Synthesis and evaluation of oligodeoxyribonucleotides containing an aryl(trifluoromethyl)diazirine moiety as the cross-linking probe: photoaffinity labeling of mammalian DNA polymerase [beta]
Synthesis and evaluation of oligodeoxyribonucleotides containing an aryl(trifluoromethyl)diazirine moiety as the cross-linking probe: photoaffinity labeling of mammalian DNA polymerase [beta]
Department of Biological Sciences, Teikyo University of Science and Technology, 2525 Yatsuzawa, Uenohara, Yamanashi 409-01, Japan
Received March 14, 1997; Revised and Accepted April 28, 1997
ABSTRACT
Photolabile 2[prime]-deoxy-E-5-[4-(3-trifluoromethyl-3H-diazirin-3-yl)styryl]uridine and its protected phosphoramidite derivatives have been synthesized and introduced into DNA oligomers through solid-phase DNA synthesis. The (trifluoromethyldiazirinyl)stylyl moiety of this nucleoside was found to be sufficiently stable for automated DNA synthesis. In addition, this moiety was found to be stable at 60°C in aqueous solution under the annealing conditions for duplex formation with complementary strands, since >95% of the photolabile nucleoside remained after heating for 1 h. The oligo(dT) 15mer analog bearing the photolabile residue was activated/decomposed by near-UV irradiation. In photoaffinity cross-linking experiments with recombinant rat DNA polymerase [beta], constituted from a 40 kDa polypeptide, using oligo(dT) 15mer analogs bearing the photolabile residue near the 3[prime]-terminus, a covalently bound complex of 45 kDa was obtained in the presence of complementary templates. Thus it was demonstrated that our method for synthesis of photolabile oligodeoxyribonucleotides may be useful for studies of DNA-related enzymes and DNA binding proteins.
INTRODUCTION
The cross-linking of target proteins or nucleic acids with oligonucleotides that contain photoreactive nucleotide residues at specific positions provides useful structural information on protein-nucleic acid (1-11) and nucleic acid-nucleic acid interactions (12-16). A satisfactory photoreactive nucleic acid can produce a covalent linkage between the photoprobe and a target molecule with minimal structural perturbation of these molecules under near-UV light irradiation. Aryl(trifluoromethyl) diazirine and arylazide derivatives are known to be candidates for such photoprobes (17-21). It has been demonstrated that aryl(trifluoromethyl)diazirines are suitably stable under various reaction conditions, whereas these photoprobes generate highly reactive carbenes upon irradiation with near-UV light (17). As nucleic acid analogs bearing a photoreactive diazirine moiety, tRNAs derivatized at the 2 or 4 position of their pyrimidine nucleotide residues (12,14) and oligodeoxyribonucleotides (ODNs) containing a diazirinylbenzoylaminoalkyl phosphonate of a thymine dimer in an oligomer (7) have been successfully used for photoaffinity labeling of rRNAs and T4 endonuclease V respectively. Such studies have provided information about specific contact points between these photoprobes and the relevant residues of the target molecules. We have selected the C-5 position of the thymidylate residue for modification, since when DNA is in its B form, the substituents at the C-5 position of pyrimidine residues are located in the major groove and do not disturb Watson-Crick base pairing. Furthermore, cross-linking by a compound which has been connected by a shorter length and more rigid spacer with the diazirine moiety may be more selective and therefore such a photoprobe should be useful for structural analysis. In a previous study, we demonstrated that the 5[prime]-triphosphate of the 2[prime],3[prime]-dideoxy analog of 11, TDSddUTP (Fig. 1), was incorporated into the 3[prime]-end of a primer by HIV-1 reverse transcriptase and the resulting photolabile primer bound to the 66 kDa subunit of the enzyme on photoirradiation (22). In this context, we have synthesized the nucleoside 11 and its phosphoramidite derivatives 13 and 17 in order to prepare ODNs containing photoreactive residues at the desired positions. Compound 17 can serve as a reagent for introducing a photolabile nucleotide at the 3[prime]-terminus of an ODN (23). The length from C-5 to the diazirine carbon is estimated to be 8.3 Å. The present paper describes the synthesis of these compounds, preparation of a photoreactive ODN using phosphoramidite chemistry and cross-linking of DNA polymerase [beta] (pol [beta]) as a model enzyme using these ODNs.
