Synthesis of New Modified DNAs by Hyperthermophilic DNA

Novel thymidine analogue triphosphates, which have an sp3-hybridized carbon at the C5 α-position with amino-linker arms, a methyl ester, or a carboxy...
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Bioconjugate Chem. 2002, 13, 309−316

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Synthesis of New Modified DNAs by Hyperthermophilic DNA Polymerase: Substrate and Template Specificity of Functionalized Thymidine Analogues Bearing an sp3-Hybridized Carbon at the C5 r-Position for Several DNA Polymerases Hiroki Sawai,* Akiko Ozaki-Nakamura, Masayuki Mine, and Hiroaki Ozaki Department of Applied Chemistry, Gunma University, Kiryu, Gunma 376-8515, Japan. Received September 12, 2001; Revised Manuscript Received December 31, 2001

Novel thymidine analogue triphosphates, which have an sp3-hybridized carbon at the C5 R-position with amino-linker arms, a methyl ester, or a carboxyl group at the C5 sidearm, were good substrates for primer-extension reactions by DNA polymerase from Pyrococcus kodakaraensis (KOD Dash DNA polymerase), yielding exclusively full-length products. The resulting modified DNA was further alllowed to react with a functional molecule such as fluorescein isothiocyanate. By contrast, only truncated products were formed from the thymidine analogue substrate bearing the amino-linker arm or the negatively charged carboxyl group using Taq, Tth DNA polymerase, or DNA polymerase I from E. coli (Klenow fragment). The results indicate either that the thymidine analogue was not accepted by the enzymes, or that the polymerases could not extend the products, once the analogue had been incorporated, depending on the type of the analogue. A conventional thymidine analogue bearing an aminopropenyl group at the C5-position was accepted by all enzymes, among which KOD Dash DNA polymerase showed the highest activity for the polymerization with this analogue. Templates bearing the thymidine analogues in place of one thymidine residue were read by KOD Dash, Taq, Tth DNA polymerases, and the Klenow fragment giving the full-length product. KOD Dash DNA polymerase could expand structural diversities of substrates that can be used to prepare modified DNAs.

INTRODUCTION

Modified oligonucleotides with a variety of functional groups are important tools for biological and biochemical studies (1-3). The C5-position of the pyrimidine ring is an ideal site for the modification, because it is located in the major groove of double-stranded DNA and does not inhibit A:T base pairing. Functional groups such as fluorophores (3, 4), enzymes (3, 4), biotin (3, 4), metal chelators (6, 7), polyamines (8), and intercalating agents (9, 10) have been attached to C5 via amino-linker arms. Previously, we reported the synthesis of thymidine analogues, C5-substituted 2′-deoxyuridines bearing aminolinker arms at the C5-position, and their introduction into oligodeoxyribonucleotides (11, 12). Usually, modified DNA is prepared chemically by using a DNA synthesizer. However, if a DNA polymerase can accept modified nucleotides as substrates, modified DNA can be prepared enzymatically. The resulting modified DNA could be used as a DNA probe by attachment of a reporter group, or as a catalytic DNA by in vitro selection. Thus, nucleotide analogues that are substrates for DNA and RNA polymerases can be powerful tools for biochemical studies. Modified ribonucleoside 5′-triphosphates are substrates for several RNA polymerases, and are used to create various functionalized RNA molecules with catalytic activity or binding activity (13-21). However, there are a few examples of functionalized DNA molecules prepared using modified deoxynucleoside 5′-triphosphates, because DNA polymerases accept a limited range of molecular structures as a substrate. Some DNA poly* Address correspondence to this author. E-mail: sawai@ chem.gunma-u.ac.jp. Tel: 81-277-30-1220. Fax: 81-277-30-1224.

merases can use 5′-triphosphates of modified 2′-deoxyuridines with a C5 side chain carrying an (E)-propenyl or propynyl group (22-31). Vent or Taq DNA polymerases can use 2′-deoxyuridine derivatives carrying a triple bond at the C5 R-position, yielding the corresponding modified DNAs (24, 26, 29). 2′-Deoxyuridine derivatives bearing a propenyl group at the C5 R-position are good substrates for Taq DNA polymerase, but poor substrates for Vent or Pfu DNA polymerase (25, 27, 30). The Klenow fragment incorporates 2′-deoxyuridine derivatives bearing a propenyl group at the C5-position, but with low efficiency (22, 23). Reduction of the double or triple bond of the C5 propenyl- or alkynyl-substituted 2′deoxyuridines significantly inhibits their function as a substrate (30). These results indicate that an sp- or sp2hybridized carbon at the C5 R-position of the C5substituted 2′-deoxyuridine is required as a substrate for some DNA polymerases (22-31). The differences in reactivity of C5-substituted 2′-deoxyuridines as a substrate may be due to the steric effect of a large side group, or to the ionic effect of a substituent group such as an amino group. The ability of the modified nucleotides as a substrate also depends on the kind of DNA polymerase. We prepared several triphosphates of thymidine analogues, which have an sp3-hybridized carbon at the C5 R-position with either amino-linker arms or an ester or carboxylic group at the terminus, and have examined for their specificity as a substrate for several DNA polymerases in the primer-extension reaction. We have found that KOD Dash DNA polymerase readily uses the thymidine analogue nucleotides as a substrate and reads a template containing the modified thymidine, but no other DNA polymerase tested, including Taq DNA polymerase,

