Bioconjugate Chem. 1003, 4, 380-385
380
End Attachment of Phenol-Oligonucleotide Conjugates to Diazotized Cellulose Marie-laurence Fontane1,t Hervk Bazin,t and Robert TBoule’J Laboratoire de Radiobiochimie, Centre d’ Etudes NuclBaires de Grenoble, DRFMC/SESAM, 85X, 38041 Grenoble Cedex, France, and Laboratoire des Sondes Molkculaires, CIS Bio International, BP32-91192 Gif-Sur-Yvette, Cedex, France. Received April 2, 1993” The synthesis of a novel phosphoramidite reagent with a hexanediol backbone is described. This reagent has been used to incorporate a phenol moiety on an oligonucleotide (ODN) directly in the course of its automated synthesis. Multiple phenol attachments can be achieved by repetitive coupling cycles. A simple and rapid immobilization method is described where phenol-modified ODNs are covalently attached to diazotized cellulose. The binding capacity of the membrane can be modulated, depending on the ODN concentration used, to ca. 180 pmol/cm2. There is at least 80 5% end attachment of the ODN through the phenol group. In addition, the phenol residue can be used as a carrier for the radiolabeling with 1251.The non-nucleosidic hexanediol derivative incorporated at the 5’-end of the ODN is recognized as a substrate by the T4 polynucleotide kinase and the terminal hydroxyl group is successfully phosphorylated allowing its 32Plabeling.
INTRODUCTION One of the most common methods for the detection of specific nucleic acid sequences typically involves the immobilization of the target nucleic acid on solid supports such as nitrocellulose or nylon membranes followed by hybridization with an appropriate labeled signal probe. The immobilization methods such as the “baking procedure= or UV irradiation suffer from a low retention efficiency and proceed by random attachment along the polynucleotide. Another approach involves the coupling of DNA to cyanogen bromide activated supports (1, 2) and to Sephadex, Sephacryl and cellulose via diazotized aromatic amines (3-5). Multiple attachments through base residue (mostly guanine) inhibit hybridization. All these methods are restricted to the immobilization of long DNA (>400 bp), but for ODNs (14-30 mers), the hybridization process can be disrupted. This led to the development of chemical covalent methods in which there is generation of a specific and stable linkage preferentially between the 5’-end of the ODN and the support. Procedures for the modification of ODNs have been reviewed (6). One of the methods is based on carbodiimidemediated end attachment of nucleic acids to polysaccharide matrixsuch as cellulose (3,Sephadex (3),or Sephacryl (8) and to hydroxyl polystyrene beads through ODN 5’phosphate group (9). Attachments of 5’-amino alkyl ODNs to carboxyl polystyrene beads (9) or controlled pore glass (CPG) derivatized with long-chain alkylamine or carboxylic Sephacryl have been reported (10). For most of these reported methods the 5’-terminal attachment efficiency is low even with long reaction times and the nonspecific binding to the support is high. The development of the automated synthesis of short ODN probes makes them readily available. They show better hybridization kinetics and high sensitivity in the detection of mutations. They have become a powerful tool either in the PCR technology and in the reverse dot hybridization format (ll), where the target nucleic acid
* To whom correspondence should be addressed. +
Centre d’Etudes Nucleaires de Grenoble.
* CIS Bio International.
e Abstract published in Aduance
1, 1993.