Figure 1 Synthetic route of photolabile nucleoside 11 and its protected phosphoramidite reagents 13 and 17, sequences of the photolabile ODNs dT14D and dT13DT and structure of TDSddUTP. D represents a modified uridylate residue bearing the aryl(trifluoromethyl)diazirine moiety. (a) n-Bu4NF, THF; (b) TsCl, pyridine; (c) DBU, ether; (d) 1, Li2PdCl4, MeOH; 2, Me2(t-Bu)SiCl, imidazole, DMF; (e) HONH2·HCl, KOH, EtOH; (f) liquid NH3, ether; (g) MnO2, ether; (h) DMT-Cl, pyridine; (i) (i-Pr)2NP(Cl)CH2CH2CN, EtN(i-Pr)2, DMAP, CH2Cl2; (j) (PhO)2POCl, DMAP, CH2Cl2.
MATERIALS AND METHODS
1H and 31P NMR spectra were recorded on a JEOL Alpha 500 (500 and 202 MHz respectively) spectrometer with tetramethylsilane or H3PO4 as the standard. Mass spectra were measured on a JMX-SX 102A spectrometer. HPLC analysis was performed using a Shimadzu LC-9A apparatus fitted with a YMC pack ODS A-302 (YMC Co. Ltd; 4.6 mm x 15 cm) reverse phase column or TSK gel DEAE-2SW (Tohso; 4.6 mm x 25 cm) or DEAE-NPR (Tohso; 4.6 mm x 3.5 cm) anion exchange column at a flow rate of 0.8 or 1 ml/min, run at 45°C. ODNs were synthesized using an ABI model 391 DNA synthesizer and the reagents were purchased from ABI. [3H]dTTP and [[gamma]-32P]ATP were purchased from Amersham. Nuclease P1 was purchased from Yamasa Shoyu. T4 polynucleotide kinase and bacterial alkaline phosphatase were purchased from Nippon Gene. Sephadex G-25 NAP-10 columns and the silver staining kit were purchased from Pharmacia Biotech. Recombinant rat pol [beta] (24) was provided by Dr Akio Matsukage (Aichi Cancer Center Research Institute).
p-Trifluoroacetylstyrene (4)
The silyl ether 1 (17) (17.9 g, 53.8 mmol) was deprotected by treatment with a tetrahydrofuran (THF) solution of 0.3 M tetrabutylammonium fluoride (200 ml) and allowed to stand for 4 h at room temperature. The solvent was evaporated under reduced pressure and the residue was partitioned between CH2Cl2 and water. The organic layer was dried over MgSO4. After evaporation of the solvent, the desilylated product was purified by column chromatography on silica gel (120 g) with CH2Cl2/CH3OH (19:1) to give 2 (11.1 g, 50.9 mmol, 94.6%) as a colorless oil. A solution of 2 (4.5 g, 20.6 mmol) and p-toluenesulfonyl chloride (TsCl) (5.2 g) in pyridine (60 ml) was stirred for 12 h at room temperature. After evaporation of the solvent, the residue was dissolved in ether and the organic solution was washed with brine and dried over MgSO4. The solvent was evaporated, and the crude tosylate (3) was purified by column chromatography on silica gel (100 g) with CH2Cl2/hexane (1:1) to give 3 (4.8 g, 14.6 mmol) as a yellowish oil. This material was used in the next step. A mixture of 3 (4.8 g) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (4.3 ml, 28 mmol) in ether was stirred vigorously for 10 h at room temperature and then the reaction was quenched by addition of water. The organic layer was separated, washed with brine and 0.5 N aqueous HCl and brine, dried over MgSO4 and the solvent evaporated to give 4 (2.19 g, 10.9 mmol, 53% from 2) as a yellowish oil. 1H NMR (CDCl3) [delta]: 5.50 and 5.94 (d × 2, 1H × 2, vinylic-CH2), 6.79 (dd, 1H, olefin, J = 11 and 17 Hz), 7.55 and 8.05 (d × 2, 2H × 2, aromatic). MS m/z: 200 (M)+.