10.1021/bc010088l CCC: $22.00 © 2002 American Chemical Society Published on Web 02/06/2002

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can accept the thymidine analogues as a substrate. KOD Dash DNA polymerase is a hyperthermophilic DNA polymerase and now commercially available. The enzyme was originally isolated from the extremely thermophilic archaeum Pyrococcus kodakaraensis, found in a hot spring on Kodakara Island in southern Japan, and is reported to have high DNA polymerization activity with high fidelity (32). This is the first report that a thymidine analogue which has an sp3-hybridized carbon at the C5 R-position can function as a substrate. The enzyme also accepts the conventional thymidine analogues bearing an aminopropenyl group more readily than other enzymes. The finding that KOD Dash DNA polymerase has broader substrate specificity than other polymerases may expand the variety of nucleotide analogues that can be used for enzymatic modified DNA synthesis. MATERIALS AND METHODS

Materials. 2′-Deoxynucleoside 5′-triphosphates (dATP, dGTP, dCTP, and TTP) were purchased from Seikagaku Kogyo. 2′-Deoxyuridine 5′-triphosphate (dUTP) was a gift from Mr. H. Kusakabe of Yamasa Co. Snake venom phosphodiesterase and alkaline phosphatase were from Worthington Biochemicals, and nuclease P1 was from Yamasa Co. Klenow fragment of E. coli DNA polymerase I and Taq DNA polymerase were purchased from Takara, and KOD Dash and Tth DNA polymerases were from Toyobo Co. [R-32P]dGTP (800 Ci/mmol) and [R-33P]ddNTP (A, G, C, and T) (1500 Ci/mmol, 450 µCi/mL) were from Amersham. C5-substituted 2′-deoxyuridine derivatives, 5-N-(6-trifluoroacetylaminohexyl)carbamoylmethyl-2′deoxyuridine (1), 5-(methoxycarbonylmethyl)-2′-deoxyuridine (2), and 5-[N-2-[N,N-bis(2-trifluoroacetylaminoethyl)amino]ethyl]carbamoylmethyl-2′-deoxyuridine (3), were prepared as described previously (11, 12). A radiolabeled DNA marker was prepared from the template and primer DNA using a thermo sequenase radiolabeled terminator cycle sequence kit obtained from USB Co. according to the method of the manufacturer. All other chemicals were reagent grade and were used without further purification. Oligonucleotides. Oligodeoxyribonulceotides were synthesized on an Applied Biosystems model 381 A DNA synthesizer using deoxyribonucleoside 2-cyanoethyl phosphoramidites. Normal nucleoside phosphoramidites were purchased from Applied Biosystems. Phosphoramidites of the thymidine analogues bearing ethylenediamine, hexamethylenediamine, and tris(2-aminoethyl)amine at the C5-position were prepared as described previously (12). Oligodeoxyribonucleotides (37mer) bearing a thymidine analogue in place of one thymidine residue were prepared along with the normal oligodeoxyribonucleotide, and used as the template for DNA polymerase reactions. Analytical Methods and Gel Electrophoresis. 1H and 31P NMR spectra were obtained with a JEOL R-500. Triphosphates of 2′-deoxyuridine derivatives for NMR measurement were passed through a Dowex 50-WX-8 (Na+ form) column to convert the triethylammonium salt to the sodium salt. Tetramethylsilane (TMS) and 85% phosphoric acid were used as the internal standards for 1 H and 31P NMR, respectively. ESI mass spectra were recorded on a PE Sciex API-100. MALDI TOF mass spectra were taken with a Shimadzu AXIMA-CF. UV spectra were obtained with a Hitachi 3200 spectrometer. High-pressure liquid chromatography (HPLC) on an ODS-silica gel column (4 mm × 250 mm) was carried out with a linear gradient elution (2-40% acetonitrile in 50 mM triethylammonium acetate, pH 7.0) over 30-40 min at a flow rate of 1.0 mL/min. Gel electrophoresis was

Sawai et al.