ACS Abstracts, September
1043-1802/93/29060300$04.00/0
probe is hybridized to a capture ODN probe. Numerous 5’-modifications of ODNs based on the phosphoramidite approach for the introduction of 5’-NHz or 5’-SH have been investigated. The reaction time required for the attachment to the support has been considerablydecreased from 20 to 3 h (12). Further improvements allowed a reaction time of 1min (131,butthe coupling efficiency was still too low. More recently, more specific reactions were used at the 5’-end of the ODN (14 1. The systems showed stable and efficient attachment a t one end of the ODN (15-18) on different solid supports: nylon beads (13, membranes (151, magnetic beads (181, and polyacrylamide (19). In this work we describe a simple and rapid method for covalent attachment of ODNs modified by a phenol group on a diazotized cellulose matrix. The phenol groups were incorporated into the ODNs by using a phenylphosphoramidite derivative according to the direct labeling procedure described by Roget et al. (20). Other authors reported the synthesis of simple non-nucleosidic phosphoramidite units: Misiura et al. (21) expanded the use of 1,2-ethanediolphosphoramidite reagents derived from a glycerol intermediate. Nelson’s reagent (22)is based on a 1,3-propanediol backbone. Both provide multiple incorporation and 3’-labeling. The use of the present 1,2ethanediol backbone is not only restricted to the immobilization but it can be used for the radiolabeling (either 1251 (26) or 32P).When the modification is introduced at the 5’-end of the ODN, it is still recognized as a substrate by the T4 polynucleotide kinase (PNK). EXPERIMENTAL PROCEDURES Iodine-125 S4 (200 mCi/mL) was a product of Cis Bio International. Adenosine 5‘-triphosphate (ATP) was purchased from Boehringer (100 mM) and [ T - ~ ~ P I A T P (10 mCi/mL) came from Amersham. Cellulose membranes (APT Cellulose) came from Schleicher and Schull. T4 polynucleotide kinase was obtained from Pharmacia. Analytical grade solvents were used, and dichloromethane stabilized with 2-methyl-2-butenewas used for synthesis. The ‘H NMR and 31P-NMR spectra were recorded respectively on AC 200 and WM 250Bruker spectrometers. The J values are reported in hertz. The mass spectra were recorded on a Fisons-VG type ZAB2-SEQ spectrom0 1993 American Chemical Society
End Attachment of Phenol-ODN Conjugates
Bioconlugate Chem., Vol. 4, No. 5, 1993
381
mL of THF) was added dropwise to a cooled (0 “C) solution of 4 (640 mg, 3.2 mmol) in THF (15 mL) with stirring. After 15 min at 0 OC and then 4 h at 25 OC, the reaction mixture was treated with water (0.4 mL), 15% aqueous NaOH (0.4mL),and water (1.2mL). The white precipitate was filtered over celite and washed with THF (2 X 10mL). The combined fitrates were evaporated and the residue was purified with a gradient of methanol in dichloromethane (5-50% 1. The title compound was obtained as an oil. Yield: 481 mg (87%1. R f = 0.16 in methanol/ dichloromethane (2/8, v/v). lH NMR (CDC13): 4.2-3.9 (m,2H, H-1 and H-21, 3.5 ( t ,J = 6.4 , lH, H-1’1, 2.7 ( t , J = 6.2 , 2H, H-6,6’), 1.72 (s, 2H, NH3, 1.7-1.1 (m,6H, 3 x CH2), 1.4 (s, 3H) and 1.32 (s,3H) (2 X isopropylidene CH3). 