Well-dried 5 (25) (6.4 g, 13.8 mmol) was suspended in CH3OH (70 ml) and then styrene 4 (3.2 g, 16.0 mmol) and a methanolic solution of 0.1 M lithium tetrachloropalladate (150 ml) was added. After being refluxed for 2 h, the mixture was filtered and the filtrate was evaporated. The brown residual syrup was chromatographed on a column of silica gel (200 g) with CHCl3/CH3OH (9:1). Fractions containing the product, which fluoresced upon irradiation with UV light (366 nm), were collected. After evaporation of the solvent, the residue was chromatographed again under the same conditions. The solvent was evaporated and co-evaporated with toluene. To a suspension of the resulting residue (3.3 g) and imidazole (3.7 g) in N,N-dimethylformamide (DMF) (30 ml) was added t-butyldimethylsilyl chloride (3.3 g, 22 mmol). After stirring for 4 h, the reaction mixture was evaporated and the residue was partitioned between CH2Cl2 and water. The organic layer was washed with water, dried over MgSO4 and the solvent evaporated. The residue was purified by column chromatography on silica gel (100 g) with CH2Cl2/ethyl acetate (5:1) to give 6 (1.9 g, 2.9 mmol, 21.0% from 5) as a yellowish gum. 1H NMR (CDCl3) [delta]: 0.09, 0.10, 0.13 and 0.14 (s × 4, Si-Me), 0.91 and 0.93 (s × 2, t-butyl), 1.95 and 2.27 (m × 2, 1H × 2, H-2[prime]), 3.7-3.9 (m, 2H, H-5[prime]), 3.92 (dd, 1H, H-4[prime]), 4.33 (m, 1H, H-3[prime]), 6.22 (dd, 1H, H-1[prime]), 6.79 and 7.60 (d × 2, 1H × 2, olefin, J = 15 Hz), 7.46 and 7.93 (d × 2, 2H × 2, aromatic), 7.77 (s, 1H, H-6). FAB-MS m/z: 677 (M+Na)+.
To a stirred mixture of hydroxylamine hydrochloride (2.0 g, 28 mmol) and KOH (0.9 g, 28 mmol) in boiling 2-propanol (40 ml) was added a solution of 6 (3.54 g, 5.40 mmol) in 2-propanol (20 ml) and the mixture was refluxed for 20 h. After evaporation of the solvent, the residue was partitioned between water and ether. The organic layer was washed with brine, dried over MgSO4 and evaporated to give crude 7 (3.3 g) as a yellowish gum. A solution of crude 7 (3.3 g) and TsCl (1.4 g, 7.3 mmol) in pyridine (25 ml) was refluxed for 2.5 h. After evaporation of the solvent, the residue was partitioned between water and CH2Cl2. The organic layer was washed with water and dried over MgSO4. The solvent was evaporated and the residual crude oxime tosylate was purified by column chromatography on silica gel (70 g) with CHCl3/ethyl acetate (2:1) to give 8 (3.20 g, 3.88 mmol, 71.9% from 6) as a yellowish gum. 1H NMR (CDCl3) [delta]: 0.09, 0.10, 0.13 and 0.14 (s × 4, Si-Me), 0.91 and 0.93 (s × 2, t-butyl), 1.97 and 2.26 (m × 2, 2H, H-2[prime]), 2.38 (s, 3H, Ar-CH3), 3.7-3.8 (m, 2H, H-5[prime]), 3.91 (dd, 1H, H-4[prime]), 4.32 (m, 1H, H-3[prime]), 6.21 (dd, 1H, H-1[prime]), 6.70 and 7.52 (d × 2, 1H × 2, olefin, J = 15 Hz), 7.2-7.4 and 7.75-7.8 (m, aromatic), 7.71 (s, 1H, H-6). FAB-MS m/z: 846 (M+Na)+.
The oxime tosylate (8) (3.10 g, 3.76 mmol) was dissolved in ether (60 ml) and cooled to -78°C. Liquid ammonia (30 ml) was added and the mixture in a sealed tube was left to stand for 20 h at room temperature. After evaporation of excess ammonia, precipitates were removed by filtration and the filter cake was washed with ether. The filtrate and the washes were combined and then condensed under reduced pressure. The residue was purified by column chromatography on silica gel with CHCl3/ethyl acetate (2:1) to give 9 (2.19 g, 3.27 mmol, 87.1%) as a yellowish gum. 1H NMR (CDCl3) [delta]: 0.09, 0.10, 0.12 and 0.13 (s × 4, Si-Me), 0.91 and 0.92 (s × 2, t-butyl), 2.04 and 2.34 (m × 2, 2H, H-2[prime]), 3.80 and 3.92 (dd × 2, 1H × 2, H-5[prime]), 4.00 (dd, 1H, H-4[prime]), 4.42 (m, 1H, H-3[prime]), 6.32 (dd, 1H, H-1[prime]), 6.76 (d, 1H, olefin, J = 15 Hz), 7.45-7.6 (m, 5H, aromatic and olefin), 7.78 (s, 1H, H-6). FAB-MS m/z: 678 (M)+.