carried out using 0.5 mm thick denaturing15% polyacrylamide gels containing 7 M urea, and run at 2000 V for 2.5-3 h. The gels were autoradiographed with Kodak BioMax MS film at -70 °C. Synthesis of 5′-Triphosphates of 2′-Deoxyuridine Derivatives. (A) 5-N-(6-Trifluoroacetylaminohexyl)carbamoylmethyl-2′-deoxyuridine 5′-Triphosphate (4). 5′Triphosphorylation of the modified nucleosides was carried out by the method described by Ludwig (33) as shown in Scheme 1. To a solution of 1 (143 mg, 0.30 mmol) in trimethyl phosphate (0.75 mL) was added phosphoryl chloride (60 µL, 0.64 mmol) with stirring on ice. After stirring for 2 h at 0 °C, the reaction mixture was added to a solution of 0.5 M tributylammonium pyrophosphate in dry DMF (3 mL) containing 0.3 mL of tributylamine, and the solution was stirred for 30 min at room temperature. The reaction mixture was poured into a 40 mL ether/acetone (1:1) solution containing NaClO4-saturated acetone (2 mL) with stirring. The precipitates were collected, dried under vacuum, and redissolved in a small volume of water, and the products were separated on a column of DEAE-Sephadex A-25 using a linear gradient (0.1-1.0 M) of triethylammonium hydrogen carbonate (pH 7.5) as an eluent. The appropriate fractions containing (4) were collected and concentrated under vacuum to give white precipitates, which were further purified by column chromatography on an ODS-silica gel column (30 × 250 mm) with a linear gradient elution of acetonitrile (0-50%) in 50 mM triethylammonium acetate buffer (pH 7.0) to remove inorganic pyrophosphate and lyophilized. The yield of 4 was 13% (346 ODU at 260 nm). UV(H2O): λmax, 267 nm. 31P NMR(D2O): δ(ppm), -10.2(d, J ) 20 Hz, Pγ), -10.9(d, J ) 20 Hz, PR), -22.6(t, J ) 20 Hz, Pβ). 1H NMR(D2O): δ(ppm), 7.81(s, 1H, H-6), 6.27(t, 1H, H-1′), 4.58(q, 1H, H-3′), 4.15(m, 3H, H-4′+H-5′), 3.28(s, 2H, CH2CO), 3.243.10(m, 4H, NCH2+CH2N), 2.34(m, 2H, H-2′), 1.51-1.25(m, 8H, (CH2)4). ESI-Mass(negative mode): found 719.2; calcd for [M-H+]-, 719.1. (B) 5-N-(6-Aminohexyl)carbamoylmethyl-2′-deoxyuridine 5′-Triphosphate (5). A portion of 4 (54 ODU at 260 nm) was dissolved in 1 M ammonium hydroxide solution (200 µL) and kept for 3 days at -20 °C to remove the protecting trifluoroacetyl group. The resulting triphosphate of 5-N-(6-aminohexyl)carbamoylmethyl-2′-deoxyuridine (5) was purified by ODS-silica gel column chromatography as described above and lyophilized. The yield was 24 ODU at 260 nm (44%). UV(H2O): λmax, 267 nm. 31P NMR(D O): δ(ppm), -5.5(d, J ) 20 Hz, P ), -10.2(d, 2 γ J ) 22 Hz, PR), -20.9(t, J ) 20, 22 Hz, Pβ). 1H NMR(D2O): δ(ppm), 7.93(s, 1H, H-6), 6.34(t, J ) 7.0 Hz, 1H, H-1′), 4.77(m, 1H, H-3′), 4.24-4.17(m, 3H, H-47+H-5′), 3.38(s, 2H, CH2CO), 3.21(t, J ) 6.4 Hz, 2H, CH2N), 2.96(t, J ) 7.5 Hz, 2H, NCH2), 2.41(m, 2H, H-2′), 1.67-1.34(m, 8H, (CH2)4). ESI-Mass(negative mode): found 623.2; calcd for [M-H+]-, 623.1. (C) 5-[N-2-[N,N-Bis(2-aminoethyl)amino]ethyl]carbamoylmethyl-2′-deoxyuridine 5′-Triphosphate (7). Preparation of 5-[N-2-[N,N-bis(2-aminoethyl)amino]ethyl]carbamoylmethyl-2′-deoxyuridine 5′-triphosphate (7) was carried out by a similar procedure to that of 4 from the nucleoside 3 (182 mg, 0.30 mmol). The yield of 7 was 20% from 3 after removing the trifluoroacetyl group by treatment with ammonium hydroxide solution (457 ODU at 260 nm). UV(H2O): λmax, 267 nm. 31P NMR(D2O): δ(ppm), -9.6(br, Pγ), -10.9(br, PR), -22.3(br, Pβ). ESI-Mass(negative mode): found 653.4; calcd for [M-H+]-, 653.5. (D) 5′-Triphosphates of 5-(Methoxycarbonylmethyl)-2′deoxyuridine (6) and of 5-(Carboxymethyl)-2′-deoxyuri-

New Modified DNAs by Thermophilic DNA Polymerase

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Scheme 1. 5-Substituted 2′-Deoxyuridine Triphosphates, 4-10a

a (a) H N(CH ) NH , MeOH; (b) CF COOEt, (dimethylamino)pyridine, MeOH; (c) POCl , (MeO) PO; (d) tributylammonium 2 2 6 2 3 3 3 pyrophosphate, DMF; (e) 2 M NH4OH; (f) triethylamine, MeOH; (g) N(CH2CH2NH2)3, MeOH; (h) 0.2 M NaOH; (i) 5% Pd-C, H2.

