfonic acid (MOPS), and 1-ethyl-3- [3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) were purchased from Calbiochem, and Sephadex G-75 was from Pharmacia. 2-Iminothiolane hydrochloride was obtained from Pierce and all other chemicals were purchased from Aldrich. Collodion bags (MW cutoff 25 000) and nitrocellulose filters were obtained from Schleicher and Schuell. Centricon-30 microconcentra- tors and Centriprep-30 concentrators (MW cutoff 30 000) were purchased from Amicon.

Plasmid pARV7A/2, constructed by inserting a cDNA copy of the HIV genome into the EcoRI site of pUC19 pBR322 (9), was obtained from D. Richman (University

Abbreviations used are as follows: BSA, bovine serum albumin fraction V; SDS, sodium dodecyl sulfate; PW, polyvinylpyr- rolidone; DE-52, DEAE-cellulose; HEPES, N-(2-hydroxyeth- y1)piperazine-N'-ethanesulfonic acid; MOPS, 3-N-morpholinopropanesulfonic acid; EDC, l-ethyl-3-[3-(dimethylamino)pro- pyllcarbodiimide hydrochloride; DAB-NiCl,, 3,3'-diaminobenzidine hydrochloride-nickel chloride; NBT, nitroblue tetrazolium; BCIP, 5-bromo-4-chloro-3-indolyl phosphate; DMF, N,N-dimethylformamide; HBsAg, hepatitis B surface antigen; EDTA, ethylenediaminetetraacetic acid; MHS, 6-maleimidohexanoic acid succinimido ester; DTT, dithiothreitol.

of California, San Diego). Oligonucleotide HIV-300 (5'- TGGTCCTGTTCCATTGAACGTCTTATTATT-3') is complementary to the region coding for the HIV sequence in plasmid pARV7A/2. Plasmid pTBO61B was constructed by inserting a 0.9-kb EcoRI fragment containing the sequence of the hepatitis B surface antigen (HBsAg) into vector pBR322 (IO). Oligonucleotide HBsAg-133 (5'- TGGCTCAGTTTACTAGTGCCATTTGTTCAG-3') is complementary to the region coding for the hepatitis B surface antigen in plasmid pTBO61B. The oligonucleotides were synthesized on an Applied Biosystems 380A automated DNA synthesizer and were purified according to the reverse-phase chromatographic conditions described by Ghosh et al. (11). The purified oligonucleotides migrated as single bands on a 20% polyacrylamide gel. Enzymatic phosphorylation of the oligonucleotides at the 5'-terminus using T4 polynucleotide kinase and cold ATP or [T-~~P]ATP was performed according to the protocol of Maniatis et al. (12).

Preparation of 5'-Cystaminyl Oligonucleotide Derivative. Reaction tubes were silanized with a freshly prepared 5 % solution of dichlorodimethylsilane in chlo- roform to prevent adhesion of the nucleic acids to the walls of the tubes. The 5'-cystaminyl derivative was prepared by the two-step procedure described by Chu et al. (13). Alternately, 5'-phosphorylated oligonucleotide (16 nmol) was treated with 3 mL of 0.1 M imidazole, 0.15 M EDC, 0.25 M cystamine, pH 6.00, at 23 "C for 16 h. The crude cystaminyl oligonucleotide derivative was isolated as a pellet by lithium chloride precipitation from an aque- ous ethanol solution and used in the next step without further purification.

Reduction of 5'-Cystaminyl Oligonucleotide De- rivative. Reduction of the disulfide linkage of the 5'- cystaminyl oligonucleotide derivative (- 16 nmol) was effected by treatment with 2.5 mL of a degassed solution of 0.1 M DTT, 0.2 M HEPES, 1 mM EDTA, pH 7.7, for 1 h at 23 "C. The 5'-(mercaptoethy1)phosphoramidate oligonucleotide derivative was isolated from excess reagent with three consecutive lithium chloride/ethanol precipitations and used immediately in the conjugation reaction or in its reaction with N,N'-1,2-phenylenedimaleimide. It is essential to use degassed buffer in the subsequent step and keep the solution of the oligonucleotide derivative under an atmosphere of argon to prevent air oxidation of the terminal thiol group.