1,2-0-Isopropylidene-N-[ 3 44-hydroxypheny1)propanoyl]-6-amino-1,2-hexanediol(6):Compound 5 (2.5 g, 14.5 mmol) dissolved in a mixture of acetonitrile (10 mL) and dichloromethane (20 mL) was reacted with N-succinimidyl 3-(4-hydroxyphenyl)propanoate (5.2 g, 19.7 mmol) in the presence of diisopropylethylamine (1.87 g, 14.5 mmol). After 3 h of stirring, the reaction mixture was diluted with dichloromethane and washed with saturated NaHC03 solution (2 X 100 mL) and water (100 mL), and the organic layer was dried over Na2S04 and evaporated. The crude compound was loaded onto a silica gel column packed in a dichloromethane petroleum ether mixture (8/2) and eluted with a stepwise gradient to dichloromethane and then to 5% methanol in dichloromethane. Yield: 4.08 g (88%). Rf = 0.63 in dichloromethane/methanol (9/1, v/v). lH NMR (CDCl3): 7.0 (d, J = 8.4 , 2H, H-2,6 arom), 6.76 (d, 2H, H-3,5 arom), 4.1-3.8 (m,2H, H-1,2), 3.5-3.3 (m,J = 5.2 , l H , H-l’), 1,2-0-Isopropylidene-60-(4-toluenesulfonyl) hex3.3-3.0 (m,2H, H-6,6’), 2.85 ( t , J = 7.4, 2H, H-2,2’, anetriol (3). Compound 2 (13 g, 74.7 mmol) was coevappropanoyl), 2.38 (t, 2H, CH-3,3’, propanoyl), 1.7-1.0 (m, orated with dry pyridine (2 X 50 mL) and dissolved in 6H, 3 X CH3, 1.39 (s, 3H) and 1.34 (s, 3H) (2 X dichloromethane (100 mL) containing diisopropylethylisopropylidene CH3). amine (9.65 g, 74.7mmol) and 4-(dimethy1amino)pyridine N-[3-(4-Hydroxyphenyl)propanoyl]-6-amino-l,2(122 mg, 1mmol). A solution of 4-toluenesulfonylchloride hexanediol(7). Compound 6 (2.9 g, 30 mmol) was treated (15.6 g, 87.2 mmol) in dichloromethane (40 mL) was added with Dowex 50 W-X12 (100-200 mesh, H+ form) in a dropwisea t 0 “C with stirring. After 7 h a t 0 OC the reaction dioxane-water mixture ( l / l , 50 mL) for 45 min at 100 OC. mixture was extracted with water (3 X 150 mL) and The ion-exchenger was filtered off and washed with a evaporated to dryness. The residue was purified over a dioxane/methanol mixture ( U l , 10 mL). The filtrate was short silica gel column using a gradient of dichloromethane evaporated and the residue was dried under vacuum over in petroleum ether (from 30 to 100%). The compound 3 P z O ~ Yield: . 2.12 g (83% 1. Rj = 0.8 in dichloromethane/ is obtained as an oil. Yield: 23.4 g (96%). R f = 0.67 in methanol (8/2, v/v). lH NMR (CDCl3): 7.0 (d, J = 8.4, ethyl acetate/hexane ( l / l , v/v). lH NMR (CDC13): 7.84 2H, H-2,6 arom), 6.76 (d, 2H, H-3,5 arom), 3.72-3.27 (m, (d, J = 8 , 2H) and 7.32 (d, 2H) (tosyl H), (4.15-3.70 (m, 3H, H-1,l’ and H-2), 3.25-2.97 (m,2H, H-6,6’), 2.78 (t, J 4H, H-1, H-2 and H-6,6’), 3.43 (pst, J = 10, l H , H-l’), 2.45 = 7.3, 2H, H-2,2’, propanoyl), 2.35 (t, 2H, H-3,3’, pro(s,3H) (tosyl CH3), 2.0-1.1 (m, 6H) 3 X CH2,1.45 (s,3H) panoyl), 1.6-1.1 (m,6H, 3 X CH3. and 1.39 (s, 3H) 2-(isopropylidene X CH3). N-[ 3-[4-(Ethoxycarbonyl)oxy] phenyl]propanoyl]1%- 0-Isopropylidene-6-azido1,2-hexanediol(4). To 6-amino-1,2-hexanediol(8).To a cold (0 “C) solution of a solution of 3 (25 g ,76 mmol) in dimethylformamide (450 7 (2.1 g, 5.4 mmol) in THF (60 mL) containing triethylmL) were added sodium azide (6 g, 92 mmol) and amine (1.2 mL, 8.4 mmol) was added a solution of ethyl ammonium chloride (5.3 g, 100 mmol). The reaction chloroformate (1.09 g, 9.8 mmol) in THF (4 mL) dropwise. mixture was kept at 120 OC for 2 h with stirring. The After 1h a t room temperature the reaction mixture was solvent was distilled off (oil pump). The residue dissolved evaporated to dryness, dissolved in ethyl acetate (10 mL), in dichloromethane (400 mL) was washed with saturated and precipitated from hexane (100 mL) as a powder. NaHC03 (3 X 300 mL) and water (400 mL). The crude Yield: 1.53 g (58%). R f = 0.47 in dichloromethane/ compound was purified on a silica gel column with a methanol (8/2, v/v). ‘H NMR (CDCl3): 7.3-6.8 (m, 4H, gradient of dichloromethane in petroleum ether (30arom), 4.28 (q,J = 7.1,2H, CH&H3), 3.6-3.45 (m, 2H, H-1 100%1. Yield: (64%1. R f = 0.6 in ethyl acetate/hexane and H-2), 3.4-3.27 (m, l H , H-l’), 3.25-3.0 (m, 2H, H-6,6’), (3/7, v/v). ‘H NMR (CDC13): 4.10-4.01 (m, lH, H-2), 4.0 2.88 (t, J =7.5,2H, H-2,2’, propanoyl), 2.38 ( t ,2H, H-3,3’, (pst, J = 7.0 and 6.0, l H , H-l), 3.44 (pst, J = 8 and 7, l H , H-l’),3,23(pst, J=6.0and7.0,2H,H-6,6’),1.68-1.10(m, propanoyl), 1.55-1.10 (m,9H, 3 X CH2 and CH3). MS (FAB+): m/e (MH+) = 354.4, (MNa+) = 376. 