Dimeric 2-amino-1,8-naphthyridine selectively binds to a G-G mismatch with high affinity (K(d) = 53 nM). We have investigated a binding mechanism of naphthyridine dimer 2 to a G-G mismatch by spectroscopic studies, thermodynamic analysis, and structure-activity studies for the thermal stabilization of the mismatch. 1H NMR spectra of a complex of 2 with 9-mer duplex d(CATCGGATG)2 containing a G-G mismatch showed that all hydrogens in two naphthyridine rings of 2 were observed upfield compared to those of 2 in a free state. The 2D-NOESY experiments showed that each naphthyridine of 2 binds to a guanine in the G-G mismatch within the pi-stack. In CD spectra, a large conformational change of the G-G mismatch-containing duplex was observed upon complex formation with 2. Isothermal calorimetry titration of 2 binding to the G-G mismatch showed that the stoichiometry for the binding is about 1:1 and that the binding is enthalpy-controlled. It is clarified by structure-activity studies that show (i) the linker connecting two naphthyridine rings was essential for the stabilization of the G-G mismatch, (ii) the binding efficiency was very sensitive to the linker structure, and (iii) the binding of two naphthyridines to each one of two Gs in the G-G mismatch is essential for a strong stabilization. These results strongly supported the intercalation of both naphthyridine rings of 2 into DNA base pairs and the formation of a hydrogen bonded complex with the G-G mismatch.
We have discovered a new molecule naphthyridine-azaquinolone hybrid (Npt-Azq) that strongly stabilized the guanine-adenine (G-A) mismatch in duplex DNA. In the presence of Npt-Azq, the melting temperature (T(m)) of 5'-d(CTA ACG GAA TG)-3'/3'-d(GAT TGA CTT AC)-5' containing a single G-A mismatch increased by 15.4 degrees C, whereas fully matched duplex increased its T(m) only by 2.2 degrees C. Npt-Azq was immobilized on the sensor surface for the surface plasmon resonance (SPR) assay to examine SPR detection of duplexes containing a G-A mismatch. Distinct SPR signals were observed when 27mer DNA containing a G-A mismatch was analyzed by the Npt-Azq immobilized sensor surfaces, whereas the signal of the fully matched duplex was approximately 6-fold weaker in intensity. The SPR signals for the G-A mismatch were proportional to the concentration of DNA in a range up to 1 microM, confirming that the SPR signal is in fact due to the binding of the G-A mismatch to Npt-Azq immobilized on the surface. Examination of all 16 G-A mismatches regarding the flanking sequence revealed that the sensor surface reported here is applicable to eight flanking sequences, covering 50% of all possible G-A mismatches.
A naphthyridine dimer that binds specifically to G-G mismatches has been used to induce hairpin formation in oligonucleotides immobilized onto chemically modified gold surfaces. Surface plasmon resonance (SPR) imaging measurements of DNA microarrays were used to demonstrate that binding of the naphthyridine dimer to G-G mismatches within the stem portion of an immobilized 42-mer oligonucleotide could be used to induce hairpin formation that prevented hybridization of DNA complementary to the loop sequence. In addition, the selectivity of the naphthyridine dimer for G-G mismatches was verified through SPR imaging measurements of the hybridization adsorption of an 11-mer oligonucleotide to a four-component DNA array of zero- and single-base mismatch sequences.
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