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 mole...

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Bibliographic Details
Published in:Biochemical and biophysical research communications 2009-12, Vol.390 (2), p.192-195
Main Authors: Urata, Hidehito, Tamaki, Chihiro, Matsuno, Miki, Wada, Shun-ichi, Akagi, Masao
Format: Article
Language:English
Subjects:
Base Sequence
Deoxyribonuclease EcoRI - chemistry
DNA
DNA - chemistry
EcoRI-promoted hydrolysis
Fluorescence Resonance Energy Transfer
FRET
Heterochiral
Hydrolysis
Kinetics
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Summary: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 Vmax and small Km values compared to the corresponding homochiral one. This chiral modification is expected to induce a preferable conformational change for binding and catalysis by EcoRI.
ISSN:0006-291X
1090-2104
DOI:10.1016/j.bbrc.2009.08.164