Preparation of Maleimide-Derivatized Oligonu- cleotide. 5'-(Mercaptoethy1)phosphoramidate oligonucleotide derivative (8 nmol) was dissolved in 2 mL of 0.2 M HEPES and 1 mM EDTA, pH 7.7, and then 0.016 mL of a 25 mM solution of N,N'-1,2-phenylenedimaleimide in CH,CN was added. The reaction mixture was kept at 23 "C for 30 min and then ethanol precipitated two times. The maleimide-derivatized oligonucleotide was used immediately for the subsequent coupling step.

Derivatization of Calf Intestine Alkaline Phos- phatase with 6-Maleimidohexanoic Acid Succinim- ido Ester. 6-Maleimidohexanoic acid succinimido ester (MHS) was prepared according to a literature procedure (14). Calf intestine alkaline phosphatase (11.3 mg, 81 nmol) in 1.13 mL of 3 M NaCl, 0.1 mM MgCl,, 0.1 mM ZnCl,, 30 mM triethanolamine, pH 7.6, was dialyzed against 0.1 M NaHCO,, 3 M NaCI, 0.02% NaN, at pH 8.5 with a collodion bag for a period of 3 h at 4 "C. A 50 molar excess of MHS (0.125 mL) as a 32 mM solution in CH,CN (10% final concentration of CH,CN in the reaction) was then added, and the reaction was allowed to proceed at 23 "C for 30 min. Excess reagent was then

Synthesis of Sensitive Oligonucleotide-Enzyme Probes

removed by dialysis against argon-degassed 50 mM MOPS, 0.1 M NaCl, pH 7.5, to provide a 1.60-mL solution of maleimide-derivatized alkaline phosphatase.

Preparation of Oligonucleotide-Alkaline Phos- phatase Conjugate. A 1.60-mL aliquot of a 50 pM solution of maleimide-derivatized calf intestine alkaline phosphatase in 50 mM MOPS, 0.1 M NaC1, pH 7.5, was added to 16 nmol of a 5'-(mercaptoethy1)phosphoramidate oligonucleotide derivative, and the conjugation reaction was allowed to proceed at 23 "C for 16 h. Gel filtration in a Bio-Rad P-100 column (1.5 X 75 cm) at 4 "C using 0.05 M Tris, pH 8.5, as an eluant separated unreacted oligonucleotide from the enzyme-oligonucleotide conjugate and excess enzyme. The enzyme fractions were pooled and applied to a DEAE-cellulose column (1 X 7.4 cm) equil- ibrated with 0.05 M Tris, pH 8.5, a t 23 "C. The column was washed with 0.1 M Tris, pH 8.5 (15 mL), and a 40- mL salt gradient of 0-0.2 M NaCl in 0.1 M Tris, pH 8.5, followed by 40 mL of 0.2 M NaCl, 0.1 M Tris, pH 8.5, to elute free alkaline phosphatase. Pure oligonucleotide- alkaline phosphatase conjugate was obtained by elution with 0.5 M NaCl and 0.1 M Tris, pH 8.5. The conjugate fractions were combined, simultaneously dialyzed and con- centrated with Centriprep-30 concentrators (Amicon), and stored in 0.1 M Tris, 0.1 M NaCl, pH 8.5, at 4 "C.

Thiolation of Horseradish Peroxidase. The thiolation of horseradish peroxidase was carried out by using a modification of a literature-described procedure (15). A 0.05-mL aliquot of a 0.12 M solution of 2-iminothiolane hydrochloride (6 wmol) in 25 mM sodium borate, pH 9.00, was added to 0.28 mL of a 0.21 mM solution of horseradish peroxidase (60 nmol) in the same buffer. The reaction mixture was kept at 23 OC for 1 h and then excess reagent was removed by dialysis against 25 mM sodium borate, pH 9.00, at 4 "C.

Preparation of Oligonucleotide-Horseradish Per- oxidase Conjugate. To -8 nmol of maleimide-derivatized oligonucleotide in 2 mL of degassed 0.2 M HEPES, 1 mM EDTA, pH 7.7, was added 0.214 mL of a 0.19 mM solution of thiolated horseradish peroxidase (40 nmol) in 25 mM sodium borate, pH 9-00. The reaction mixture was kept under an argon atmosphere, and the conjugation was allowed to proceed for 16 h at 23 "C. A Sephadex G-75 column (1.5 X 44 cm) was used to sepa- rate unreacted oligonucleotides from the mixture of conjugates and excess thiolated enzyme. The conjugates were then purified by DEAE ion-exchange chromatography with the conditions described in the previous section.

