ABSTRACT
The divergent synthesis of bDNA structures is described. This new type of branched DNA contains one unique oligonucleotide, the primary sequence, covalently attached through a comb-like branching network to many identical copies of a different oligonucleotide, the secondary sequence. The bDNA comb molecules were assembled on a solid support using parameters optimized for bDNA synthesis. The chemistry was used to synthesize bDNA comb molecules containing 15 secondary sequences. The bDNA comb molecules were elaborated by enzymatic ligation into branched amplification multimers, large bDNA molecules (a total of 1068 nt) containing an average of 36 repeated DNA oligomer sequences, each capable of hybridizing specifically to an alkaline phosphatase-labeled oligonucleotide. The bDNA comb molecules were characterized by electrophoretic methods and by controlled cleavage at periodate-cleavable moieties incorporated during synthesis. The branched amplification multimers have been used as signal amplifiers in nucleic acid quantification assays for detection of viral infection. It is possible to detect as few as 50 molecules with bDNA technology.
Nucleic acids have joined antigen and antibodies as key molecular targets for human diagnostic tests. Of particular importance, due to the development of a variety of technical capabilities, microorganisms can now be detected and quantified at unprecedented low levels in clinical specimens. Many forms of target amplification have now been introduced since the first reports of the polymerase chain reaction (1 ). In contrast, we have utilized signal amplification for direct analysis of nucleic acids. The key molecule in the signal amplification method is a branched DNA (bDNA) molecule for specific incorporation of many labels. bDNA signal amplification technology has been applied to the quantification of many organisms and mRNAs. For example, as few as 50 molecules of the human immunodeficiency virus type 1 (HIV-1) genome have been quantified in human plasma samples (2 ).
In the accompanying article (3 ) we reported our investigation of synthesis of bDNA oligomers of the `comb' type. Briefly, a linear oligonucleotide was synthesized by standard phosphoramidite chemistry on a solid support and a special `branch point monomer', the BM nucleoside, which contained a dimethoxytrityl (DMT)-protected 5'-hydroxyl function and an additional protected sidechain hydroxyl function, was incorporated using standard DMT extension. After completion of linear synthesis, selective deprotection of the sidechain hydroxyl protecting group opened up reactive sites for continued DNA synthesis. By multiple incorporation of BMs several secondary sequences were synthesized directly on the linear sequence. We found that N-4-(6-hydroxyhexyl)-5-methyl-2'-deoxycytidine, the BM nucleoside, in which the sidechain hydroxyl was protected as the levulinate (LEV) was the most suitable branch point monomer (see Scheme 1 A). We implemented several important modifications to standard phosphoramidite chemistry in order to achieve high quality bDNA in good yields and a number of bDNA oligomers were synthesized using the new protocols.
In this communication we describe the synthesis of bDNA comb oligomers with 15 secondary sequences (15× bDNA) and their elaboration into large bDNA molecules, branched amplification multimers (15×3 bAM), containing >1000 nt. Using enzymatic ligation a long linear 60mer oligonucleotide [comprising three consecutive 18mer hybridization sites complementary to an alkaline phosphatase-labeled probe (AP probe)] was added to each 6mer secondary sequence of the bDNA comb oligomer. The assembled 15×3 bAMs were characterized by electrophoretic methods and by controlled cleavage at periodate-cleavable moieties incorporated during synthesis. The hybridization properties of a 15×3 bAM were assessed using a hybridization-dependent fluorescent quenching assay which showed that an average of 36 out of 45 theoretical sites were available for hybridization. Large 15×3 bAMs have been used as signal amplifiers in quantitative assays for detection of hepatitis B virus DNA, hepatitis C virus RNA and HIV-1 RNA (2 ,4 ).
All chemicals and biochemicals were reagent grade or better and were used without further purification. O-(2-Cyanoethyl)-N,N-diisopropylphosphoramidites of dA, dC, dG and T were purchased from Glen Research (Sterling, VA). Anhydrous acetonitrile (<30 p.p.m. water content) was from either Baxter or Fisher. All other DNA synthesis ancillary reagents were purchased from Applied Biosystems (a Division of Perkin Elmer, Foster City, CA). Standard protocols for the ABI 380B DNA synthesizer as provided by the manufacturer were used unless otherwise indicated. The oligomer 5'-AAG TAC GAC AAC CAC ATC-3'-BODIPY FL, where the dye is attached to the oligomer through a C3 spacer, was prepared by Molecular Probes (Eugene, OR). Fluorescence measurements were performed on a Perkin Elmer LS-50B spectrofluorometer. Ion spray mass spectrometry (ESI) measurements were run on a Perkin-Elmer PE SCIEX API III electrospray quadrupole instrument. NMR spectra were recorded on a Varian 300 MHz instrument. The 31P NMR spectra were run in CH3CN with d6-DMSO for a lock and aqueous 85% H3PO4 as an external reference.