6H) 3 X CH2, 1.39 (s, 3H) and 1.26 (s, 3H) (2 X isopropylidene CH3). l-O-(4,4’-Dimethoxytrityl)-N-[3-[4-[ (ethoxycarbonyl)oxy]phenyl]propanoyl]-6-amino-1,2-hexane diol 1,2-O-Isopropylidene-6-amino1,a-hexanediol (5). (9). Compound 8 (1.4 g, 4 mmol) was coevaporated with Lithium aluminium hydride (366 mg, 37.9 mmol) in 10
eter using a cesium gun. Short-column chromatography was run on silica G60 (Merck) and TLC analysis was performed on plastic sheets (60F(254), 0.2 mm layer, Merck). The HPLC analysis were performed on a Varian 5000 equipped with a Lichrospher RP-18 (5 pm) column (Merck) working a t a flow rate of 1 mL/min (gradient 1,10% to 50% B in 20 min and then 50% to 100% B in 30 min, where A = 5% CH3CN in 25mM triethylammonium acetate (TEAAc) and B = 50% CH3CN in 25mM TEAAc) and on a Mono Q HR 5/5 anion-exchange column (Pharmacia),working at a flow rate of 1mL/min (gradient 2,0-2 min 0% B, 5 min 20% B, 40 min 40% B, where A = 20% CH3CN in 40 mM sodium acetate, pH 5.5, and B = 20% CH3CN in 40 mM sodium acetate, 1.5 M lithium chloride). 1,2-O-Isopropylidene-l,2,6-hexanetriol(2). A solution of 1,2,6-hexanetriol (20 g, 149 mmol) in a mixture of acetone (45 mL) and petroleum ether (45 mL) containing 4-toluenesulfonic acid was heated in a round-bottom flask fitted with a Vigreux column, a Dean-Stark apparatus, and a condenser (Drierite guard). After 22 h under reflux the cooled reaction mixture was neutralized with saturated aqueous NaHC03 (25mL) and concentrated in vacuo. The residue was taken up in dichloromethane (100 mL) and washed with saturated NaHC03 (3 X 100 mL) and water (100 mL). The organic phase was evaporated and coevaporated with carbon tetrachloride. Compound 2 was obtained as an oil. Yield: 21.5 g (83%). Rf = 0.46 in chloroform/methanol(90/10,v/v).lH NMR (CDCl3): 4.06 ( P S ~J, = 6.33, l H , H-2), 4.03 (pst, J= 7.35 and 6.33, l H , H-l), 3.63 (pst, J = 6.39 and 6.48, 2H, H-6,6’), 3.51 (pst, J= 7.33 and 7.35, l H , H-1’), 1.74-1.20 (m,6H, 3 X CH2, 1.38 (s, 3H) and 1.32 (s, 3H) (2 X isopropylidene CH3).
Fontanel et al.
382 Bioconjugate Chem., Vol. 4, No. 5, 1993
dry pyridine (2 X 20 mL), dissolved again in pyridine (35 mL), and 4,4’-dimethoxytrityl chloride (1.7 g, 5 mmol) in pyridine (10 mL) was added at 0 “C. After 4 h at 0 “C, water (20 mL) was added and the reaction mixture was partitioned between dichloromethane (100 mL) and saturated NaHC03 solution (100 mL). The organic layer was washed with NaHC03 (100 mL) and water (2 X 100 mL). After evaporation and coevaporation with toluene the residue was purified on a short silica gel column packed in a petroleum etheddichloromethane mixture (6/4) containing 5 % of triethylamine, eluting successively with a gradient of dichloromethane in petroleum ether and then 1%methanol in dichloromethane. Yield 2.24 g (86%). Rj = 0.28 in dichloromethane/methanol(9/1). ‘H NMR (CDCl3): 7.6-7.0 (m,13H, arom DMT), 6.82 (d, J = 8.6, 4H, DMT), 4.28 ( 4 , J = 7.1, 2H, CHZCH~), 3.95-3.60 (m, l H , H-2), 3.8 (s, 6H, 2 X CH30, DMT), 3.35-2.90 (m,6H, H-l,l’, H-2,2’, H-6’6’, and H-2,2’, propanoyl), 2.4 ( t ,J = 7.35, 2H, H-3,3’, propanoyl), 1.65-1.10 (m,9H, 3 X CHz and 3 X CH3). MS (FAB+): (MNa+) = 678.7. 