Preparation of Oligonucleotide-&Galactosidase Conjugate. The conjugation of maleimide-derivatized oligonucleotides to @-galactosidase was performed under conditions identical with those for thiolated horseradish peroxidase. The purification of the conjugate was carried out following the protocol for the isolation of the oligonucleotide-alkaline phosphatase.

Alkaline Phosphatase Assay. The enzymatic activity of alkaline phosphatase and the oligonucleotide-alkaline phosphatase conjugates was assayed at 23 "C by following the hydrolysis of 0.1 mM p-nitrophenyl phosphate in 0.1 M Tris, 0.1 M NaCl, 0.01 M MgCl,, pH 9.5, a t 410 nm.

Horseradish Peroxidase Assay. The enzymatic activity of horseradish peroxidase and ita conjugate was assayed with a 3,3'-diaminobenzidine hydrochloride-nickel chloride (DAB-NiC1,) solution (0.5 mg/mL DAB in 0.05 M Tris, pH 7.6, containing 0.04% NiC1,).

&Galactosidase Assay. The enzymatic activity of 0- galactosidase and its conjugate was assayed a t 37 "C by

Bioconjugate Chem., Vol. 1, No. 1, 1990 73

following the release of 4-nitrophenolate at 410 nm with a solution of 4-nitrophenyl-/3-~-galactoside (1.57 mg/ mL) in 50 mM potassium phosphate, 1 mM MgCl,, 0.1 M 2-mercaptoethanol, pH 7.8.

Sensitivity of Conjugates for Nitrocellulose-Im- mobilized Target DNA: (i) Oligonucleotide-Alka- line Phosphatase Conjugate, Varying amounts of plasmid pARV7A/2, 1 pg of Escherichia coli DNA, 100 ng of pBR322 and 10 pg of human DNA were denatured under alkaline conditions at 65 OC (0.2 M NaOH, 15 min) and then neutralized with an equal volume of 2 M ammo- nium acetate. The DNA samples were immobilized onto a nitrocellulose membrane using a Schleicher and Schuell Minifold I1 slot-blot apparatus, and the nucleic acids were fixed to the nitrocellulose by UV radiation (16). The filter was prehybridized in 5X SSC, 0.5% BSA, 0.5% PVP, and 0.1% SDS for 10 min at 50 "C, followed by hybridization in the same buffer with 2 pg/mL of oligonucleotide-alkaline phosphatase conjugate for 1 h at 50 "C. After three washes with l x SSC containing 0.1% SDS at 23 "C and a stringency wash in the same buffer a t 50 OC, the filter was rinsed three times with developing buffer (0.1 M Tris, 0.1 M NaC1, 0.01 M MgCl,, pH 9.5). Color development was allowed to proceed for 4 h with 0.33 mg/mL nitroblue tetrazolium (NBT) and 0.16 mg/mL 5-bromo-4-chloro-3-indolyl phosphate (BCIP) in developing buffer containing 0.33% v/v DMF.

(ii) Oligonucleotide-Horseradish Peroxidase Con- jugate. The prehybridization and hybridization steps were identical with the protocol described above. After the stringency wash at 50 "C, the filter was washed with 0.05 M Tris, pH 7.6. The detection assay was then carried out with DAB-NiC1, solution.

RESULTS

Preparation of Derivatized Oligonucleotides. A number of methods (13,17-19) have been reported which describe the introduction of sulfhydryl groups at the ter- mini of synthetic oligonucleotides. The strategy of Chu et al. (13) was used for modification of our oligonucleotide probes, since it conveniently allows 32P labeling of the 5'-end of the nucleic acid, thereby enabling us to mon- itor the efficiencies of the conjugation reactions. In addition, the method has the advantage of permitting the func- tionalization of any unprotected oligonucleotide or RNA sequence which can be made available by chemical or enzymatic syntheses.