dine (8). Triphosphorylation of compound 2 (90 mg, 0.3 mmol) was carried out by a similar procedure to that used for 1 as described above. Compound 6 was obtained in 7% yield from 2 (186 ODU at 260 nm). UV(H2O): λmax, 267 nm. 31P NMR(D2O): δ(ppm), -5.5(br, Pγ), -10.4(br, PR), -22.6(br, Pβ). 1H NMR(D2O): δ(ppm), 7.81(s, 1H, H-6), 6.21(dd, J ) 6.7 Hz, J ) 13.5 Hz, 1H, H-1′), 4.104.07(m, 2H, H-3′+H-4′), 4.03(m, 2H, H-5′), 3.60(s, 3H, CH3), 3.40(s, 2H, CH2CO), 2.28(m, 2H, H-2′). ESI-Mass(negative mode): found 539.0; calcd for [M-H+]-, 539.0. To a solution of 6 (2.4 ODU at 260 nm, 0.27 mmol) in 15 µL of water was added 15 µL of 0.2 M NaOH solution, and the reaction mixture was kept overnight at 4 °C for hydrolysis of the methyl ester. The resulting triphosphate of 5-(carboxymethyl)-2′-deoxyuridine (8) was purified by HPLC on ODS-silica gel as described above. The yield was 2.0 ODU at 260 nm (0.23 µmol, 85%). UV(H2O): λmax, 267 nm. ESI-Mass(negative mode): found 526.2; calcd for [M-H+]-, 526.0. (E) 5′-Triphosphates of 5-(E)-(Aminopropenyl)-2′-deoxyuridine (9) and 5-(Aminopropyl)-2′-deoxyuridine (10). Compound 9 was prepared from dUTP according to the published procedure (22). Hydrogenation of 9 (155 ODU at 260 nm) was carried out in the presence of 5% Pdcarbon (8 mg) in 2 mL of methanol/H2O (9:1) containing 100 µL of 4 M acetic acid for 2 days under an atmospheric pressure of hydrogen. The reaction mixture was filtered through Celite, and the Celite was rinsed with 2 mL of methanol. The filtrate was evaporated under vacuum.

The residue was separated by HPLC on ODS-silica gel as described above and lyophilized, giving 10 in 57% yield (88 ODU at 260 nm). UV(H2O): λmax, 268 nm. 31P NMR(D2O): δ(ppm), -4.6(br, Pγ), -10.2(br, PR), -20.4(br, Pβ). 1H NMR(D O): δ(ppm), 7.86(s, 1H, H-6), 6.38(t, J ) 1.1 2 Hz, 1H, H-1′), 4.69(m, 1H, H-3′), 4.27(br, 2H, H-4′+H5′), 4.18(br, 1H, H-5′), 3.01(m, 1H, CH), 2.86(m, 1H, CH), 2.48(m, 1H, CH) 2.40(t, J ) 5.8 Hz, 2H, H-2′), 2.2(m, 1H, CH), 1.93(m, 2H, CH2). ESI-Mass(positive mode): found 523.8 and 546.0; calcd for [M+H+]+ and [M+Na+]+, 524.2 and 546.2, respectively. Enzymatic Incorporation of Thymidine Analogues into DNA with KOD Dash, Taq, Tth DNA Polymerase, or DNA Polymerase I (Klenow Fragment). DNA polymerase reactions were performed using a 17mer oligodeoxyribonucleotide, 5′-TAATACGACTCACTATA-3′, as a primer and a 37mer oligodeoxyribonucleotide, 3′-ATTATGCTGAGTGATATCCTGTACTCCTAATGGGTAC-5′, as a template. The primer-template complex was prepared by heating a 1:1 mixture of the primer and the template strands in TE buffer (10 mM Tris-HCl and 0.1 mM EDTA, pH 8.0) to 90 °C for 2 min and then cooling. KOD Dash DNA polymerase reaction was carried out in a mixture (10 µL) containing 0.5 unit of the enzyme, 0.2 µM primer-template complex, 20 µM substrates (dATP + dCTP + TTP or TTP analogue), 5 µM dGTP, and 1 µCi (800 Ci/mmol) of [R-32P]dGTP in the buffer supplied by the maker or in 120 mM Tris-HCl (pH 8.0),