1-0(4,4’-Dimet hoxytrity1)-2-O[[(2-cyanoethy1)oxy1(N,N-diisopropylamino)phosphino]-N-[ 3-[ 4-[ (ethoxycarbonyl)oxy]phenyl]propanoyl]-6-amino-1,2-hexanediol (10). Compound 9 (1.7 g, 2.6 mmol) was coevaporated with dry acetonitrile and taken up in dichloromethane (22 mL) under argon. Diisopropylammonium tetrazolide (263mg, 1.5 mmol) and 2-cyanoethylN,N,”,”-tetraisopropylphosphorodiamidite (1.07 mL, 3.4 mmol) were added. After 3 h the reaction mixture was diluted with dichloromethane (50 mL) and washed with saturated NaHC03 (2 X 20 mL) and brine (2 X 100 mL). The organic layer was dried over Na2S04 and evaporated. The residue was loaded onto a short silica gel column eluting with a gradient from hexane/dichloromethane/ triethylamine (90/9/1) to hexane/dichloromethane/triethylamine (50/49/1). The relevant fractions were evaporated, coevaporated with toluene and lyophilized from benzene in several serum flasks fitting on the DNA synthesizer. Yield: 1.55 g (70%). Rf = 0.6 and 0.54 in hexane/dichloromethane/triethylamine (45/45/10). ‘H NMR (CD3COCD3): 7.6-7.1 (m,9H, DMT, 7.28 (d, J = 8.0, 2H, H-2,6, arom), 7.1 (d, 2H) H-3,5 (arom), 6.90 (2d, J = 9.0,4H, DMT, 4.28 ( 4 , J = 7.1,2H, CHzCHs), 3.9-3.5 (m,5H, 2 X CH(CH3)2,H-2, CHZCH~CN), 3.80 (2s, 6H, 2 X C&o, DMT), 3.3-3.1 (m,2H, H-l,l’), 3.0-2.85 (m,4H, H-6,6’ and H-2,2’, propanoyl), 2.58 ( t , J = 6.2, 2H, CHzCHzCN), 2.55-2.35 (m,2H, H-3,3’, propanoyl), 1.51.1 (m,21H, 3 X CHz and 5 X CHd. General Protocol for the Attachment of Oligonucleotides. Disks (0.5 cm2) were cut out of the APTcellulose membrane with a punch. They were activated as follows (volumes given for each square centimeter). Activation. The disks were soaked for 5 min in 1mL of cold 1.2 M HC1 solution (0 “C). A solution of sodium nitrite (freshly prepared) was added (30 pL at 10 mg/mL) and the disks (bright yellow color) were incubated for 30 min a t 0 “C with gentle shaking. Washings. The membranes were washed with ice cold sodium acetate buffer 50 mM, pH 4 (2 X 1min) and with ice-cold water (1 min). Fixation. The disks were immediately transferred and incubated in the fixation solution (50 mM phosphate buffer, pH 7) containing the phenol-ODN conjugate at a concentration of 0.5 OD units/mL. For the determination of the binding capacity, the corresponding 5’-32P-labeled oligonucleotide was added (ca. 105cpm/sample, 700 cpm/ pmol).
Inactivation of the RemainingDiazonium Groups. The membranes were washed with 0.1 N NaOH (3 X 1 mL) and water (1mL). General Protocol for Hybridization. The disks (0.5 cm2)of cellulose-bound ODNs were incubated for 2.5 h at 50 “C in 150 pL of 5X SSPE solution (NaC1 750 mM, NaHzP04 50 mM, EDTA 5 mM, pH 7.4) containing the complementary ODN (ca. 2 X lo5 cpm, specific activity: 3 X 104-105 cpm/pmol). Iodination of the Phenol-Modified ODN. An 11pmol portion of ODN 12 (Scheme 11) in 10 pL of 0.25 mM phosphate buffer pH 8.5 was added to 1pL of iodine (200 pCi) in 10 pL of phosphate buffer. Then 10 pL of freshly prepared chloramine T (5 mg/mL of phosphate buffer) was added and after 1 min the reaction was quenched with 65 pL of aqueous sodium metabisulfite (2.4 mg/mL phosphate buffer). This iodinated ODN was purified by gel filtration on a NAP 10column (Pharmacia)equilibrated in water. After the void volume, the fraction (900 pL) containing the labeled ODN was collected. Action of the T4 Polynucleotide Kinase (PNK) on Modified ODN with 1,2-Ethanediol Moiety. Each reaction mixture (20 pL) contained kinase buffer (50mM Tris-HC1, pH 7.5, 5 mM dithiothreitol, 10 mM MgClz), 450 pmol of ODN, 6800 pmol of ATP, and 10 units of polynucleotide kinase. The samples were incubated at 37 “C for 30 min and then analyzed by reverse phase HPLC. For the radiolabeling of the ODN, the incubation previously described was realized in the presence of 0.6 pmol of [T-~~PIATP (5000 mCi/mmol). RESULTS AND DISCUSSION
