Fluorescence detection of the endonuclease III reaction using modified oligonucleotides.
We have developed a method for the detection of the endonuclease III reaction by fluorescence. The probes were 13-base-pair hairpin-shaped oligonucleotides containing one of the isomers of thymine glycol or 5,6-dihydrothymine as a damaged base at the center, and had a fluorophore and a quencher at the 5' and 3' ends, respectively. Fluorescence was detected when the probe was cleaved by the enzyme, because the short fragment bearing the fluorophore could not be hybridized to the quencher strand at the incubation temperature. The substrate specificity was shown using Escherichia coli and human enzymes.
Matsumoto
Naoyuki
N
Division of Chemistry, Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka 560-8531, Japan.
Hayashi
Ryosuke
R
Himoto
Masayuki
M
Kuraoka
Isao
I
Morita
Sayuri
S
Hagiwara
Fuki
F
Katayanagi
Katsuo
K
Ide
Hiroshi
H
Iwai
Shigenori
S
D004249:DNA Damage; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D015345:Oligonucleotide Probes / Q000737:chemistry*; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli; C468347:NTHL1 protein, human