Polyacrylamide gel electrophoresis (PAGE). PAGE was carried out using 10% cross-linked slab gels (1 mm thick) with or without added urea with the following running buffer: 100 mM Tris-borate, 1 mM EDTA, pH 8.3 (diluted from a 10× stock) (5 ). Bromophenol blue was used as tracking dye. Gels were transferred to a thin layer chromatography plate containing a F254 indicator (20 × 20 cm; EM Sciences) and DNA bands were visualized under UV light.
Capillary electrophoreses. All capillary electrophoreses were performed on a Beckman P/ACE 2050 automatic CE system (Beckman Instrument, Fullerton, CA) with System Gold control software (version 7.11).
Method I: gel-filled capillary electrophoresis. Analysis was performed in a fused silica capillary column of 75 µm i.d. and 375 µm o.d. filled with 5% T, 5% C polyacrylamide gel (µPAGE-5 gel; J&W Scientific, Folsom, CA) in a buffer of 100 mM Tris-borate, 7 M urea, pH 8.3 (µPAGE buffer; J&W Scientific). The same µPAGE buffer was used for both inlet and outlet vials. The effective length of the capillary was 30 cm (inlet to detector). The samples were injected at 100 or 135 V/cm for 4-6 s and separated by electrophoresis at 250 V/cm.
Method II: polymer network capillary electrophoresis. To prepare a polymer network solution, 2 g hydroxypropyl cellulose (HPC) powder (average mol. wt 100 000; Aldrich) was added to 50 ml µPAGE buffer. The mixture was gently stirred at room temperature for 16-24 h until all HPC was hydrated and the solution became clear. The 4% HPC viscous solution was then filtered through a polycarbonate filter device with 5 µm pores (µPrep filter disc; Poretics, Livermore, CA). The solution was stored at 4°C and discarded after 3 months. For the CE with polymer network solution a coated capillary (DB-17, 375 µm o.d., 100 µm i.d. with a 0.1 µm thickness of bonded film; J&W Scientific) was used with an inlet to detector length of 20 cm and a total length of 27 cm. A 5 mm section of polyimide coat was carefully removed with either a sharp razor or a capillary burner (Euramark, Mt Prospect, IL) to serve as a detection window. The polarity of electrophoresis was reversed for all DNA-related analyses, with the inlet electrode negative. A 260 nm detector was used and the capillary was maintained at 25°C using the liquid cooling system supplied with the instrument. The commercial µPAGE buffer was used for all electrophoreses and both inlet and outlet buffers were replaced after every 4-8 runs. Prior to use on the CE all buffers and polymer solutions were degassed by brief sonication or centrifugation. The capillary was filled with 4% HPC solution under 20 p.s.i. pressure (inlet to outlet) for 10 min, followed by pre-running at 200 and 300 V/cm for 5 and 10 min respectively, when the current becomes stabilized. When not in use the capillary was rinsed first with µPAGE buffer, followed by deionized water, methanol and blown dry with helium gas before storage. The bonded capillary was reused over a period of 3 months. For routine analysis the oligonucleotide samples were diluted with deionized water to a concentration of 0.2 (purified) or 2 (crude) A260 units/ml and injected electrokinetically into the capillary for 4-6 s at 4 kV. Electrophoresis was performed at 200-250 V/cm for 40 min.
Periodate cleavage of bDNA. Purified bDNA (0.1-0.3 A260 units) containing 1,2-diol cleavable monomers (CM2) were treated with freshly prepared aqueous sodium periodate solution at a concentration of 25 mM in a total volume of 12 µl for 60 min at room temperature. Excess periodate was reacted with 4 µl aqueous 1 M glycerol solution. The mixture was analyzed by high performance capillary electrophoresis (HPCE) directly without further treatment. The residual salt in the mixture reduced the amount of sample which could be electrokinetically injected into the HPCE (estimated to be 10-fold less), however, the resolution of the capillary gel appeared unaffected.
