Here we report on a significant enhancement of the binding affinity of naphthyridine-based fluorescence ligands that can selectively bind to cytosine opposite an AP site in a DNA duplex (5'-TCC AGX GCA AC-3'/3'-AGG TCC CGT TG -5', X = AP site, C = target). We have previously reported that 2-amino-7-methyl-1,8-naphthyridine (AMND) binds to cytosine with a dissociation constant of 370 nM (pH 7.0, I = 0.11 M, at 20 degrees C). In this work, AMND is modified simply by introducing two methyl groups to the naphthyridine ring. The present ligand, 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND), shows a stronger binding affinity for cytosine, and a dissociation constant reaches 56 nM. ATMND is effectively applicable to the analysis of cytosine-related mutation of PCR products. These promising abilities of ATMND are presented based on the examination by melting temperature (T(m)) and fluorescence measurements.
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