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10 mM KCl, 6 mM (NH4)2SO4, 0.1% Triton X-100, 0.01 mg/mL BSA, and 1.0 mM MgCl2. Either unmodified TTP or a thymidine analogue triphosphate (4, 5, 6, 7, 8, 9, or 10) was used in each reaction. Control reactions without TTP were carried out under the same conditions. Reaction mixtures were incubated at 50 °C for 5 or 30 min, and quenched by addition of 6 µL of 90% formamide containing 0.1% xylenecyanol, 0.1% bromophenol blue, and 20 mM EDTA (pH 8.0). The solutions were heated for 3 min at 95 °C for denaturation, and then cooled. The reaction products were separated by electrophoresis in a 15% denaturing polyacrylamide gel containing 7 M urea. The gels were visualized by autoradiography. Taq DNA polymerase reactions were conducted as described above in reaction mixture (10 µL) containing the primer-template complex, the substrates, 0.5 unit of enzyme in 10 mM Tris-HCl (pH 8.3), 50 mM KCl, and 1.5 mM MgCl2 at 50 °C for 5 or 30 min. Tth DNA polymerase reactions were conducted as described above in reaction mixture (10 µL) containing the primer-template complex, the substrates, 0.5 unit of enzyme in 10 mM Tris-HCl (pH 8.0), 1.5 mM MgCl2, 80 mM KCl, 500 µg/mL of bovine serum albumin, 0.1% sodium cholate, and 0.1% Triton X-100 at 50 °C for 5 or 30 min. Reactions with the Klenow fragment were performed by a similar method as used for the KOD Dash DNA polymerase except for the reaction conditions. Each reaction mixture (10 µL) contained 0.5 unit of the enzyme, the primer-template complex, the substrates in 10 mM Tris-HCl (pH 8.0), and 5 mM MgCl2. Reaction mixtures were incubated at 37 °C for 30 min. Preparation of the Modified DNA in Large Quantity from the Thymidine Analogue 5, Its Characterization with a MALDI-TOF Mass Spectrometer, and Reaction with Fluorescein Isothiocyanate. A reaction mixture (1 mL) containing 5 units of KOD Dash DNA polymerase, a 0.1 µM sample of the 37mer DNA template, a 2 µM aliquot of the 17mer primer, 0.2 mM substrates (dATP + dCTP + dGTP + 5), and the buffer supplied by the maker was divided in 10 portions, and each 100 µL solution was transferred in a 0.5 mL tube. The polymerization reaction was carried out with a thermal cycler using the following program: 20 cycles of 20 s at 94 °C, 10 s at 40 °C, 30 s at 60 °C. The resulting 37mer DNA was extracted with PCI solution [phenol saturated with TE buffer/chloroform/isoamyl alcohol (25: 24:1) twice, and then with CI solution (chloroform/ isoamyl alcohol, 24:1)] and precipitated with ethanol. The product was further purified by HPLC on an ODS-silica gel column, giving 0.28 ODU at 260 nm of the DNA 37mer, whose mass spectrum was taken by a Shimazu AXIMA-CF MALDI-TOF mass spectrometer. Two peaks were present in the mass spectrum. One is due to the template DNA (found 11 370; calcd for [M+H+]+, 11 368), and the other is due to the modified DNA produced by the polymerization (found 11 937; calcd for [M+H+]+, 11 932). The DNA 37mer (0.1 ODU) was reacted with fluorescein isothiocyanate (5 mM) in the mixture (50 µL) containing 0.2 M sodium hydrogen carbonate buffer (pH 9.5) and 20% DMF at 37 °C for overnight. The reaction mixture was analyzed by HPLC monitoring by UV (260 nm) and fluorescence (excitation at 490 nm and emission at 520 nm). Template Activity of Oligodeoxyribonucleotides Containing Thymidine Analogues. DNA polymerase reactions were carried out by the same method as described above using a 37mer modified oligodeoxyribonucleotide template and a 17mer oligodeoxyribonucleo-

Sawai et al. Scheme 2. Sequence of the Primer, the Template, and the Full-Length DNA Producta

aTemplates were Prepared Chemically, and T represents either standard thymidine (I) or one of three modified thymidines (II-IV). In the product, T indicates the positions where the polymerase has to incorporate thymidine residues (modified or unmodified).

tide primer (Scheme 2). Templates contained, at position 31, either normal thymidine or a thymidine analogue bearing hexamethylenediamine, tris(2-aminoethyl)amine, or ethylenediamine at the C5 sidearm (X position). KOD Dash, Taq, and Tth polymerase reactions were performed at 50 °C for 1, 2, 5, or 20 min in reaction mixture (10 µL) containing 0.5 unit of DNA polymerase, 0.2 µM primertemplate complex, 20 µM unmodified substrates (dATP, dCTP, and TTP), 5 µM dGTP, and 1 µCi (800 Ci/mmol) of [R-32P]dGTP in the buffer for KOD Dash, Taq, and Tth DNA polymerases described above. The reactions with the Klenow fragment were performed at 37 °C for 5, 10, 30, and 60 min in the reaction mixture (10 µL) containing 0.5 unit of enzyme, 0.2 µM primer-template complex, 20 µM unmodified substrates (dATP, dCTP, and TTP), 5 µM dGTP, and 1 µCi (800 Ci/ mmol) of [R-32P]dGTP in 10 mM Tris-HCl (pH 8.0) and 5 mM MgCl2. The polymerase reactions were stopped by addition of 3 µL of formamide dye, denatured, and analyzed by gel electrophoresis as described above. RESULTS

A series of C5-substituted 2′-deoxyuridine derivatives were prepared from 3′,5′-di-O-acetyl-5-methoxycarbonylmethyl-2′-deoxyuridine, which was synthesized easily according to the method described previously (11). The 5′-triphosphorylation of the nucleosides was carried out by the one-pot method described by Ludwig (32) with a slight modification as shown in Scheme 1. The terminal amino-protecting trifluoroacetyl group of the triphosphate from 1 or 3 was removed by treatment with concentrated aqueous ammonia quantitatively at low temperature. The resulting terminal amino group can be further modified by reaction with a functional molecule such as fluorescein isothiocyanate. Conventional modified substrates, 5′triphosphates of 5-(E)-(aminopropenyl)-2′-deoxyuridine (9) and 5-(aminopropyl)-2′-deoxyuridine (10), were also prepared to compare the substrate specificity for the DNA polymerases. Enzymatic Incorporation of the Thymidine Analogues by DNA Polymerases. Compounds 4, 5, 6, 7, 8, 9, and 10 were tested as substrates for DNA poly-