Compound CM2 (33 mmol) was silylated with t-butyldimethylsilyl chloride (TBDMS-Cl, 19.8 g, 132 mmol) in the presence of N,N-dimethylaminopyridine (100 mg) and triethylamine (27 ml, 200 mmol). After 18 h the reaction mixture was concentrated and diluted with ethyl acetate (250 ml). The organic phase was washed with 250 ml 5% NaHCO3 and then 250 ml 80% saturated aqueous NaCl solution. After drying over solid Na2SO4 the solvent was removed in vacuo. Crude TBDMS2-CM2 in pyridine was treated with benzoyl chloride (132 mmol) at room temperature for 18 h and then subjected to aqueous work-up as described above. Without purification TBDMS2-CM2(Bz2) (30 mmol) was desilylated with glacial acetic acid (100 ml)/tetrabutylammonium fluoride (100 ml, 1 M in THF) at 4°C for 18 h. Most of the solvent was then removed in vacuo and the residue in ethyl acetate was treated with solid NaHCO3 to neutralize excess acid, then washed and dried as described above to give CM2(Bz2) (30 mmol, 17.0 g). A sample was purified by silica gel chromatography to yield pure CM2(Bz2). 1H-NMR (CDCl3): [delta] 2.8 (t, 4H: C6H4-CH2-C-OH), 3.8 (t, 4H: C6H4-C-CH2-OH), 4.4 (d, 4H: O-CH2-C), 6.0 [m, 2H: C-CH(OH)-C], 6.9 (d, 4H: aromatic H), 7.15 (d, 4H: aromatic H), 7.4 (m, 4H: aromatic H), 7.55 (m, 2H: aromatic H), 8.05 (d, 4H: aromatic H) p.p.m. Calculated for C34H34O8, C 71.56, H 6.01; found, C 71.24, H 6.18.
The crude product was directly tritylated using standard procedures and purified on a large silica gel column using CH2Cl2/1% triethylamine as solvent system, to yield pure DMT-CM2(Bz2) (13.3 g, 15 mmol). 1H NMR (CDCl3): [delta] 2.8 (m, 4H), 3.2 (t, 2H), 3.8 (s, 6H), 4.4 (m, 4H), 6.0 (s, 2H), 6.8-6.9 (m, 8H), 7.0-7.6 (m, 31H), 8.0 (d, 4H) p.p.m.
Purified DMT-CM2(Bz2) (15 mmol) was converted to the BCE phosphoramidite using a published phosphitylation procedure (6 ) to give a white foam of pure DMT-CM2(Bz2)-BCE phosphoramidite (14.2 g, 13 mmol). 31P NMR: 148 p.p.m. Analysis, calculated for C64H69O11N2P, C 71.63, H 6.48, N 2.61; found, C 71.21, H 6.43, N 2.41.
Oligodeoxynucleotides were synthesized by standard solid phase chemistry using 2-cyanoethyl phosphoramidite monomers. The phosphorylating reagent 2-((2-((4,4'-dimethoxytrityl)oxy)ethyl) sulfonylethyl-2-cyanoethyl-N,N-diisopropylphosphoramidite (Phostel) was used to synthesize 5'- phosphorylated oligomers (7 ).
General procedure for synthesis of 15 site bDNA comb oligomer.
Synthesis of the primary sequence. The linear sequence 5'-(BM-TT)14-T18-GAC ACG GGT CCT ATG CCT-3' was synthesized on a controlled pore glass (CPG) solid support (2000 Å pore size) derivatized with 14.8 µmol/g DMT-thymidine through a N-succinyl-aminopropyl linker (40.5 mg, 0.6 µmol) which was packed into a column (ABI). The primary sequence was synthesized on an automated DNA synthesizer (model 380B; Applied Biosystems Division, Perkin Elmer, Foster City, CA). The detritylation step used two 7 s pulses of 3% trichloroacetic acid (TCA) in toluene/CH2Cl2 (1:1 v/v), each followed by a 4 s pause, then the column was flushed out. This process was repeated twice more. The phosphoramidite reagent was used at 18 µmol for each coupling of A, G, C or T, however, for BM phosphoramidite 20 µmol was used for each coupling.
