## 3 Results and Discussion  
First, we examined the binding ability of ClNaph (500 nM) to target orphan nucleobases in model 21-meric AP-DNA duplexes (500 nM; 5\({}^{\prime}\)-d(GCA GCT CCC AXA GTC TCC TCG)-3\({}^{\prime}\)/3\({}^{\prime}\)-d(CGT CGA GGG TNT CAG AGG AGC)-5\({}^{\prime}\), \(\underline{\textbf{X}}\) = AP site (Spacer C3, a propyl residue), \(\underline{\textbf{N}}\) = target nucleobase; G, C, A or T) by the fluorescence measurements. As shown in Figure 2, ClNaph showed the emission with the maximum at 393 nm in the absence of DNAs. The addition of AP-DNA duplexes caused a decrease in the fluorescence intensity of ClNaph. The largest fluorescence quenching response was observed for target C, which indicates that ClNaph shows the preferential binding to target C compared to target G, A and T. The binding affinity of ClNaph was assessed by the fluorescence titration experiments (Inset of Figure 2). ClNaph showed the quenching response for target DNA duplexes in a concentration-dependent manner and the resulting titration curves were analyzed by a 1:1 binding model for the determination of the dissociation constants (\(K_{\mathrm{d}}\)). The \(K_{\mathrm{d}}\) value for C was obtained as 70 \(\pm\) 5.3 nM. Significantly, this affinity is much stronger compared to the parent ADMND and the previously developed CF\({}_{3}\)-AMND and is almost comparable to ATMND (Table 1). In addition, CINaph showed useful binding selectivity toward target C, in which the \(K_{\mathrm{d}}\) value for target C was two orders of magnitude smaller than those for other target nucleobases (\(K_{\mathrm{d}}\)/nM: T, 2020 \(\pm\) 240, G and A \(>\) 5000). The observed selectivity for C over T is much superior to ADMND and ATMND [3] whereas it is slightly moderate in comparison with CF\({}_{3}\)-AMND [24]. We reason that this is due to the more favorable protonation of CINaph at the N1 position for the complementary base-pairing with orphan C compared to the N8 protonation (Figure 1B), where the electron-withdrawing effect of the chloro group would be essential as was observed for CF\({}_{3}\)-AMND [24]. We note that the type of the AP site affects the binding of CINaph. The use of Spacer C3 (propyl residue) allowed the stronger binding to target C compared to the tetrahydrofuranyl residue (dSpacer), presumably due to less steric hindrance (Figure S3). The observed strong and highly selective binding properties for target C should render CINaph a useful APL unit in the conjugate with TO unit. one order of magnitude stronger than CINaph itself (cf. Table 1). Apparently, the conjugation with the TO unit led to the enhanced binding affinity for target C in the AP-DNA duplexes. It should be noted that CINaph-TO conjugate has high binding selectivity for target C over other three nucleobases (Figure S4: \(K_{\text{d}}\) values for T, G and A \(>\) 1500 nM). Considering that TO lacks the selectivity for the orphan nucleobases [19], the CINaph unit is responsible for the observed C-selectivity of the conjugate. This was confirmed by the fluorescence quenching response of the CINaph unit with high selectivity to target C (Figure S5). These results showed the useful binding and light-up functions of the CINaph-TO conjugate for the detection of C-related mutations in target DNA sequences. It is also noteworthy that the strong emission of CINaph-TO conjugate for target C can be seen even with the naked eyes under UV light irradiation (Figure 3B). Fluorescence response for target C is clearly distinguishable from those for other target nucleobases, which thus facilitates a simple and rapid analysis of target DNA sequences. We found that the kind of heterocycles of the TO unit, benzothiazole or quinolone rings, that connect to CINaph unit in the conjugate affected the binding and fluorescence signaling abilities for the binding to AP-DNA duplexes.