A mixture of diaziridine 9 (2.10 g, 3.14 mmol) and MnO2 ( 5.8 g) in ether (45 ml) was stirred vigorously for 30 min at room temperature. MnO2 was removed by filtration and the filtrate was evaporated to dryness. The residue was purified by column chromatography on silica gel (70 g) with CHCl3/ethyl acetate (4:1) to give 10 (1.64 g, 2.46 mmol, 78.3%) as a yellowish gum. To a solution of this material 10 (1.60 g, 2.40 mmol) in THF (100 ml) was added 1 M tetrabutylammonium fluoride/THF (6.5 ml) and the mixture was stirred for 2 h at room temperature. After evaporation of the solvent the desired compound (11) was purified by column chromatography on silica gel (50 g) with CHCl3/C2H5OH (19:1) to give 11 (0.62 g, 1.41 mmol) as a yellowish powder. UV (H2O) [lambda]max 315 nm ([epsis] 22 000), (50 mM NaOH) [lambda]max 320 nm ([epsis] 25 000). 1H NMR (CDCl3) [delta]: 2.16-2.18 (m, 2H, H-2[prime]), 3.62 and 3.68 (m × 2, 1H × 2, H-5[prime]), 3.82 (dd, 1H, H-4[prime]), 4.29 (dd, 1H, H-3[prime]), 6.18 (dd, 1H, H-1[prime]), 6.96 and 7.42 (d × 2, 1H × 2, olefin, J = 16.5 Hz), 7.20 and 7.42 (d × 2, 2H × 2, aromatic), 8.26 (s, 1H, H-6). HR-FAB-MS m/z: 461.1052 (M+Na)+. Calculated for C19H17F3N4O5Na: 461.1049.
To a solution of dimethoxytrityl ether 12 (0.4 g, 0.54 mmol), diisopropylethylamine (0.4 ml) and DMAP (0.02 g) in CH2Cl2 was added N,N-diisopropylchlorophosphoramidite (0.2 ml) and the mixture was stirred for 15 min at room temperature. The reaction was quenched by addition of ethyl acetate and water. The organic layer was separated, washed with brine and dried over MgSO4. After evaporation of the solvent, the residue was purified by column chromatography on silica gel (30 g) with CH2Cl2/ ethyl acetate (9:1) containing 1% triethylamine to give 13 (0.40 g, 0.43 mmol, 78%) as a colourless foam. 31P NMR (CDCl3) [delta]: 148.47, 148.91. FAB-MS m/z: 963 (M+Na)+.
To a solution of p-bromophenethyl alcohol (10 g, 49.7 mmol) and imidazole (7.8 g) in DMF (100 ml) was added t-butyldiphenylsilyl chloride (12.9 ml, 49.7 mmol). After stirring for 1 h at room temperature, the reaction mixture was evaporated and the residue was partitioned between water and ether. The organic layer was washed with water, dried over MgSO4 and the solvent was evaporated. The residue was purified by column chromatography on silica gel (100 g) with hexane/CH2Cl2 (9:1). The fractions containing the product were combined, and the solvent was evaporated to give 4-[2-[(t-butyldiphenylsilyl)oxy]ethyl]bromobenzene (20.3 g) as a colorless oil. This material (20.0 g, 49.4 mmol), magnesium turnings (1.20 g, 49.4 mmol) and THF (100 ml) were placed in a round-bottomed flask. A drop of iodomethane was added and the mixture was heated at 80°C with stirring until most of the magnesium turnings had dissolved. The reaction mixture was then cooled to -10°C. Dried CO2 gas was introduced into the reaction mixture below -5°C with stirring and the procedure was continued until the exothermic reaction had ceased. The mixture was hydrolyzed with saturated aqueous NH4Cl (10 ml). Water (80 ml) was added and the pH of the aqueous layer was adjusted to 4 by addition of 6 N HCl. After addition of ether (80 ml), the organic layer was separated and dried over MgSO4. After evaporation of the solvent, the residue was purified by column chromatography on silica gel (100 g) with CHCl3/CH3OH (19:1) to give 14a (11.1 g, 27.4 mmol, 55.5% from p-bromophenethyl alcohol ) as colorless fibers, mp 105- 107°C (ether/hexane). 1H NMR (CDCl3) [delta]:1.01 (s, 9H, t-butyl), 2.90 and 3.87 (t × 2, 2H × 2, methylenes), 7.25-8.00 (m, 14H, aromatic). Calculated for C25H28O3Si: C, 74.22; H, 6.98. Found: C, 74.25; H, 7.00.