Synthesis of the Phenol Phosphoramidite 10. The 1,Bdiol system of 1 was protected as its isopropylidene derivative by reaction with acetone in the presence of toluenesulfonic acid as catalyst in 83% yield (Scheme I). The isopropylidene 2 was treated with toluene sulfonyl chloride giving 3 with 95 % yield. The azido derivatives 4 was prepared in 64 % yield by nucleophilic displacement of the tosylate 3 with sodium azide in DMF. The azide 4 was reduced with lithium aluminium hydride (LAH) to give the amino derivative 5 (87 % yield) which was treated with the N-succinimidyl3-(4-hydroxyphenyl)propanoate to give the amide 6 with 81% yield. The isopropylidene group on 6 was removed by treatment with Dowex H+ resin in dioxane-water mixture, the diol 7 was isolated in 84% yield. The phenol functions are reactive toward phosphitylation reagents and toward the iodine oxidation mixture used on the DNA synthesizer, therefore the phenol group must be masked. After some preliminary studies, the ethoxycarbonyl group was selected as being stable throughout the synthetic cycle and readily cleaved during the final ODN deprotection in aqueous ammonia. The phenolic hydroxyl of 7 was protected by reaction with ethyl chloroformate in the presence of triethylamine in dichloromethane, giving the ethoxy carbonate 8 in 58% yield. The primary hydroxyl of 8 was selectively protected by reaction with dimethoxytrityl chloride in anhydrous pyridine (85% yield). The resulting dimethoxytrityl ether was phosphitylated with bis(diisopropy1amino)[(2-cyanoethyl)oxy]phosphine in the presence of diisopropylammonium tetrazolide to give the fully protected phosphoramidite 10 in 70% yield, which upon freeze-drying from benzene was obtained as an oil. Synthesis of an Oligonucleotide-Phenol Conjugate and Its Complementary Sequence. The phosphoramidite 10 in 0.1 M acetonitrile solution was coupled four times on a CPG support bearing the ODN 11 (Scheme 11)
~ n Attachment d of
Bioconjugate Chem., Vol. 4, No. 5, 1993 383
Phenol-ODN Conjugates
Scheme I. Synthesis of Phosphoramidite Reagent Bearing a Phenol Residue M e M e
o x o
Y
o
1 B . ~
(
C
G’ R =
-3
H
Z
q w ) .N H C o ( c ~ ) , - + $ + 1
OTS Reagents:
Figure 1. Effect of the pH on the immobilization in sodium phosphate buffer 50 mM, pH 5-8, and citrate, borate, phosphate buffer, p H 9: (A) ODN 11 modified with a phenol residue, (B) unmodified ODN with the sequenceof ODN 11,negative control.
a. TsOH /Acetone b.TsCl/DMAP/ElsNICH2Ck
c. NaN 3 / NH,Cll DMF
DMTO
. ’ 0
0 CE
q(ci+,),
NHCO(CH,),+
OCOOEt
lo Scheme 11. Oligonucleotides Modified with a Phenol Residue PhOH
. ?P=OI
I
u
0\5’011gmucieotlde
’*
(sequence = 5‘GAA TCA TAC CTA CCG TGT GGT3’) according to the phosphoramidite procedure. The 5’terminal dimethoxytrityl (DMT) group was not removed, in order to act as a lipophilic “handle” on reverse-phase HPLC. In a separate experiment, the coupling efficiencies for 10 were higher than 95%, as determined photometrically (498nm) from the release of dimethoxytritylcation (0.1M toluenesulfonic acid in acetonitrile). After deprotection in 25% aqueous ammonia and evaporation, the crude compound ODN 11 was purified over HPLC using gradient 1. The main peak ( t =~22 min) was collected and the corresponding compound was detritylated (acetic acid/water 812). The fully deprotected ODN 11 showed a single peak ( t =~15 min) on HPLC (gradient 1). The complementary ODN 13 (sequence = 5’ACC ACA CGG TAG GTA TGA TTC3’)and ODN 14 (sequence = 5’TGT ACC TGA ATC GTC CGC CAT3’) were synthesized and deprotected in ammonia as described above and purified on HPLC giving a main peak at t~ = 17 min (gradient: from 25% to 50% B in 30 min).
