Preparation of oligodeoxynucleotide-alkaline phosphatase conjugates and their use hybridization probes

Volume 14 Number 15 1986 Nucleic Acids Research Preparation of oligodeoxynucleotide-alkaline phosphatase conjugates and their use as hybridization probes Edward Jablonski, Ellen W.Moomaw, Richard H.Tullis and Jerry L.Ruth Molecular Biosystems, Inc., 11180-A Roselle Street, San Diego, CA 92121, USA Received 23 April 1986; Accepted 10 July 1986 INTRODUCTION The most common method for the detection of nucleic acid hybrids is autoradiography of probes radiolabeled with P. This method can reproducibily visualize 0.5-5 pg of target DNA in an overnight exposure (1). Recently, nonradiometric detection methods for cloned, doublestranded nucleic acids have been reported. These are predominantly "indirect" methods which require the detection of biotinylated (2-7) or hapten-labeled (8-10) probes by protein complexes. Although other methods have been reported (11-14), the most successful approach uses complexes of alkaline phosphatase, which catalyze dye precipitation (4), to detect biotinylated nucleic acids. Sensitivities have generally been 0.5-5 pg target DNA. Direct, nonradioactive detection systems have seen little use in DNA hybridization studies. One group reported coupling of microperoxidase to DNA as a label (15). Renz and Kurz (16) have described a direct detection system based on enzymes covalently linked to long DNA. An enzyme complex © IR L Press Limited, Oxford, England. 6115 ABSTRACT Short synthetic oligonucleotides have been covalently cross-linked to alkaline phosphatase using the homobifunctional reagent disuccinimidyl suberate. The oligomers, twenty-one to twenty-six bases in length, are complementary to unique sequences found in herpes simplex virus, hepatitis B virus, Campylobacter jejuni and enterotoxigenic Escherichia coli. Each oligomer contains a single modified base with a 12-atom"linker arm" terminating in a reactive primary amine. Cross-linking through this amine results in oligomer-enzyme conjugates composed of one oligomer per enzyme molecule that have full alkaline phosphatase activity and can hybridize to target DNA fixed to nitrocellulose within 15 minutes. The hybrids are detected directly with a dye precipitation assay at a sensitivity of 10 6 molecules (2 x l(T 18 mol) of target DNA in 4 hours development time. The enzyme has no apparent effect on selectivity or kinetics of oligonucleotide hybridization and the conjugates can be hybridized and melted off in a conventional manner. Nucleic Acids Research was first prepared by attaching peroxidase or alkaline phosphatase to a polyethyleneimine core followed by coupling the residual free amines to DNA via glutaraldehyde. ratio (about The resulting complex had a large protein to DNA mass 30) and a detection sensitivity similar biotinylated probes. have to nick-translated Nonradiometric detection of oligonucleotide probes been limited to enzymatic end-labeling with biotin (17) or chemical end-labeling with either biotin (18,19) or fluorescent moieties (20,21). dodecamer containing an internal fluorescent base has also (22). Ruth (23) has reported a method for been chemically A described incorporating modified bases with functionalized " linker arms" into synthetic oligomers has described the synthesis of biotinylated and fluorescent oligomers. This approach is more general and offers control over location and number of labeling sites. We have used this latter method to prepare several complexes composed of alkaline phosphatase and active covalent synthetic oligonucleotides. The usefulness of these conjugates in the detection of complementary target DNA is demonstrated by hybridization and visualization of target DNA fixed to membranes in both dot-blot and Southern blot formats. of The sensitivity colorimetric detection of these conjugates ( 2 attomol) is better optimum overnight exposure with times than P-labeled oligomers (5 attomol) and 5-100 more sensitive than either end-labeled [400-2000 attomol (18,19)] or internally-labeled [10 attomol (23)] biotinylated oligomers. processing steps and time are significantly reduced. In addition, Total hybridization and assay times are 2-5 hours. MATERIALS AND METHODS Calf intestine alkaline phosphatase (E.C. 3.1.3.1), enzyme immunoassay grade, was obtained from Boehringer Mannheim. Disuccinimidyl suberate (DSS) was from the Pierce Chemical Company. bromo-4-chloro-3-indoyl Nitro blue tetrazolium (NBT) and 5- phosphate (BCIP) were gifts from American Research Products Company. Bovine serum albumin fraction V (BSA), was purchased from Miles Laboratories. and Polyvlnylpyrrolidone (PVP), 4-methylumbelliferyl phosphate came from Acrylamide, catalysts, sodium dodecyl sulfate obtained from Bio-Rad Laboratories. Chemicals with a material 6116 and phosphate Company. Chemical (SDS) and Bio-Gel P-100 were Sephadex G-25 was from Pharmacia Fine DEAE cellulose (DE-52) came from Whatman. molecular were p-nitrophenyl Sigma weight exclusion of 25,000 and Collodion nitrocellulose purchased from Schleicher and Schuell. Gene Screen bags filter Plus and Nucleic Acids Research nylon membranes radiolabeled Oligomers were with32P-ATP from ICN using T4 polynucleotide kinase from were obtained from New England Nuclear. New England Biolabs. BamHI was supplied by Bethesda Research Laboratories. Preparation of linker arm oligonucleotides Protected linker arm nucleoside 3'-phosphoramidite [5'-0- dimethoxytrityl-5-[N(7-trifluoroacetylaminoheptyl)-3-acrylamido]-2'-deoxyuridine 3'-0-(methyl N,N-diisopropyl) phosphoramidite] was prepared by the method of Ruth (24). The linker arm monomer was incorporated directly into automated oligonucleotide synthesis (23) using an Applied Biosystems Model 380A synthesizer. DNA Crude oligomers were analyzed and purified to (0.46 cm x 25 cm), eluting with a 60 min linear gradient of acetonitrile in 100 mM ammonium acetate, pH 7.1. analyzed electrophoresis, by 20% polyacrylamide gel 7% to 35% Isolated oligomers were HPLC, and by hybridization of -^^P-labeled oligomers against target and control DNA. A total of five specific oligomers were designed for four different infectious agents and extensively tested using a radioisotopic label. conjugation to enzymes, the oligomers were constructed linker arm nucleoside replacing a thymine base. were tested against known targets, Table 1. with For one The linker arm amine oligomers and have the characteristics shown in Hybridization and radioisotopic detection results for some of the oligomers have been described elsewhere [HSV (25); ETEC (26)]. Preparation of target and control DNA The following DNA targets were used: for HSV, pHSV106 (a 7.7Kb plasmid containing a 3.4 Kb insert of HSV1 thymidine kinase gene in pBR322, from Bethesda Research Laboratories); for HBV, containing the pPClOX 1.9 Kb insert in pBR322 purchased (a entire 3.2 Kb HBV (...truncated)