To a solution of 14a (0.40 g, 0.99 mmol) and DMAP (1.2 g) in CH2Cl2 (60 ml) was added diphenylphosphoryl chloride (0.27 g, 1.3 mmol). The mixture was stirred for 15 min at room temperature, then 12 (0.63 g, 0.85 mmol) was added. Stirring was continued for 12 h at room temperature and the reaction was quenched by addition of saturated aqueous NaHCO3. The organic layer was washed with brine and dried over MgSO4. After evaporation of the solvent, the residue was purified by column chromatography on silica gel (50 g) with CH2Cl2/ethyl acetate (9:1) to give 15 (0.59 g, 0.52 mmol, 61%). To a solution of 15 (0.59 g, 0.52 mmol) in THF (10 ml) was added 1 M tetrabutylammonium fluoride/THF (0.68 ml) and the mixture was stirred for 1 h at room temperature. After evaporation of the solvent, the residue was dissolved in CH2Cl2, washed with water and dried over MgSO4. After evaporation of the solvent, the residue was purified by column chromatography on silica gel (50 g) with CH2Cl2/ethyl acetate (1:2) containing 1% triethylamine to give the desilylated product 16 (0.22 g, 0.25 mmol) as a pale yellowish gum. Phosphoramidite 17 was prepared in a manner analogous to 13, except that the reaction mixture was stirred for 5 min at room temperature; 0.17 g (0.15 mmol, 29% from 15) of 17 was obtained as a colorless foam. 31P NMR (CDCl3) [delta]: 147.50. FAB-MS m/z: 1090 (M+H)+.
Synthesis of ODNs
Oligo(dT) 15mer analogs dT14D and dT13DT were synthesized on a 1 [mu]mol scale using an automated DNA synthesizer. The phosphoramidites 13 and 17 were used as 0.12 M solutions in acetonitrile. The oligomer bearing the photoreactive residue at the 3[prime]-terminus, dT14D, was synthesized using 17 and a commercially available solid support as starting materials. The cleavage of ODN from the solid support and deprotection of the cyanoethyl and acyl protecting groups were performed by treatment with 40% methylamine/concentrated ammonium hydroxide (1:1 v/v) for 35 min at 37°C, as reported by Reddy et al. (26). Deprotection of the dimethoxytrityl (DMT) group and simplified purification of ODN were performed using a reverse phase OPC cartridge (ABI). Further purification was carried out on a TSK gel DEAE-2SW column using a linear gradient of 0.2-1.1 M ammonium formate containing 20% acetonitrile over 60 min. The fractions containing ODNs were combined and concentrated to half volume under reduced pressure. The solution was diluted with 3 vol. 1 M NaCl and desalted using an OPC cartridge. A solution of dT14D in 10 mM acetate buffer, pH 5.3, was treated with nuclease P1, which cleaves polynucleotides to give 5[prime]-monophosphates. Furthermore, it was treated with alkaline phosphatase in 50 mM Tris-HCl, pH 8.0, containing 1 mM MgCl2 and analyzed by HPLC. As expected, TDSdU (11) was shown to be released.
For preparation of the annealed template/primer, an aqueous mixture of template and primer was heated at 60°C for 5 min with subsequent slow cooling to room temperature over 1 h. This mixture was used for assay or cross-linking experiments.
Assay for pol [beta] activity
The reaction mixture (25 [mu]l) comprised 50 mM Tris-HCl, pH 8.8, 1 mM dithiothreitol, 100 [mu]g/ml bovine serum albumin, 100 mM KCl, 0.5 mM MnCl2, 50 [mu]g/ml poly(A)/oligo(dT)12-18 or poly(A)/synthetic ODN (4:1 w/w), 50 [mu]M [3H]dTTP (110 c.p.m./ pmol) and 1 [mu]l enzyme preparation. When poly(dA) was used as the template, poly(A) was omitted and 4 mM MgCl2 instead of 0.5 mM MnCl2 was added. Incubation was carried out for 20 min at 25°C and the radioactivity in the polynucleotides was determined (24).
End-labeling of synthetic ODNs dT14D and dT13DT
The ODNs were phosphorylated at the 5[prime]-end using [[gamma]-32P]ATP and T4 polynucleotide kinase (27). After the reaction, the mixture was extracted with phenol and low molecular weight materials were removed by gel filtration using a Sephadex G-25 NAP-10 column. The eluate was concentrated under reduced pressure at 35°C.