Diazotized Cellulose Filter as Oligonucleotide Support. A suitable immobilization support should have a high capacity, should be adequately rigid and durable and should not interfere with hybridization of DNA. For solid phase type, hybridization membranes are a suitable substrate. Cellulose membranes are easy to handle, reasonablyresistant, and convenient for both hybridization and detection procedures. Cellulose membrane functionalized with amino phenyl thioether groups (APT paper) are commercially available. This aryl amine substituted cellulose is stable, can be stored a t 4 OC, and is activated just before use in ice cold sodium nitrite hydrochloric acid mixture. The immobilization described herein is based on the general method used for coupling DNA to activated supports bearing diazotized aromatic amines (3-5). Such a method applied to a short ODN would strongly affect its hybridization properties. The introduction a t one end (3’ or 6’) of the ODN of a group more reactive than guanosine should favorize the end attachment rather than random binding. The ODN are functionalized by introducing a phenol group a t their 3‘ or 5‘end during automated synthesis by mean of the phosphoramidite 10 described in Scheme I. Optimized Immobilization Conditions. We gained optimal immobilization in phosphate buffer, pH 6-7 (25 to 50mM), within 15-30 min (Figure 1). We have observed an additional 10% fixation by the pretreatment of the buffer with diazo paper. Followingthe activation the paper should be washed in sodium acetate buffer, pH 4, and then with cold H20 (2 X 1 min) in order to keep the diazonium reactivity. Effect of the Number of Phenol Residue. The capacity of covalent binding has been improved by increasing the number of phenol residues introduced on the ODN: 2-5 times with 4-10 phenol residues. Not only the higher number of potential site of attachment can explain this increase but probably the phenol linkers themselves can act as a spacer between the binding site and the ODN chain. Specificity of the Attachment. It is well-knownthat the diazonium salts react mainly with the electron-rich center at the C-8 position of the guanine residues and to a lesser extent with adenine residues which gives rise to unstable products (24). The phenol group incorporated a t the 3’- or 5’-end of the ODN is a better center for electrophilic attack than the purine bases and should develop faster kinetics. Moreover the linker attached to the phenol favors its accessibility and should allow a selective reaction. The amount of immobilized ODN waa determined by 32P-labeling of the same ODN (17). The quantity of covalently immobilized ODN is proportional to the quantity of ODN reacted to the activated cellulose support (Figure 2).
384
Fontanel et el.
Bloconlugate Chem., Vol. 4, No. 5, 1993 250
1
1
Capacity pmoles I cm2 ,
,
0 0
10
.
)fD*,
1
20 30 Time (hrs)
Figure 3. Kinetics of hybridization: ODN 11 immobilized on
the cellulose: (C) complementary ODN 13, (D)noncomplementary ODN 14. 0
1
2
concentration (OD/ mL) Figure 2. Immobilization capacity of the cellulose.
3
A covalent binding efficiency of 20-30% was achieved after 15-30 min of immobilization. The percentage of end attachment through the phenol linker was calculated taking into account the extent of binding of an unmodified ODN. We obtained about 87 % specific end attachment. The extent of passive adsorption was evaluated using a phenol modified ODN and inactivated (NaOH treatment) diazoaryl cellulose. A capacity of 200 pmol/cm2 for 2 OD units/mL loaded was evaluated (Figure 2). This binding capacity was of the same order as those previously reported for the hybridization formats involving the use of a membrane. Zhang (15) immobilized ODNs tethered at their 5’-end with aminopolyethylene glycol linker on carboxylic nylon membrane at more than 50 pmol/cm2 and 90% end attachment. 5’-NH2 ODNs were attached to carboxylic nylon membrane at 200 pmol/cm2 (16). An original format with miniaturized matrix was successfully applied to DNA sequencingand automated. Polyacrylamidegel on a glass plate is derivatized with hydrazide groups that react with ODN 3‘-uridine treated with NaI04 (19).However it failed as concerns the criteria of durability and easy handling. Other types of formats (nylon (13,magnetic latex (181, and glass (25) beads) have been developed for the hybridization of target nucleic acids. In the case of Van Ness nylon beads (17) coated with PEI, the non-immobilized activated triazinyl-ODN cannot be recycled. Solid phase synthesis has been proposed as a way for definitive immobilization: the ODN remains tethered to the glass beads after ammonia deprotection (25). HybridizationAssay Performance of the Cellulose Matrix. Our work is based on a simple model using a capture probe (21mere) immobilized on a cellulose matrix. Hybridizations were carried out with the complementary labeled probe. A non complementary labeled ODN 14 was used as a negative control. The melting temperature was first evaluated in solution by a spectroscopic method and confirmed by monitoring the hybridization on the support. Maximum rate of hybridization occurs at 25 “C below the T,. Reactions were carried out a t 20 OC below the T, at low stringency (26). Washes were performed at increasing stringency (low salt) until the cpm values of the negative control attained lower values than those of the target. In a reported hybridization study of Sephacrylbound ODN with a purified target nucleic acid, hybridization was complete after 1-2 h (27). Our experiment showed similar results. the extent of hybridization was sufficient within 2 h (Figure 3). Hybridization efficiency has been estimated to 60% with a non specific binding less than 0.5 96. Hybridization with Denhart’s reagent (BSA,volume exclusionreagents, Ficoll, and PVP) showed