Removal of LEV protecting group of BM. The CPG column from the primary synthesis was attached to a 10 ml plastic syringe and rinsed with 10 ml acetic acid/pyridine (1:1 v/v). Approximately 10 ml HPAA (1:1) reagent (0.5 M hydrazine hydrate in acetic acid/pyridine 1:1 v/v) was periodically pushed through over a period of 90 min at room temperature. The solid support was then rinsed with 10 ml acetic acid/pyridine (1:1 v/v) and detached from the syringe, followed by extensive rinsing with acetonitrile before brief drying under vacuum. A small portion (2 mg) of the CPG support was removed for HPCE analysis and the filters on the column were replaced.
Synthesis of the secondary sequence. All the reagents used in the secondary synthesis were the same as in the primary synthesis, however, their amounts and step times differed. The detritylation step used three 8 s pulses of 3% TCA in toluene/CH2Cl2 (1:1 v/v), each followed by a 4 s pause, followed by a 20 s rinse (a 15 s pulse with a 5 s pause) with toluene/CH2Cl2 (1:1 v/v). This process was repeated twice more. Nucleoside phosphoramidite (96 µmol) was used for each coupling of A, C, G or T. The coupling process consisted of an 8 s addition of activator and an 8 s addition of activator and phosphoramidite, followed by a 30 s wait. This was performed a total of eight times. The capping and oxidation processes each used two 10 s pulses of reagents separated by a 5 s pause and then followed by a 60 s wait step. 5'-End phosphorylation of the secondary sequence was performed by coupling twice with Phostel phosphoramidite (107 µmol each at 100 mM) with no capping between couplings. Secondary sequence 5'-p-TGA-CTG-3'.
Deprotection and purification of the 15 site branched comb DNA. The detritylated bDNA was cleaved from the CPG support with 2 ml 30% NH4OH for 60 min at room temperature. The supernatant was collected in a 4 ml glass vial, capped and heated in a 60°C oven for at least 16 h, then dried under vacuum. The crude bDNA was purified on a 7% T, 5% C polyacrylamide gel (20 × 40 × 1.5 cm) containing 7 M urea. The gel was run until the bromophenol blue dye migrated to within 2-3 cm of the bottom. The product band was excised and soaked in 100 mM Tris-HCl, pH 8.0, 0.5 M NaCl and 5 mM EDTA for at least 24 h with agitation. The salt was removed by loading the bDNA solution onto a short C-18 column (Sep-Pak cartridge; Millipore Corp., Bedford, MA) and rinsing with water. The purified bDNA was eluted from the column with methanol/H2O (1:1 v/v). After being dried, the bDNA was precipitated from ethanol/0.3 M aqueous potassium acetate (3:1 v/v). The product was routinely analyzed by HPCE.
The 15× bDNA comb oligomer (1 nmol), with 5'-p-TGA CTG-3' secondary sequences and a linear DNA sequence 5'-(GAT GTG GTT GTC GTA CTT)3-GCG TAG-3' (60mer, 23.44 nmol), was combined with the linear DNA linker 5'-CAG TCA CTA CGC-3' (12mer, 18.75 nmol) in a reaction tube in a total volume of 140 µl water and 25 µl ligation buffer (10× buffer: 500 mM Tris, pH 7.5, 100 mM MgCl2, 20 mM spermidine) was added. The mixture was heated in a closed tube to 95°C before being slowly cooled down to room temperature for hybridization to occur. To the mixture was added ATP (5 µl 0.1 M solution), DTT (5 µl 0.5 M solution), polyethyleneglycol 8000 (70 µl 50% solution) and T4 ligase (6.7 U/µl, 50 U; Pharmacia 27-0870-04). This reaction mixture was incubated at room temperature overnight. Sodium chloride was added (16.5 µl 4 M solution) and ice-cold ethanol (800 µl) was added. The mixture was kept at -20°C for 30 min and then centrifuged at 12 000 g for 30 min. The supernatant was decanted off and the precipitate first gently dried in vacuo and then resuspended in water. The product was gel purified using the same procedure as described above for 15× bDNA comb oligomer.
The amount of single stranded oligomer in pmol (10-12 mol) was 109 000/n × A260, where n is the number of nucleotides and analogs in the oligonucleotide and A260 is absorbance units at 260 nm.