New Modified DNAs by Thermophilic DNA Polymerase

Figure 1. Autoradiogram of the products of primer extension reactions using KOD Dash DNA polymerase, and either normal TTP or modified as substrates. The reactions were done at 50 °C for 5 min (A) or 30 min (B) under the conditions described under Materials and Methods. Lane 1, marker DNA; lane 2, positive control (TTP+dATP+dCTP+dGTP); lane 3, 4+dATP+ dCTP+dGTP; lane 4, 5+dATP+dCTP+dGTP; lane 5, 6+dATP+ dCTP+dGTP; lane 6, 7+dATP+dCTP+dGTP; lane 7, 8+dATP+ dCTP+dGTP; lane 8, negative control (dATP+dCTP+dGTP); lane 9, 9+dATP+dCTP+dGTP; lane 10, 10+dATP+dCTP+dGTP.

merase I from E. coli (Klenow fragment), Taq, Tth, and KOD Dash DNA polymerases using the same amount of standard enzyme activity for comparison of activities against the modified substrate. The polymerization reactions with KOD Dash, Taq, and Tth DNA polymerases were carried out at 50 °C for 5 and 30 min, because the melting temperature of the primer-template complex was estimated to be a little lower than 50 °C under the reaction conditions (34). The polymerization reactions in the case of the Klenow fragment were done at 37 °C for 30 min. DNA polymerases extend the primer on the template (Scheme 2). Thus, when unmodified substrates and the same enzyme activities were used, all enzymes formed 37mer full-length DNA at the same rate. However, the ability of the modified thymidine triphosphates to function as a substrate depended on the type of modification, and on the polymerase. KOD Dash DNA Polymerase Reactions. Figure 1A and Figure 1B show the KOD Dash DNA polymerase reactions for 5 and 30 min, respectively. The 37mer fulllength DNA was the major product from the unmodified substrate (lane 2). The result of the control reaction with no TTP is shown in lane 8: the polymerization was completely stopped before the first T position, forming a 22mer DNA. In contrast to the other enzymes, KOD Dash DNA polymerase tolerated the modification, and readily accepted the modified substrates forming the full-length DNA. Thus 37mer DNAs were formed from compounds 4, 5, and 6 (lanes 3, 4, and 5) when the polymerization reaction was carried out for 30 min (B), although some short-chained DNAs were observed in the 5 min reaction (A). The 37mer DNA product containing trifluoroacetylprotected hexamethylenediamine (compound 4, lane 3) or hexamethylenediamine (compound 5, lane 4) modified thymidine showed lower electrophoretic mobility than DNA containing unmodified thymidine (lane 2) or meth-

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oxycarbonyl-modified thymidine (compound 6, lane 5). Attachment of a bulky substituent and a cationic protonated amino group presumably retarded the mobility of these modified DNAs. The structure of the modified 37mer obtained from compound 5 was confirmed by MALDI-TOF mass spectroscopy. The observed molecular weight of the modified 37mer, prepared in a large scale and isolated by HPLC, was 11 937, while the calculated mass number ([M+H])+ of the enzymatic product was 11 932. The modified DNA from 5 bearing a terminal amino group was further reacted with fluorescein isothiocyanate to furnish the fluorescence-labeled DNA. Treatment of the 37mer obtained from compound 4 with concentrated ammonium hydroxide removed the terminal trifluoroacetyl groups, giving the modified DNA with hexamethylenediamine, whose electrophoretic mobility was the same as the modified DNA obtained from compound 5. KOD Dash DNA polymerase also accepted as a substrate the highly modified compound 7 (lane 6), which bears a bulky branched polyamine with two cationic protonated amino groups at the C5 sidearm. Incorporation of compound 7 and chain-elongation took place at the first T-23 position. However, the enzyme could not tolerate two successive incorporations of compound 7, stopping at the T-30 position (lane 6). This enzyme could tolerate the substrate analogue 8 with an anionic carboxyl, yielding the full-length 37mer, although the 36mer was the main product in the case of the long reaction time (lane 7). The enzyme also accepted a conventional modified substrate, 9, bearing an (E)aminopropenyl group easily giving the corresponding 37mer DNA (lane 9). The analogue 10 bearing an aminopropyl group was partially accepted as a substrate. Small amounts of the full-length 37mer DNA were formed, although the main product was a truncated DNA 30mer in which the chain-elongation stopped at the two successive T positions (lane 10 of Figure 1B). The results indicate that, in contrast to the other DNA polymerases, the KOD Dash DNA polymerase can accept C5-substituted 2′-deoxyuridine with an sp3-hybridized carbon at the R-position as a substrate. Taq and Tth DNA Polymerase Reactions. Polymerization reactions using Taq and Tth DNA polymerases are shown in Figure 2. Both enzymes showed similar behavior with the modified substrates. The full-length 37mer was exclusively formed after 5 min when the natural substrate TTP was used (Figure 2A, lane 2), while chain-elongation was completely stopped at the first T position, forming 22mer DNA in the control reaction without TTP (Figure 2A, lane 8). Small amounts of 38mer and 23mer DNAs were formed after 30 min due to the terminal transferase activity of the enzymes both with and without TTP, respectively (Figure 2B, lanes 2 and 8). Compounds 4 and 5 slowed elongation, forming mainly the 22mer DNA after 5 min (lanes 3 and 4). However, the enzymes accept compounds 4 and 5 slowly, and extend the polymerization reaction after the first single T position, and a substantial amount of 28mer DNA was formed after 30 min. The incorporation was completely inhibited at the two successive TT positions. The main polymerization product when using compound 6 was 28mer DNA after 5 min (Figure 2A, lane 5). Chain elongation took place after 30 min, forming 29mer and full-length 37mer DNAs (Figure 2B, lane 5). On the other hand, these enzymes could not use compounds 7 and 8, giving only 22mer and 23mer DNA, although very small amounts of 28mer DNA were formed after 30 min (Figure 2B, lanes 6 and 7). The enzymes use compound 9, giving full-length 37mer DNA after 30 min in accordance with