Photoaffinity cross-linking of pol [beta] with photolabile ODNs
The reaction mixture (7.5 [mu]l) comprised 50 mM Tris-HCl, pH 8.8, 1 mM dithiothreitol, 100 mM KCl, annealed template/synthetic ODN [40 [mu]g/ml poly(A) and photolabile ODN (dT14D or dT13DT)], 40 ng (1 pmol) pol [beta] and 0.5 mM MnCl2. When poly(dA) was used as the template instead of poly(A), 4 mM MgCl2 instead of 0.5 mM MnCl2 was added. Incubation was carried out for 10 min at 25°C. After chilling at 0°C, the reaction mixture was irradiated with a near-UV lamp (UVP Inc., model BA100AF) at a distance of 8 cm (11 000 [mu]W/cm2, 365 nm) for 10 min in an ice bath. The product was analyzed by SDS-PAGE (10% separation gel) (28) and detected by autoradiography or silver staining.
RESULTS
Synthesis and characterization of the ODNs
The synthesis of photolabile nucleoside 11 and its protected phosphoramidites 13 and 17 is outlined in Figure 1. The silyl protecting group of the substituted trifluoroacetophenone (1) (17) was removed and the resulting hydroxyethyl group of 2 was converted to a vinyl group to obtain styrene analog 4. Introduction of a styryl group at C-5 of 2[prime]-deoxyuridine was performed through a palladium-catalyzed coupling reaction of 5 with 4 (25,29), followed by protection with t-butyldimethylsilyl to yield compound 6 in 21% yield in two steps. Conversion of 6 to 10 was achieved in 49% yield in four steps according to a conventional method (17,30-32), but with slight modifications. After removal of the silyl protecting group of 10, the desired nucleoside TDSdU (11) was obtained. Proton NMR spectra of 11 showed that the coupling constant (J) between the olefin protons of the styryl moiety was 16.5 Hz, indicating that the styryl substituent was trans. Standard dimethoxytritylation and phosphitylation of 11 yielded phosphoramidite 13. In order to introduce the photolabile nucleotide at the 3[prime]-terminus of ODNs, the 3[prime]-hydroxyl group of 12 was protected with a functionalized benzoyl group as reported previously (23). However, in the present study silyl ether 14a instead of benzyl ether 14b was introduced at the 3[prime]-OH of 12, followed by treatment with tetrabutylammonium fluoride to give 16, since the diazirine ring may be sensitive to the hydrogenation necessary for debenzylation. Further activation of the resulting primary alcohol of 16 as the phosphoramidite afforded compound 17. The photolabile oligo(dT)15 analogs dT14D and dT13DT were obtained by automated synthesis. After purification by HPLC, almost pure dT14D and dT13DT were obtained, as shown in Figure 2.
Figure 2 HPLC profiles of dT14D. Samples ([sim]0.005 OD260 units) were analyzed by HPLC on a TSK gel DEAE-NPR column and detected at 260 nm; elution was with 20 mM Tris-HCl, pH 8.8, and a linear gradient of 0.2-1.0 M NaCl for 15 min. (a) dT14D purified by an OPC cartridge and (b) further purified by HPLC on a TSK gel DEAE-2SW column.
Figure 3 Heat stability of TDSdU (11) at various temperatures. After incubation of an aqueous solution of 11 for various periods at 60 (a), 70 (b), 80 (c) or 90°C (d), [sim]0.01 OD310 units of sample were analyzed by HPLC on a YMC pack ODS A302 column and detected at 310 nm; elution was with 50 mM Et3NHOAc, pH 7.0, containing 45% CH3CN.
It is desirable for photolabile ODN to be stable during annealing for preparation of a complementary duplex. Therefore, the heat stability of the (trifluoromethyldiazirinyl)styryl group in aqueous solution was investigated using TDSdU (11) as a model compound. Although TDSdU (11) was immediately decomposed at 80°C, it was found to be sufficiently stable at 60°C, since >95% of 11 remained after heating for 1 h (Fig. 3), and thus applicable for various cross-linking experiments. In addition, 97% of the initial TDSdU (11) remained after heating in concentrated ammonium hydroxide for 8 h at 55°C or 40% methylamine/concentrated ammonium hydroxide (1:1 v/v) for 2 h at 37°C (data not shown). Treatment of 11 for 2 h at room temperature with the reagent (0.02 M iodine/pyridine/THF/water) for the oxidation step during DNA synthesis resulted in no detectable decomposition, as confirmed by TLC analysis (data not shown).