Thus, the 5'-cystaminyl oligonucleotide derivatives were obtained through conversion of the 5'-terminal phosphates to the activated phosphorimidazolides, followed by nucleophilic displacement of the leaving group with cystamine. The same modification was achieved in a single step with cystamine and EDC in imidazole buffer. Product analysis by 20 % polyacrylamide gel electrophore- sis indicated a 6.570% yield for both procedures (data not shown). Reduction of the disulfide linkage with DTT resulted in quantitative formation of the 5'-(mercapto- ethy1)phosphoramidate oligonucleotide derivatives.

The thiolated oligonucleotides can be used directly in conjugation reactions with maleimide-modified enzymes (Figure 1). Alternately, the sulfhydryl groups can be functionalized with the bifunctional linker N,N'-1,2-phenylenedimaleimide to provide maleimide-derivatized oligonucleotides, thereby allowing the converse conjugation reaction with thiolated enzymes or enzymes possessing free cysteines (Figure 2). The reaction of 5'-(mercapto- ethy1)phosphoramidate oligonucleotide derivatives with N,"-1,2-phenylenedimaleimide was rapid and was essen-

74 Bioconjugate Chem., Vol. 1, No. 1, 1990

0 5 -

0 4 -

2 P 0 3 -

0 2 -

: s

0 1 -

0 0 -

Ghosh et al.

s 15001

0

Figure 1. Conjugation of 5’-thiolated oligonucleotides with calf intestine alkaline phosphatase using 6-maleimidohexanoic acid succinimido ester.

Figure 2. Cross-linking of oligonucleotides to enzyme using N,N’-1,2-phenylenedimaleimide.

tially complete in 30 min as determined by polyacrylamide gel analysis (data not shown).

Modification of Reporter Enzymes. Maleimide groups were introduced in calf intestine alkaline phosphatase by utilizing a 50-fold excess of the heterobifunctional linker 6-maleimidohexanoic acid succinimido ester (MHS) (13). T h e extent of the modification was followed by treatment of the derivatized enzyme with excess DTT, followed by titration of the sulfhydryl groups with 5,5’-dithiobis(2-nitrobenzoic acid) (20). An average of 6.2 maleimide residues per enzyme molecule was estimated by this procedure.

Thiolation of horseradish peroxidase with 2-iminothiolane (15) was performed by using a modification of a literature procedure. The modified enzyme was found t o possess an average of 1.9 sulfhydryl groups per mole of protein.

Synthesis of Oligonucleotide-Alkaline Phos- phatase Conjugate. It is not essential to purify the 5’- (mercaptoethy1)phosphoramidate oligonucleotide derivative prior to the conjugation reaction, since unreacted phosphorylated oligonucleotides are inert toward maleimides. Typically, a &fold molar excess of maleimide- modified calf intestine alkaline phosphatase was reacted with 5’-(mercaptoethy1)phosphoramidate oligonucleotide derivative (Figure 1). Excess enzyme and conjugate were separated from the unreacted oligonucleotide with gel filtration (Figure 3A). The efficiency of the conjugation can be followed by 32P labeling of the 5’-phos- phorus of the oligonucleotide derivative. Since crude, thiolated oligonucleotides are used in the reaction, alkaline phosphatase hydrolysis of residual 5’-kinased oligonucleotides occurs in the conjugation step. Thus, a late- eluting, 32P-labeled, inorganic phosphate peak is also observed in the cpm elution profile (not shown in Fig- ure 3A). This allowed an independent determination of the efficiency of 5’-(mercaptoethy1)phosphoramidate oli-

407 60003

Fraction Number

051 3000,

B. 2500

2000 4 1000

500

0 0 10 20 30 40 50 60 70 80

Fraclion Number

1

0 5

0 4

03 z 9

0 2 =

0 1

00

Figure 3. Purification of oligonucleotide-alkaline phosphatase conjugates: (A) Gel filtration chromatography of a conjugation reaction mixture on a Bio-Rad P-100 column [1.5 X 75 cm, 0.88 mL/reaction; (0) cpm; (0 ) absorbance], (B) ion- exchange chromatography of pooled reactions from the first peak in A on a DEAE-cellulose column (1 X 7.4 cm, 1.1 mL/ reaction).

gonucleotide formation (- 70%), which compares favor- ably with the results from the polyacrylamide gel elec- trophoresis discussed earlier. In addition, the distribu- tion of radioactivity in t h e oligonucleotide-enzyme