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Figure 3. Autoradiogram of the products of primer extension reactions using DNA polymerase I from E. coli (Klenow fragment), with various mixtures of nucleoside triphosphates (normal and/or modified). The reactions were done at 37 °C for 30 min under the conditions described under Materials and Methods. Lane numbering is identical to that in Figure 1.

Figure 2. Autoradiogram of the products of primer extension reactions using Taq (I) and Tth (II) DNA polymerase, with various mixtures of nucleoside triphosphates (normal and/or modified). The reactions were done at 50 °C for 5 min (A) or 30 min (B) under the conditions described under Materials and Methods. Lane numbering is identical to that in Figure 1.

previous report (25, 30), although 28mer and 29mer DNAs were the main products in the 5 min reaction (lane 9). Compound 10 was accepted by the enzyme at the first T-23 position, and chain-elongation continued beyond this point, but not at the two successive TT position giving the 28mer and 29mer DNAs (lane 10). Polymerization Reactions with DNA Polymerase I from E. coli (Klenow Fragment). Figure 3 shows results of polymerization reactions with DNA polymerase I from E. coli (Klenow fragment). The 37mer full-length DNA was the major polymerization product when the unmodified substrate was used, although small amounts of 38mer and 36mer DNAs were also formed due to the

presence of terminal transferase and 3′-5′ exonuclease activities of the enzyme (lane 2). In the control reaction in which TTP was absent (lane 8), polymerization was completely stopped before the first T position, forming 22mer DNA, although small amounts of the 23mer were formed by the terminal transferase activity. Klenow fragment accepted compounds 4 and 5, and extended the chain elongation at the first T-23 position. The enzyme could tolerate two successive incorporations of 4 or 5 at T-29 and T-30, but further chain-elongation was completely stopped, forming 30mer DNA (lanes 3 and 4). The enzyme also accepted compound 6 producing full-length 37mer DNA, along with substantial amounts of 36mer and 29mer, which were terminated at the T-36 and T-29 positions, respectively (lane 5). Compound 7 completely inhibited the enzyme. Thus, the main product was 22mer DNA which ended before the first T position, although very small amounts of 28mer DNA, in which chainelongation was stopped before the double T position, were also formed (lane 6). Compound 8 was accepted to some extent by the enzyme, with formation of 28mer DNA, which passed through the T-23 but stopped before the two successive T (lane 7). With the conventional substrate analogue 9, full-length 37mer DNA was formed as a sole product (lane 9). With compound 10, only a truncated DNA 30mer was formed (lane 10). The results with compounds 9 and 10 are consistent with the previous report (22, 23) in which the Klenow fragment accepts 2′deoxyuridine derivatives bearing a propenyl group at the C5-position, but reduction of the double bond inhibits their function as a substrate. The formation of short DNAs with compounds 7 and 8 is due to inhibition of incorporation of the modified substrate. Inhibition of chain-elongation after incorporation results in the formation of the truncated DNA in the cases of compounds 4, 5, 6, and 10.

New Modified DNAs by Thermophilic DNA Polymerase

Figure 4. Autoradiogram of the products of primer extension on a DNA template bearing at position 31 either a normal thymidine residue or a C5-substituted 2′-deoxyuridine. The reactions were done at 50 °C for 5 min for Taq, Tth, and KOD Dash DNA polymerases, and at 37 °C for 30 min for Klenow fragment and under the conditions described under Materials and Methods, and unmodified 2′-deoxyribonucleoside triphosphates as substrates (TTP+dATP+dCTP+dGTP). Lane 1, marker DNA; lane 2, positive control (unmodified template I); lane 3, modified template II, bearing hexamethylenediamine at C5; lane 4, modified template III, bearing tris(2-aminoethyl)amine at C5; lane 5, modified template IV, bearing ethylenediamine at C5.

