## 3 Results and Discussion
When the C10-linker was appended to the benzothiazole ring, the resulting conjugate (ClNaph-TO2) showed the selective light-up response for target C in the AP-DNA duplexes (Figure S6); however, the degree of the response was smaller compared to CINaph-TO (cf. Figure 3A). This is attributable to the reduced binding affinity of CINaph-TO  
Figure 3: **(A)** Fluorescence response of CINaph–TO conjugate (500 nM) to target AP–DNA duplexes (500 nM). Excitation, 504 nm. Temperature, 20 \({}^{\circ}\)C. **(B)** Images of fluorescence emission of CINaph–TO conjugate (500 nM) in the absence and presence of target AP–DNA duplexes (500 nM) under excitation light with the wavelength of 365 nm, obtained by a digital camera at room temperature. Other solution conditions are the same as those given in Figure 2.  
(\(K_{\text{d}}=71\) nM). This indicates that the conjugation of CINaph unit into the benzothiazole ring hinders the effective intercalation of the TO unit for DNA duplexes as reported in the literature [27]. In addition, we observed moderate selectivity of CINaph-TO2 conjugate for target C over other three nucleobases in AP-DNA duplexes (Figure S6) while the reason for this is unclear yet. Hence, the connection of CINaph unit to the quinolone ring of the TO unit is effective for the strong binding and large light-up response of the conjugate for target C in the AP-DNA duplexes. As described above, the CINaph-TO conjugate can serve as a useful light-up probe for target C in the AP-DNA duplexes. Meanwhile, we noticed weak binding of the conjugate for AP-RNA duplexes (Figure 4A and Figure S7). The binding affinity was estimated as \(>\) 1300-nM for target C in the AP-RNA duplex (5'-r(GCA GCU CCC AXA GUC UCC UCG)-3'/3'-r(CGU CGA GGG UUCu CAG AGG)-5', \(\mathbf{\chi}=\) AP site (Spacer C3), \(\mathbf{\underline{C}}=\) target nucleobase), which was three orders of magnitude larger than that for the AP-DNA duplex (cf. Figure S4). We consider that this arises from the preferential binding of the CINaph-TO conjugate to B-formed AP-DNA duplexes relative to A-formed AP-RNA duplexes [6]. However, when targeting C in single stranded RNA, the use of AP-DNA probe is highly effective. The resulting duplex is the hybrid between RNA and AP-DNA ((5'-d(GCCA GCT CCC AXA GTC TCC)-3'/3'-r(CGU CGA GGG UUCu CAG AGG AGC)-5', \(\mathbf{\chi}=\) AP site (Spacer C3), \(\mathbf{\underline{C}}=\) target nucleobase) that can adopt A-form/B-form intermediate structure [28], under the same measurement conditions used to examine AP-RNA duplexes. As shown in Figure 4B, we observed the significant light-up response of the conjugate for the AP-DNA/RNA hybrid, in which the response was 22-fold larger compared to that for the AP-RNA duplex. The \(K_{\text{d}}\) for target C in the hybrid was obtained as 22 nM (Figure S7), where the affinity is much superior to that for AP-RNA duplexes. Importantly, the CINaph-TO conjugate retains its selectivity for target C over U opposite the AP site in the DNA/RNA hybrid, as was observed in AP-DNA duplexes (cf. Figure 3B). These results suggest the potential use of CINaph-TO conjugate for the detection of microRNAs in combination with AP-DNA hybridization probe (cf. Figure 1A). We performed the preliminarily experiments for detection of microRNAs based on the binding-induced light-up response of CINaph-TO conjugate for AP-DNA/RNA hybrids. A 21-meric AP-DNA probe was designed for the selective detection of the let-7d sequence among let-7 family [29], as shown in Figure 5A. Hybridization between this probe and let-7d allows the construction of the AP-DNA/RNA hybrid containing an orphan C opposite an AP site. Meanwhile, the hybridization with other let-7 sequences leads to the formation of orphan U-containing hybrids with several mismatch base pairs (Table S1). We observed the significant light-up response of CINaph-TO for the let-7d-containing hybrid (Figure 5A).