Solution hybridization with fluorometer detection. In a series of Eppendorf tubes were mixed 15×3 bAM (1 pmol) and 5'-AAG TAC GAC AAC CAC ATC-3'-BODIPY FL (0-100 pmol) in a final volume of 10 µl 1× SSC (150 mM NaCl, 15 mM Na citrate, pH 7). The samples were annealed in a water bath at 60°C for 15 min, then removed and cooled to room temperature over 15 min. Individual reactions were diluted into 3 ml 40 mM Tris-acetate, 2 mM EDTA, pH 8, 0.5 M NaCl and the relative fluorescence determined in a Perkin-Elmer 50B spectrofluorometer using a 485 nm excitation filter and a 500 nm emission filter. A control curve of free probe was constructed by diluting 5'-AAG TAC GAC AAC CAC ATC-3'-BODIPY FL (total 0-100 pmol) into 3 ml 40 mM Tris-acetate, 2 mM EDTA, pH 8, 0.5 M NaCl and the relative fluorescence recorded.
The main focus of this work was the synthesis of 15× bDNA comb oligomers with T2 (Ny = 2) spacing between BMs and their subsequent elaboration into 15×3 bAMs. The 15× bDNA comb oligomer is shown in Figure 1 A. Without spacing between BMs (Ny = 0) the bDNA comb oligomer was expected to be quite congested. With more spacing between BMs (Ny = T6) synthesis was less practical, since it required a greater number of condensations during synthesis, resulting in substantially reduced overall yields.
We have developed a signal amplification scheme based on bDNA amplification multimers (bAM) to achieve greater sensitivity in DNA-based probe assays. Amplification in the assay is realized because the bDNA contains a unique sequence, the primary sequence, covalently connected to many secondary hybridization sites, each of which can hybridize with an alkaline phosphatase-labeled probe. This unique signal amplification system should be capable of amplifying a single hybridization event 10 000-fold.
The use of bDNA to amplify the signal for nucleic acid detection and quantification has permitted investigators to demonstrate several unique features of infectious diseases. Ho and co-workers (16 ) have been able to show the dynamics of HIV-1 infection, while Mellors has reported a correlation of HIV-1 RNA with the likelihood of progression of AIDS (17 ). Both studies employed bDNA assays for quantification of HIV-1 RNA. Lau and colleagues (18 ) have reported on the prognostic value of HCV RNA quantification using bDNA assays for monitoring interferon treatment of infected individuals. The bDNA signal amplification method has been used in a variety of other studies involving several other organisms and mRNA of human origin (reviewed in 19 ).
We wish to acknowledge the excellent technical support of Yougen Gee in synthesis of BM reagents and the expert technical assistance of David Ahle, Jennifer Clyne, Tim Fultz, Sarah Hamren and Joyce Running for construction of bAMs by enzymatic ligation. We thank Dr Celine Hu for helpful suggestions during manuscript preparation, Dr F.Masiarz for ion spray mass spectometry measurements and Dr Linda Wuestehube for editorial assistance.
This article has been cited by other articles:
Synthesis of 2-(4-(4-(4-(2-dimethoxytrityloxy)ethyl-)phenoxy- 2,3- di(benzoyloxy)-butane-oxy)phenyl)ethyl-2-cyanoethyl-N,N-diisopropyl phosphormidite (CM2 cleavable site monomer). To a mixture of 2-(4-hydroxyphenyl)-1-ethanol (21.4 g, 155 mmol) and 1,4-dibromo-2,3-butane diol (19.3 g, 78 mmol) dissolved in 400 ml absolute ethanol was added NaOH (26 ml of a 6 M solution in water). The reaction mixture was kept at gentle reflux for 18 h. After cooling the reaction mixture was concentrated to 200 ml and this solution added dropwise to 1000 ml water with rapid stirring. The precipitate was filtered off and dried extensively in a vacuum desiccator over solid NaOH to give DL-1,4-bis-(4-(2-hydroxyethyl)phenoxy)-2,3-butanediol (CM2, 11.9 g, 33 mmol). A sample was purified by silica gel chromatography to yield pure CM2.ESI MS, mol. wt calculated for C20H26O6 362.4; found 362.3.1H-NMR (CD3OD): [delta] 2.75 (t, 4H: 2C6H4-CH2-C-OH), 3.7 (t, 4H: C6H4-C-CH2-OH), 4.0-4.2 (m, 4H: O-CH2-C), 4.0-4.2 [m, 2H: C-CH(OH)-C], 6.9 (d, 4H: aromatic H), 7.15 (d, 4H: aromatic H) p.p.m. Analysis, calculated for C20H26O6, C 66.28, H 7.23; found, C 66.00, H 7.39.
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