no significant effect whereas prehybridization with this reagent for 1.5 h at 50 OC improved the hybridization efficiency 2-fold. This is consistent with the fact that dextran sulfate could displace from the surface adsorbed ODN probes covalently attached at their 5’-ends by disrupting the adsorbtion forces holding the ODN to the surface of the support. This adsorption could interfere with their ability to hybridize target DNA from the solution (27). Higher capture yields were observed with increasing amounts of ODNs. Iodination of the Phenol Moiety of the Modified ODN. Radioiodination of ODN can be done through a two step procedure where ODNs bearing a free amino group are reacted in a postsynthetic step with a 4-methoxyphenyl isothiocyanate (28)or a N-succinimidyl3-(4-hydroxypheny1)propanoate(23). Sauvaigoet al. (23)developed asimple method where a tyramine residue was incorporated into the ODN during the automated synthesis. The conjugated ODN is able to be radioiodinated with 200 pCi of [12511NaI according to the reported chloramine-T oxidation procedure. This method was tested for the phenol modified ODN 12 (Scheme 11). The yield of iodine incorporation was 20% and the specific activity was 3.4 Ci/kmol. Synthesis of Oligonucleotide Models. (dT)lo, X(dT)lo, and pX(dT)lo (phosphorylation was carried out with Phostel, a phosphoramidite purchased from American Bionetics, Inc. that allowed chemical phosphorylation) were synthesized and purified as described above. 5’-DMT (dT)loand X(dT)lo ODNs were purified by reverse phase HPLC. X(dT)lo: Rt = 26.7 min (gradient 1)and t~ = 12.2 min after detritylation (gradient: from 10% to 40% B in 20 min). pX(dT)lo was purified by an anion exchange column HPLC. The main peak, t~ = 11.5 min, was collected (gradient 2). LiCl was eliminated by precipitation with 5 volumes of ethanol and acetone mixture (l/l). Action of the Ta Polynucleotide Kinase (PNK) on Modified ODN with 1,2-Ethanediol Moiety. One or four 1,Zethanediol units were incorporated at the 5’-end of (dT)lo (X and X4(dT)lo) (Scheme 11). Both labeled ODNs were tested for their substrate capacity toward the phosphorylating activity of PNK. HPLC showed clearly that, within 30 min, XdTlo incorporated a phosphate group; pX(dT)lo, being more hydrophilic than its precursor, has a shorter retention time on the reverse-phase column (19.7 vs 10.8 min). The same trend was observed for X4(dT)lo (19.7 vs 21.7 min). pX(dT)lo coelutes with authentic material obtained by chemical phosphorylation. In the case of the use of [ Y - ~ ~ P I A Tand P PNK, pX(dT)lo was found to be radioactive. Both the incorporated radioactivity and the peak areas calculated from HPLC indicated a 50% yield of phosphorylation within 30 min at 37 OC. The unreacted X(dT)lo from HPLC was again allowed to react with ATP in the presence of PNK without any success. The results reported here show that a (dT)lo modified on the 5’-position with one or more non-nucleosidic
End Attachment of Phenol-ODN Conjugates
ethanediol moieties is still recognized as a substrate by the PNK. The ethanediol derivative includes a 4-[3-(4hydroxyphenyl)propionamidol butyl chain in the C-2 position. The stereochemistry of the conjugates (2R or 2 s ) determines two diastereoisomers after the incorporation in dTlo. We determined the extent of phosphorylation to be 50%, which led us to think that only one of the two diastereoisomers is recognized as a substrate by the enzyme. This supports the hypothesis of a steric hindrance that would prevent the enzyme to bind to the first internucleotide phosphate group (29). Concluding Remarks. In this study we propose the synthesis of a novel phosphoramidite reagent based on a unique 1,2-ethanediol backbone that possesses many advantageous features. It can conveniently be used in automated DNA synthesis for the direct incorporation of conjugates in oligonucleotide chains even internally at any position of the ODN. The conjugate is the substrate of choice for either radiolabeling (both iodination (26) and 5’-end phosphorylation with [ T - ~ ~ P I A Tand P Tq PNK) or immobilization on a diazotized cellulose matrix. The immobilization is site specific rather than a random attachment in photochemical cross-linkings (UV).The immobilization process is short (less than 30 min), the azo linkage is stable. Moreover, the ODN in excess during the immobilization procedure can be recycled. The capacity is higher than that previously described for the membrane attachment methods. This type of support is not compatible with colorimetric detection systems but a chemiluminescent detection can be performed. With the reverse dot procedure it may be applied in the screening of different sequences or the detection of point mutations (PUS,c-myc genes). LITERATURE CITED (1) Siddell, S. G. (1978) RNA hybridization to DNA coupled
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