A R T I C L E S
Hennig et al.
(MBP) into the pMAL digested with EcoR1 and HindIII using phage
T4 DNA ligase (New England Biolabs), incubating for 2 h at 25 °C.
The ligation reaction mixture containing the resulting expression
plasmid pUDK1 was used to transform E. coli JM109 cells by standard
procedures.11
transcription reactions without further purification. Data: 1H NMR (600
3
5
MHz, D2O) δ (mult.) 8.11 (d, JHF ) 7.8 Hz, H-6), 5.98 (dd, JHF
)
1.5 Hz, H-1′), 4.41 (dd, H-3′), 4.37 (dd, H-2′), 4.29 (dt, H-4′), 4.26
(dt, H-5′); 13C NMR (150 MHz, D2O) δ (mult) 162.0 (s, C-4), 152.9
(s, C-2), 143.4 (d, C-5), 128.0 (d, C-6), 91.1 (d, C-1′), 86.1 (d, C-4′),
76.4 (d, C-2′), 72.2 (d, C-3′), 67.7 (t, C-5′); 19F NMR (564 MHz, D2O)
δ (mult.) -86.15 (dd, F-5).
Preparation of Enzymes Used in the Synthesis of 5-Fluoro-
Substituted NTPs. Ribokinase (rbsK, EC 2.7.1.15),12 5-phospho-D-
ribosyl-R-1-pyrophosphate synthetase (prsA, EC 2.7.6.1),12 and uracil
phosphoribosyltransferase (uraP, EC 2.4.2.9)13 were all purified from
overexpressing strains as described previously. Uridine kinase was
prepared from inducible strain JM109/pUDK1 grown in Luria-Bertani
broth (per liter: 10 g of Tryptone, 5 g of yeast extract, 10 g of NaCl,
200 µg/mL ampicillin, Fisher Scientific) until mid log phase (A600 of
0.8) at 37 °C. The cells were induced for 4 h with the addition of 1
mM isopropyl-â-D-thiogalactopyranoside (IPTG) at 37 °C. After 4 h
the cells were harvested by centrifugation at 6000g for 15 min (6000
rpm for a Sorval SLA-300 rotor). The cell pellet was resuspended in
20 mL of lysis buffer (50 mM potassium phosphate (pH 7.8), 5 mM
2-mercaptoethanol), and the cells were subjected to sonic disruption
lysis. Cell debris was removed by centrifugation at 20 000g for 30 min
(13 000 rpm for a Sorval SS-34 rotor). The clarified udk lysate was
loaded to an amylose column preequilibrated with lysis buffer, and
the partial purified MBP-udk was eluted with elution buffer (50 mM
potassium phosphate (pH 7.8), 5 mM 2-mercaptoethanol, 10 mM
maltose). Fractions containing the MBP-udk fusion were combined,
an equal volume of glycerol was added, and the enzyme was stored at
-20 °C.
Uridine Kinase Assay. Uridine kinase (udk, EC 2.7.1.48) activity
was determined by spectrophotometric assay coupling consumption of
ATP to NADH oxidation in a manner similar to that of the assay
described previously for adenine phosphoribosyltransferase.13 To the
equilibrated assay solution (50 mM potassium phosphate, pH 7.5, 20
mM dithiothreitol, 10 mM MgCl2, 5 mM ampicillin, 5.0 mM phos-
phoenolpyruvate, 0.5 mM ATP, 0.2 mM NADH, 0.1 mM kanamycin,
2 units of lactate dehydrogenase, 2 units of pyruvate kinase), udk was
added to begin the assay. The change of absorbance at 340 nm that
occurs when NADH is oxidized to NAD+ was monitored as a function
of time, with the activity being determined using a ∆ꢀ340 of 6220 cm-1
mol-1 for NADH.
Synthesis of 5-Fluorocytidine-5′-triphosphate. In a round-bottom
flask, 5-fluorocytidine (100 mg, 0.38 mmol, ICN Biomedicals) was
dissolved in 75 mL of the same synthesis buffer described above for
the synthesis of 5F-UTP. The synthesis was started with the addition
of 50 units of uridine kinase (udk, EC 2.7.1.48), 1 unit of nucleoside
monophosphate kinase (pyrH, EC 2.7.4.4), 75 units of pyruvate kinase
(pyrF, EC 2.7.1.40), 150 units of 3-phosphoglycerate mutase (yipO,
EC 5.4.2.1), and 50 units of enolase (eno, EC 4.2.1.11). The reaction
was monitored by HPLC methods described elsewhere.13 After 72 h,
the synthesis of 5-fluorocytidine-5′-triphosphate appeared to be com-
plete. The 5-fluorocytidine-5′-triphosphate reaction was worked up in
the same manner as that described above for 5F-UTP. The 5-fluoro-
cytidine-5′-triphosphate (0.3 mmol, 78% isolated yield) was dissolved
in 6 mL of H2O, the pH was adjusted to 7.6 with the addition of 1 M
HCl, and it was used in transcription reactions without further
3
purification. Data: 1H NMR (600 MHz, D2O) δ (mult.) 8.22 (d, JHF
5
) 6.3 Hz, H-6), 5.95 (dd, JHF ) 1.5 Hz, H-1′), 4.40 (dd, H-3′), 4.34
(dd, H-2′), 4.31 (dd, H-5′), 4.30 (ddd, H-4′), 4.28 (dd, H-5′′); 13C NMR
(150 MHz, D2O) δ (mult) 159.0 (s, C-4), 155.3 (s, C-2), 139.4 (d, C-5),
129.2 (d, C-6), 92.2 (d, C-1′), 85.7 (d, C-4′), 77.0 (d, C-2′), 71.7 (d,
C-3′), 67.2 (t, C-5′); 19F NMR (564 MHz, D2O) δ (mult.) -86.93 (dd,
F-5).