Template Activity of Oligodeoxyribonucleotides Containing Thymidine Analogues. Figure 4 illustrates the results of polymerization of unmodified substrates, dATP, dCTP, dGTP, and TTP, on modified DNA templates by KOD Dash, Taq, and Tth DNA polymerases and the Klenow fragment. The 37mer template DNA contains the thymidine analogue at the 31st position in place of a thymidine residue. All the DNA polymerases examined in this study read through the modified thymidine in the template, and accepted the complementary substrate and continued the chain-elongation until the end of the template, forming the full-length 37mer DNA. DISCUSSION

We have examined the substrate activity of TTP analogues, 4, 5, 6, 7, and 8 along with the conventional 9 and 10, for several DNA polymerases. Compounds 4-8 and 10 have an sp3-hybridized carbon at the C5 R-position with or without a bulky sidearm. Compounds 5 and 7 have one or two positively charged amino groups in the sidearm. Compound 8 bears a negatively charged carboxyl group. Compound 6 has a small noncharged substituent. Compound 9, which has an (E)-aminopropenyl group at the C5-positon of 2′-deoxyuridine, is reported to be a good substrate for several DNA polymerases (22, 23, 25, 27, 30). On the other hand, the thymidine analogue 10, the reduced product of 9, is reported to be devoid of substrate activity for the enzymes (27, 30). We found that KOD Dash DNA polymerase can accept various TTP analogues, 4, 5, 6, 8, and 9, readily as substrates and the corresponding full-length 37mer DNA was formed. The enzyme also accepts compound 10, although the full-length DNA was formed in modest yield. The enzyme, however, could not tolerate two successive incorporations of compound 7. KOD Dash DNA polymerase is a hyperthermophilic DNA poly-

Bioconjugate Chem., Vol. 13, No. 2, 2002 315

merase from thermophilic archaeum and is now commercially available. The enzyme is reported to have higher DNA polymerization activity with higher fidelity compared with other thermophilic DNA polymerases such as Taq DNA polymerase (32). In contrast, compounds 4, 5, 6, 7, and 8 were poor substrates for Klenow fragment and Taq and Tth DNA polymerases. Compounds 4 and 5 gave only truncated DNA. A modest to substantial amount of the full-length 37mer DNA was obtained in addition to the truncated one when using compound 6 with these polymerases. The results of the DNA polymerization reaction by the Klenow fragment and Taq and Tth DNA polymerase confirm and extend the results reported previously (22-30). The 2′deoxyuridine derivatives bearing a propenyl or propynyl linker at the C5 R-position are reported to function as substrates for several DNA polymerases, but the reduced product does not, indicating the possible requirement for an sp2- or sp-hybridized carbon at the C5 R-position (2230). These results demonstrate that the ability of modified thymidine triphosphates to function as a substrate for DNA polymerases depends on the type of modification of the substrate and on the kind of polymerase. KOD Dash DNA polymerase accepts the thymidine analogues which have an sp3-hybridized carbon at the C5 R-position, while other DNA polymerases cannot accept these analogues. The results are somewhat in contrast to the previous report on modified 2′-deoxyuridines, in which a propenyl or propynyl group at the C5 sidearm is required for the compound to be a substrate for DNA polymerase (22-30). Thus, this is the first example that C5-substituted 2′-deoxyuridine derivatives bearing an sp3-hybridized carbon at the C5 R-position are incorporated into DNA. All DNA polymerases examined in this study read through the thymidine modification in the template, giving the full-length DNA. KOD Dash DNA polymerase may expand the range of structural modified substrates for enzymatic synthesis of modified DNAs. The resulting modified DNA can be further functionalized by reaction with a functional molecule such as fluorescein isothiocyanate. KOD Dash DNA polymerase and the thymidine analogues will be useful for preparing large quantities of modified DNA by PCR, and then in vitro selection of the functionalized DNA. The functionalized DNA synthesis by PCR using KOD Dash DNA polymerase and various thymidine analogues will be published elsewhere. LITERATURE CITED (1) Luyten, I., and Herdewijin, P. (1998) Hybridization properties of base-modified oligonucleotides within the double and triple helix motif. Eur. J. Med. Chem. 33, 515-576. (2) Verma, S., and Eckstein, F. (1998) Modified oligonucleotides: Synthesis and strategy for users. Annu. Rev. Biochem. 67, 99-134. (3) Ruth, J. L. (1991) Oligodeoxyribonucleotides with reporter groups attached to the base. In Oligonucleotides and Analogues (Eckstein, F., Ed.) pp 255-282, IRL Press, Oxford, U.K. (4) Tesler, J. K., Cruickshank, A. L., Morrison, E., and Netzel, T. L. (1989) Synthesis and characterization of DNA oligomers and duplexes containing covalently attached molecular labels: Comparison of biotin, fluorescein, and pyrene labels by thermodynamic and optical spectroscopic measurements. J. Am. Chem. Soc. 111, 6966-6976. (5) Jablonski, E. E., Moomaw, W., Tullis, R. H., and Ruth, L. L. (1986) Preparation of oligodeoxynucleotide-alkaline phosphatase conjugates and their use as hybridization probes. Nucleic Acids Res. 14, 6115-6128. (6) Dreyer, G. B., and Dervan, P. B. (1985) Sequence-specific cleavage of single-stranded DNA: oligodeoxynucleotide-EDTAFe(II). Proc. Natl. Acad. Sci. U.S.A. 82, 968-972.

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