FRET-based kinetic analysis of highly reactive heterochiral DNA toward EcoRI endonuclease.
We found unusual reactivity of a heterochiral dodecadeoxynucleotide toward EcoRI that contains an unnatural l-nucleotide residue at the 3'-flanking site of the hexameric EcoRI recognition sequence. In this study, the kinetic parameters for the reaction were determined by means of a heterochiral molecular beacon that contains the EcoRI recognition sequence in the stem region and possesses the fluorescent and quenching dyes at the 5'- and 3'-termini, respectively. We found that the heterochiral molecular beacon showed large V(max) and small K(m) values compared to the corresponding homochiral one. This chiral modification is expected to induce a preferable conformational change for binding and catalysis by EcoRI.
Urata
Hidehito
H
Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan. urata@gly.oups.ac.jp
Tamaki
Chihiro
C
Matsuno
Miki
M
Wada
Shun-ichi
S
Akagi
Masao
M
D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D015246:Deoxyribonuclease EcoRI / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D006868:Hydrolysis; D007700:Kinetics
D004247:DNA; D015246:Deoxyribonuclease EcoRI