Synthesis of Sensitive Oligonucleotide-Enzyme Probes Bioconlugate Chem., Vol. 1, No. 1, 1990 75

A Target Target

I n

loop

1P

II

loq sq

lr 322

i. - - coli

,Br 322

4uman DNA

Figure 4. Detection of complementary target plasmid pARV7A/ 2 on nitrocellulose membranes: (A) Dilutions of the target plasmid and controls were immobilized on the membrane and hybrid- ized with an HIV-300-alkaline phosphatase conjugate. Target DNA was visualized colorimetrically by using a dye precipitation assay. (B) the sensitivity of detection of target DNA using 32P-labeled HIV-300 (4.4 X lo6 cpm/nmol).

conjugate and unreacted 5’-(mercaptoethy1)phosphoramidate oligonucleotide peaks indicated an efficiency of 85% for the coupling reaction.

The appropriate protein fractions from the gel filtration step were subjected to DEAE-cellulose ion- exchange chromatography for the final purification of oligonucleotide-alkaline phosphatase conjugate. A combination of gradient and isocratic salt elutions successfully resolved the conjugate from two enzyme species, which presumably differ in their degree of modification with maleimide residues (see absorbance profile in Figure 3B). The conjugate was determined to have a composition of a 1:l molar ratio of oligonucleotide and alkaline phosphatase by using the spectroscopic analysis of Li et al. (6). The conjugate was assayed colorimetrically with p - nitrophenyl phosphate and was found to retain 80445% of the unmodified enzyme’s activity.

Synthesis of Oligonucleotide-Horseradish Perox- idase Conjugate. A 5-fold excess of thiolated horseradish peroxidase was reacted with crude, 32P-labeled, maleimide-derivatized oligonucleotide, and the conjugate was isolated by a combination of Sephadex G-75 gel filtration and DEAE-cellulose ion-exchange chromatography. Based on a 60% yield for the formation of the maleimide-derivatized oligonucleotide, the efficiency of the conjugation reaction was estimated to be 58% from the cpm elution profile of the gel filtration step. A DAB-NiCl, assay of the conjugate indicated a 70-75% retention of the original enzymatic activity.

Synthesis of Oligonucleotide-&Galactosidase Con jugate. The coupling of @-galactosidase to maleimide-derivatized oligonucleotide was identical with the procedure described for horseradish peroxidase. The conjugate was isolated in 65% yield and was 75% as active as the unmodified enzyme.

Sensitivities of Conjugates in Detecting Comple- mentary DNA Sequences. Figure 4A shows the sensitivity of the HIV-300-alkaline phosphatase conjugate for plasmid pARV7A/ 2 immobilized on a nitrocellulose membrane by using a dye precipitation assay. The conjugate detected 100 pg (12 amol) of target DNA in 1 h and was capable of detecting 50 pg (6 amol) with 4 h of color development. No cross-hybridization was observed with plasmid pBR322, E. coli DNA, or human genomic DNA, and filter background was completely absent. With use of the same hybridization conditions, 32P-labeled HIV-300 detected 100 pg (12 amol) of target DNA after 18 h of autoradiography (Figure 4B).

Horseradish peroxidase was conjugated to oligonucleotide HBsAg-133 and was used as a probe for plasmid pTBO61B. The level of sensitivity was found to be 1 ng

(300 amol) with DAB-NiC1, for color development and was 40-fold higher than the limit of detection for the corresponding alkaline phosphatase conjugate.

We were unsuccessful in our attempts to use the HBsAg- 133-@-galactosidase conjugate for the detection of nitrocellulose-immobilized target DNA. The conjugate exhib- ited a high nonspecific background for the membrane in an assay with 5-bromo-4-chloro-3-indolyl-/3-~-galactopy- ranoside and nitroblue tetrazolium.

DISCUSS ION An important consideration in our choice of the con-

jugation chemistry was the ability to manipulate any oligonucleotide or RNA sequence, synthesized enzymati- cally or by solid phase procedure, for subsequent covalent attachment to the reporter enzymes. The procedure of Chu et al. (13) for the introduction of reactive thiol functionalities a t the 5’-terminal phosphates of unprotected oligonucleotides was therefore well-suited for our purpose. In this respect, our previous paper (4) and this report differ from the other approaches (5-3, which require the use of linker-modified nucleotide analogues to replace one of the standard bases in the automated synthesis of the oligonucleotides. The linkers are functionalized with terminal amine groups, which provide a chemical handle for the conjugation chemistry.