The photodecomposition reaction of dT14D was studied by plotting the change in the UV spectrum of this photolabile ODN in aqueous solution at different irradiation times. This ODN was rapidly decomposed by near-UV light irradiation, as shown in Figure 4. On the basis of this result, it was decided to perform photoirradiation for 10 min at 0°C in the present study.
Figure 4 UV spectra of dT14D after different periods of near-UV irradiation. An aqueous solution of dT14D (10 [mu]g/ml) was photolyzed by near-UV light (365 nm) at 0°C. Irradiation times were: (a) 0 min; (b) 2 min; (c) 5 min; (d) 10 min.
We investigated whether pol [beta] utilizes synthetic photolabile oligomers as primers in the presence of template poly(A) or poly(dA) (Fig. 5). The ODN bearing the bulky photolabile moiety at the 3[prime]-terminus showed low primer activity when poly(A) was used as the template. The ability of dT13DT to act as a primer was shown to be 75-80% that of oligo(dT)16 in the case of both poly(A) and poly(dA).
Figure 5 Primer utilization of recombinant rat pol [beta]. The DNA polymerase activity was measured under the conditions described in Materials and Methods. Base ratio, A to T, was 4. The 100% value was defined as the quantity of dTMP incorporated by the enzyme when poly(A)/oligo(dT)16 was used as the template/primer. The data represent the average of three separate experiments.
Cross-linking of recombinant rat pol [beta] with the photoprobes dT14D and dT13DT
The labeling abilities of the newly synthesized photoprobes (dT14D and dT13DT) and their 32P 5[prime]-end-labeled derivatives were investigated using recombinant rat pol [beta] (24), which showed a molecular mass of 40 kDa when analyzed by SDS-PAGE. Each of the 32P 5[prime]-end-labeled ODNs and the template were mixed with rat pol [beta] and bovine serum albumin (BSA) (1:1 w/w) and irradiated with near-UV light. Autoradiography of the radioactive products resolved by SDS-PAGE showed a shift in the molecular mass of the labeled enzyme species to the 45 kDa position in both cases (Fig. 6a). The 5 kDa increase in molecular mass is consistent with a 40 kDa polypeptide cross-linked to an oligonucleotide of [sim]4800 molecular weight. BSA in the presence of the enzyme was not cross-linked with 32P-labeled ODNs (Fig. 6a, lanes 1, 3, 5 and 6). Poly(dA) was less effective for production of the 45 kDa photoadduct than poly(A). As shown in Figure 6b, the 45 kDa photolabeled product could also be detected by silver staining without using a radioactive probe and [sim]1-2% of the enzyme protein appeared to react with photoprobe dT14D, judging from the staining intensity after SDS-PAGE analysis. Production of the 45 kDa band was more effective in the presence of 10 [mu]g/ml poly(A) than in its absence.
Figure 6 Photoaffinity cross-linking of rat DNA pol [beta] with the photolabile ODNs and their 32P 5[prime]-end-labeled derivatives and the results obtained in the presence of increasing poly(A) and poly(dA)/oligo(dT)16. Photoaffinity cross-linking experiments were carried out as described in Materials and Methods. Concentrations of templates and photolabile ODNs used were 40 [mu]g/ml poly(A) or poly(dA) and 0.2 [mu]g/ml 32P 5[prime]-end-labeled ODNs in (a) and the indicated concentrations of poly(A) and 8 [mu]g/ml dT14D in (b). In (c), 5 [mu]g/ml poly(dA) and 1 [mu]g/ml 32P 5[prime]-labeled dT14D (i) or dT13DT (ii) in the presence of the indicated concentrations of poly(dA)/oligo(dT)16 (5:1) were used. The products were analyzed by SDS-PAGE and detected by autoradiography (a and c) or silver staining (b).
Cross-linking of pol [beta] with poly(dA)/photolabile ODN was inhibited by addition of the natural template/primer, poly(dA)/ oligo(dT)16 (Fig. 6c). This means that poly(dA)/photolabile ODN might act as a template/primer for pol [beta]. In cross-linking experiments in the absence (Fig. 6a, lanes 2 and 4) or presence of the enzyme (Fig. 6a, lanes 1, 3, 5 and 6), another major band was obtained at a size of >200 kDa. Photolabile ODNs dT14D and dT13DT seemed to react with the templates, although the details were not investigated.
