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. (C) 2009 Elsevier Inc. All rights reserved.