The reaction of maleimides with sulfhydryl groups is fairly rapid compared to their reaction with amino and hydroxyl groups (21). We have exploited this reactivity to develop highly efficient methods for the conjugation of reporter enzymes to oligonucleotides. In the first strategy, calf intestine alkaline phosphatase was functionalized with maleimide groups and the heterobifunctional linker MHS and subsequently reacted with a 5‘- (mercaptoethy1)phosphoramidate oligonucleotide derivative. Our choice of MHS was influenced by the pres- ence of a suitable alkyl spacer in the reagent, thereby ensuring minimal interference of the reporter moiety in the hybridization reaction of the oligonucleotide- enzyme conjugate.

The second approach was designed for thiolated enzymes and enzymes with free cysteines and utilized N,W-1,2- phenylenedimaleimide to effect the coupling with a 5’- (mercaptoethy1)phosphoramidate oligonucleotide derivative. Thiolated horseradish peroxidase and @- galactosidase were utilized in these conjugation reactions. Lower efficiencies of conjugation were obtained with this cross-linker when compared to the MHS-based procedure.

The conjugates retain greater than 70% of their original enzymatic activity and are stable indefinitely when stored a t 4 “C. The oligonucleotide-alkaline phosphatase conjugate was extremely sensitive in nitrocellulose-based hybridization reactions, while the oligonucleotide-horseradish peroxidase conjugate was 40-fold less sensitive than its corresponding alkaline phosphatase conjugate. This observation is in accordance with our previous results using hydrazone-linked oligonucleotide-alkaline phosphatase and oligonucleotidehorseradish peroxidase conjugates (4).

The high sensitivities afforded by chemiluminescence have recently been exploited with acridinium ester labeled DNA probes (22), which were shown to detect target nucleic acid sequences in the 10-17-10-18 mol range. In contrast to the single chemiluminescent molecule per oligonucleotide probe in this system, the efficacy of oligonucleotide- enzyme conjugates derives from the signal amplification by the reporter group. Hence, even higher sensitivities of detection should be attainable with chemilumines-

76 Bioconjugste Chem., Vol. 1, No. 1, 1990

cent substrates. Indeed, oligonucleotide-alkaline phosphatase conjugates have been demonstrated t o detect 7 X mol of target DNA when used in conjunction with the dioxetane-based chemiluminescent substrate AMPPD ( 2 3 ) . Alternatively, the recently described, dual- enzyme, cascade amplification system (24) can be utilized as a colorimetric assay for the conjugates. We are currently focusing on the application of these sensitive methods with our alkaline phosphatase conjugates to the detection of target nucleic acids using a sandwich detection format.

ACKNOWLEDGMENT

We are indebted to Drs. Thomas Gingeras, Ulrich Merten, and Eoin Fahy for critical reading of the manuscript and to Linda Blonski, Claire Lynch, and Kris Blum- eyer for technical assistance. We are grateful to Janice Doty for preparation of the manuscript.

Ghosh et al.

LITERATURE CITED

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Registry No. Oligonucleotide HIV-300, 124041-88-9; oligonucleotide HBsAg-133, 124041-87-8; oligonucleotide HIV-300, 5’-cystaminyl derivative, 124041-92-5; oligonucleotide HBsAg- 133,5’-cystaminyl derivative, 124041-91-4; oligonucleotide HIV- 300,5’-(mercaptoethy1)phosphoramidate derivative, 124041-90- 3; oligonucleotide HBsAg-l33,5’-(mercaptoethyl)phosphoramidate derivative, 124041-89-0; oligonucleotide HIV-300, 5’- (mercaptoethy1)phosphoramidate derivative reacted with N,N’- 1,2-~henylenedimaleimide, 124041-94-7; oligonucleotide HBsAg-133, 5’-(mercaptoethy1)phosphoramidate derivative reacted with N,N’-1,2-phenylenedimaleimide, 124041-93-6; cystamine, 51-85-4; N,N’-phenylenedimaleimide, 13118-04-2.

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Use of Maleimide-Thiol Coupling Chemistry for Efficient Syntheses Of