As described above, a photoaffinity cross-linking experiment with pol [beta] showed that poly(dA) was less effective than poly(A) as the template. This result was inconsistent with the finding that pol [beta] activity preferentially utilized poly(dA) rather than poly(A) (Fig. 5). Presumably, pol [beta] does not possess its reactive sites near the carbene generated from dT14D or dT13DT, which is in its B form duplex with a DNA template. Another possibility is that the generated carbene molecule reacts preferentially with poly(dA) rather than with pol [beta].
DISCUSSION
In a previous paper we reported that a (trifluoromethyldiazirinyl)- styryl group was introduced into the 5 position of 2[prime],3[prime]-dideoxyuridine by the coupling reaction of p-(trifluoromethyldiazirinyl)- styrene and 5-chloromercuriuridine derivative (33). However, the isolated yield of the desired compound was low (<10%), perhaps due to instability of the diazirine ring during the coupling reaction. Furthermore, it was difficult to separate the desired compound from various unidentified products which showed a similar chromatographic behavior. In this paper a reliable synthesis of TDSdU (11) has been described. However, introduction of a trifluoroacetylstyryl group to the 2[prime]-deoxyuridine derivative (5) was carried out in low yield and the procedure remains to be improved.
For introduction of the modified residue at the 3[prime]-terminus of ODNs, we designed and synthesized a new phosphoramidite 17, which connected the 3[prime]-hydroxyl group and phosphoramidite moiety by an alkaline-labile linker (23). After automated DNA synthesis, simultaneous removal of cyanoethyl and acyl protecting groups and cleavage of the ester bond between the nucleotide and the linker with ammonium hydroxide gave an ODN carrying the photolabile residue at the 3[prime]-terminus. Instead of 3[prime]-O-succinylation and a two-phase coupling reaction of DMT-TDSdU (12) to an amino-functionalized solid support, commercially available supports (normal 2[prime]-deoxynucleoside-attached CPGs) can be used as a starting material in the present method.
The labeling abilities of the newly synthesized photoprobes (dT14D and dT13DT) were confirmed using recombinant rat pol [beta]. In a previous study we demonstrated that TDSddUTP (Fig. 1) was incorporated into the 3[prime]-end of the primer strand by HIV-1 reverse transcriptase and that the resulting photolabile primer bound effectively to the large subunit of the enzyme on photoirradiation (22). However, rat pol [beta] was not labeled by this method using TDSddUTP in the presence of poly(A)/oligo(dT) and Mg2+. It seems likely from the result of the present study using dT14D that pol [beta] may not be able to utilize TDSddUTP effectively as a substrate.
Based on a cross-linking experiment using another enzyme, we have already reported preliminary results obtained with HIV-1 reverse transcriptase and poly(A)/dT14D. We demonstrated that the 66 kDa subunit of the p66/p51 heterodimeric enzyme protein reacted selectively with the oligomer (34).
In general, photolabile moieties, aryl(trifluoromethyl)diazirines or arylazides have been introduced lastly into ODNs bearing a reactive functionalized group (amino or thiol) or incorporated enzymatically via a synthetic route. Herein, the (trifluoromethyldiazirinyl)styryl moiety was found to be sufficiently stable under the required conditions for DNA synthesis and cross-linking experiments and we have demonstrated that direct synthesis of photolabile ODNs was actually possible using phosphoramidite 13 and/or 17 as starting materials. Similarly, the present synthetic method may be applicable for the preparation of ODNs containing photolabile nucleosides which can be synthesized from 5-aminoalkyldeoxyuridines (2,35) and the succinimide ester of 3-(trifluoromethyl)diazirin-3-ylbenzoinc acid (30,31). These should facilitate the preparation and use of photolabile ODNs and expand the study of DNA and protein interactions.
ACKNOWLEDGEMENTS
We would like to thank Dr Akio Matsukage, Aichi Cancer Center Research Institute, for kindly providing recombinant rat pol [beta]. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan, to T.Y. This paper constitutes Part 39 of the series `Synthetic Nucleosides and Nucleotides'. [Part 38: Yamaguchi,T. and Saneyoshi,M. (1996) Nucleic Acids Res., 24, 3364-3369.] This paper is dedicated to Professor Shiro Ikegami on the occasion of his 60th birthday.
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Y. Mizushina, T. Ohkubo, T. Date, T. Yamaguchi, M. Saneyoshi, F. Sugawara, and K. Sakaguchi Mode Analysis of a Fatty Acid Molecule Binding to the N-terminal 8-kDa Domain of DNA Polymerase beta . A 1:1 COMPLEX AND BINDING SURFACE
J. Biol. Chem.,
September 3, 1999;
274(36):
25599 - 25607.
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