In Vitro Transcription of RNA. Transcription reactions were
carried out under the following conditions: 40 mM Tris-HCl (pH 8.1),
1 mM spermidine, 10 mM dithiothreitol, 0.01% Triton X-100, 80 mg/
mL polyethyene glycol, 17 mM MgCl2, 2 mM each nucleotide
triphosphate, 0.3 µM template DNA (Operon), 0.3 µM promoter DNA
(Operon), 2 unit/mL inorganic pyrophosphatase, 80 units/mL RNAse
inhibitor (Promega), and 3000 units/mL T7 RNA polymerase.14-16 The
reactions were incubated for 4 h in a water bath at 37 °C, quenched
with 0.1 volume of 0.5 M EDTA (pH 8.0), and extracted with an equal
volume of phenol/chloroform (Fisher Scientific) equilibrated with TE
buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The aqueous layer
was ethanol precipitated with the addition of 0.1 volume of 3 M sodium
acetate, 3 volumes 100% ethanol at -20 °C. The crude RNA precipitate
was first washed with 70% ethanol to remove extra salt, resuspended
in 80% formamide stop buffer,17 and purified on a 20% (29:1)
acrylamide/bisacrylamide denaturing electrophoresis gel in TBE buffer
(90 mM tris-borate, 2 mM EDTA, pH 8.1). The product band was
identified by UV shadowing, excised, and elutrapped at 4 °C into TBE
buffer for 4-6 h at 200 V with fractions taken each 1.5 h. The RNA
was again ethanol precipitated. The purified RNA samples were
concentrated and desalted using Centricon concentrators with 3 kDa
molecular weight cutoff (Millipore). Two single 40 mL transcription
reactions using single-stranded DNA templates yielded 1.73 and 0.88
µmol of 5F-Ura- and 5F-Cyt-substituted HIV-2 TAR RNA, respectively.
The RNA samples were dissolved in the final NMR buffer.
Synthesis of 5-Fluorouridine-5′-triphosphate. In a round-bottom
flask, D-ribose (150.1 mg, 1 mmol) and 5-fluorouracil (130.1 mg,
1 mmol) were dissolved in 200 mL of synthesis buffer (0.1 mM
kanamycin, 0.5 mM ATP, 5 mM ampicillin, 10 mM MgCl2, 20 mM
dithiothreitol, 50 mM sodium 3-phosphoglycerate, 50 mM potassium
phosphate, pH 7.5) at 37 °C. The synthesis was initiated by the addition
of 50 units of ribokinase (rbsK, EC 2.7.1.15), 2 units of 5-phospho-
D-ribosyl-R-1-pyrophosphate synthetase (prsA, EC 2.7.6.1), 2 units of
uracil phosphoribosyltransferase (uraP, EC 2.4.2.9), 2 units of nucleo-
side monophosphate kinase (pyrH, EC 2.7.4.4), 75 units of adenylate
kinase (plsA, EC 2.7.4.3), 150 units of pyruvate kinase (pyrF, EC
2.7.1.40), 300 units of 3-phosphoglycerate mutase (yipO, EC 5.4.2.1),
and 100 units of enolase (eno, EC 4.2.1.11). The reaction was monitored
by HPLC methods described elsewhere.13 After 110 h, the synthesis
of 5-fluorouridine-5′-triphosphate appeared to be complete. The solvent
was removed in vacuo, the reaction was redissolved in 35 mL of 1 M
triethylamine bicarbonate (pH 9.5), and proteins/salts were precipitated
over 12 h at 4 °C. The insoluble material was removed by centrifugation
at 20 000g (14 000 rpm for a Sorval SS-34 rotor), decanted, and solvent
was removed in vacuo. The 5-fluorouridine-5′-triphosphate (0.8 mmol,
80% isolated yield) was dissolved in 10 mL of H2O, the pH was
adjusted to 7.6 with the addition of 1 M HCl, and it was used in
NMR Spectroscopy. All 19F NMR spectra were recorded on either
400 or 600 MHz Bruker Avance spectrometers equipped with 5 mm
quadruple-resonance (1H,13C-19F,31P -QXI) probes with shielded triple
axis gradients or on a Varian Inova 600 MHz spectrometer equipped
with an actively shielded z-gradient triple-resonance (1H-13C,31P) probe
(14) Milligan, J. F.; Groebe, D. R.; Witherell, G. W.; Uhlenbeck, O. C. Nucleic
Acids Res. 1987, 15, 8783-8798.
(15) Milligan, J. F.; Uhlenbeck, O. C. Methods Enzymol. 1989, 180, 51-62.
(16) Wyatt, J. R.; Chastain, M.; Puglisi, J. D. BioTechniques 1991, 11, 764-
769.
(17) Sambrook, J.; Fritsch, E. F.; Maniatis, T.; Irwin, N.; Maniatis, T. Molecular
Cloning: a laboratory manual, 2nd ed.; Cold Spring Harbor Laboratory
Press: Cold Spring Harbor, NY, 1989.
(11) Hanahan, D. J. Mol. Biol. 1983, 166, 557-580.
(12) Tolbert, T. J.; Williamson, J. R. J. Am. Chem. Soc. 1996, 118, 7929-
7940.
(13) Scott, L. G.; Tolbert, T. J.; Williamson, J. R. Methods Enzymol. 2000, 317,
18-38.
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14912 J. AM. CHEM. SOC. VOL. 129, NO. 48, 2007