Although deoxyribozymes (DNAzymes) have
been widely used as biosensors for the detection of their
cofactors and the targets of related aptazymes, it is desirable to
expand their range of analytes to take advantage of the
DNAzyme-based signal ampli
fi
cation for more sensitive
detections. In this study, the activity of uracil-DNA glycosylase
(UNG) was successfully detected and quanti
fi
ed by deoxyur-
idine-modi
fi
ed DNAzymes that underwent UNG-dependent
deactivation or activation. In one design, the indispensable
thymidine T2.1 in the 8
−
17 DNAzyme was replaced with a
deoxyuridine, resulting in minimal change of the DNAzyme
’
s
activity. Since UNG is capable of removing uracils from single- or double-stranded DNAs, the modi
fi
ed DNAzyme was
deactivated when the uracil at the indispensable thymidine site was eliminated by UNG. In another design, introducing a
deoxyuridine to the 3
′
position of the deoxycytidine C13 in the catalytic core of the same DNAzyme caused signi
fi
cant decrease
of the activity. The removal of the interfering deoxyuridine by UNG, however, activated the DNAzyme. By monitoring the
activity change of the DNAzymes through the
fl
uorescence enhancement from the DNAzyme-catalyzed cleavage of DNA
substrates labeled by a
fl
uorophore and quencher pair, the UNG activity was measured based on UNG-dependent deactivation
and activation of the DNAzymes. This method was found to be able to detect UNG activity as low as 0.0034 U/mL. Such a
method can be applied to the detection of other nucleotide-modifying enzymes and expand the analyte range of DNAzyme-based biosensors.