Simple and rapid enzymatic method for the synthesis of single-strand oligonucleotides containing trifluorothymidine.
To investigate the mechanism of trifluorothymidine (TFT)-induced DNA damage, we developed an enzymatic method for the synthesis of single-strand oligonucleotides containing TFT-monophosphate residues. Sixteen-mer oligonucleotides and 14-mer 5'-phosphorylated oligonucleotides were annealed to the template of 25-mer, so as to empty one nucleotide site. TFT-triphosphate was incorporated into the site by DNA polymerase and then ligated to 5'-phosphorylated oligonucleotides by DNA ligase. The synthesized 31-mer oligonucleotides containing TFT residues were isolated from the 25-mer complementary template by denaturing polyacrylamide electrophoresis. Using these single-strand oligonucleotides containing TFT residues, the cleavage of TFT residues from DNA, using mismatch uracil-DNA glycosylase (MUG) of E.coli origin, was compared with that of 5-fluorouracil (5FU) and 5-bromodeoxyuridine (BrdU). The TFT/A pair was not cleaved by MUG, while the other pairs, namely, 5FU/A, 5FU/G, BrdU/A, BrdU/G, and TFT/G, were easily cleaved from each synthesized DNA. Thus, this method is useful for obtaining some site-specifically modified oligonucleotides.
Tokushima Research Center, Taiho Pharmaceutical Co. Ltd., 224-2 Hiraishi-ebisuno, Kawauchi, Tokushima, Japan. suzuki@taiho.co.jp
Norihiko
N
Suzuki
Masakazu
M
Fukushima
D001345:Autoradiography; D001973:Bromodeoxyuridine / Q000737:chemistry; D004249:DNA Damage; D011088:DNA Ligases / Q000737:chemistry; D017931:DNA Primers / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D058645:Denaturing Gradient Gel Electrophoresis; D005472:Fluorouracil / Q000737:chemistry; D009841:Oligonucleotides / Q000138:chemical synthesis* / Q000737:chemistry; D044502:Thymine DNA Glycosylase / Q000737:chemistry; D014271:Trifluridine / Q000737:chemistry*
D017931:DNA Primers; D009841:Oligonucleotides; D004259:DNA-Directed DNA Polymerase; D044502:Thymine DNA Glycosylase; C113921:mismatch-specific thymine uracil-DNA glycosylase; D011088:DNA Ligases; D001973:Bromodeoxyuridine; D014271:Trifluridine; D005472:Fluorouracil