Hly proportional in turn to the oxidation potentials of their unpaired bases C, T, A, 15900046 and G, again revealing that the bound SG’ emission is somewhat affected by the possible electron transfer between the excited state SG and the unpaired bases opposite the AP site. From the above results, we can conclude that SG shows a sequence-dependent binding at the AP site. Usually, the specific interaction of small molecules with DNA base pairs will affect the DNA thermodynamic stability. In order to verify the occurrence of effective stacking interactions of SG with the AP-DNAs, DNA Table 3. Melting temperatures of 5 mM DNA2s in the absence and presence of 5 mM SG.(16.34 ) (76.62 )11.75 10.29 13.b a b(83.66 ) (23.38 )1.006 1.019 1.039 1.FM-DNA3.25 2.a bFM2 With SG/uC Without SG/uC DT/uC 63.0 62.3 0.DNA2-A 52.1 49.1 3.DNA2-C 52.5 47.1 5.DNA2-G 52.7 49.1 3.DNA2-T 52.7 47.4 5.(72.47 )7.b(27.53 )1.The lifetimes were measured at 415 nm (a) and 586 nm (b) with excitation at 336 nm. The lifetime measurement was not applicable for DNA3-Ys and DNA4Ys due to the strong fluorescence quenching. doi:10.1371/journal.pone.0048251.tDdoi:10.1371/journal.pone.0048251.tDNA Abasic Site BinderTable 4. Melting temperatures of 5 mM DNA3s in the absence and presence of 5 mM SG.Table 5. Melting temperatures of 5 mM DNA4s in the absence and presence of 5 mM SG.FM3 With SG/uC Without SG/uC DT/uC 69.0 68.4 0.DNA3-A 60.3 57.1 3.DNA3-C 61.0 56.5 4.DNA3-G 59.1 56.7 2.DNA3-T 60.3 54.6 5.7 With SG/uC Without SG/uC DT/uCFM4 69.5 69.0 0.DNA4-A 60.6 57.8 2.DNA4-C 58.1 52.9 5.DNA4-G 59.5 57.1 2.DNA4-T 59.3 54.4 4.doi:10.1371/journal.pone.0048251.tdoi:10.1371/journal.pone.0048251.tmelting (Tm) experiments were conducted by measuring the 260 nm absorbance as a function of the solution temperature. As shown in Table 2 to 5, the presence of SG stabilizes DNAn-C and DNAn-T with the Tm increasing of 4.4?.4uC and 3.8?.7uC, while DNAn-A and DNAn-G Mirin web induce the Tm increasing of 1.9?3.2uC and 1.9?.6uC, respectively. Thus, the small-sized pyrimidines opposite the AP site allow for an effective stacking interaction, which is predicted to result in the observed greater emission enhancements for DNAn-C and -T than DNAn-G and A (n = 1, 2), and higher emission quenchings for DNAn-C and -T than DNAn-G and -A (n = 3, 4) (Figure 3 and S1). MedChemExpress CAL120 However, SG slightly stabilizes the FM-DNAs with the Tm increasing only of 0.5?.8uC. Therefore, it is reasonably concluded that SG can enter into the hydrophobic helix interior in the presence of the AP site and the AP site is believed to play an important role for the occurrence of the stacking 
interaction, implying a binding mode different from that for the FM-DNAs. This effective p stacking interaction should efficiently prevent the converted SG from contacting with water and induce a pronounced fluorescence alteration, and thus favor the formation of the emissive iminium form when the AP site neighbors are bases other than guanines. Finally, a comparison was made between SG binding to the AP site and to the mismatch site. With DNA1-C as an example, although we observed that the presence of a mismatch site (DNA1, X = T, Y = C, one of the most unstable mismatch sites [47]) also enhanced SG fluorescence, the enhancement arising from the AP site binding was still overwhelming (Figure S4), indicating the high selectivity of SG binding to the AP site. Additionally, a noticeable increase in the fluorescence response could be distinguished even with DNA1-C concentration.Hly proportional in turn to the oxidation potentials of their unpaired bases C, T, A, 15900046 and G, again revealing that the bound SG’ emission is somewhat affected by the possible electron transfer between the excited state SG and the unpaired bases opposite the AP site. From the above results, we can conclude that SG shows a sequence-dependent binding at the AP site. Usually, the specific interaction of small molecules with DNA base pairs will affect the DNA thermodynamic stability. In order to verify the occurrence of effective stacking interactions of SG with the AP-DNAs, DNA Table 3. Melting temperatures of 5 mM DNA2s in the absence and presence of 5 mM SG.(16.34 ) (76.62 )11.75 10.29 13.b a b(83.66 ) (23.38 )1.006 1.019 1.039 1.FM-DNA3.25 2.a bFM2 With SG/uC Without SG/uC DT/uC 63.0 62.3 0.DNA2-A 52.1 49.1 3.DNA2-C 52.5 47.1 5.DNA2-G 52.7 49.1 3.DNA2-T 52.7 47.4 5.(72.47 )7.b(27.53 )1.The lifetimes were measured at 415 nm (a) and 586 nm (b) with excitation at 336 nm. The lifetime measurement was not applicable for DNA3-Ys and DNA4Ys due to the strong fluorescence quenching. doi:10.1371/journal.pone.0048251.tDdoi:10.1371/journal.pone.0048251.tDNA Abasic Site BinderTable 4. Melting temperatures of 5 mM DNA3s in the absence and presence of 5 mM SG.Table 5. Melting temperatures of 5 mM DNA4s in the absence and presence of 5 mM SG.FM3 With SG/uC Without SG/uC DT/uC 69.0 68.4 0.DNA3-A 60.3 57.1 3.DNA3-C 61.0 56.5 4.DNA3-G 59.1 56.7 2.DNA3-T 60.3 54.6 5.7 With SG/uC Without SG/uC DT/uCFM4 69.5 69.0 0.DNA4-A 60.6 57.8 2.DNA4-C 58.1 52.9 5.DNA4-G 59.5 57.1 2.DNA4-T 59.3 54.4 4.doi:10.1371/journal.pone.0048251.tdoi:10.1371/journal.pone.0048251.tmelting (Tm) experiments were conducted by measuring the 260 nm absorbance as a function of the solution temperature. As shown in Table 2 to 5, the presence of SG stabilizes DNAn-C and DNAn-T with the Tm increasing of 4.4?.4uC and 3.8?.7uC, while DNAn-A and DNAn-G induce the Tm increasing of 1.9?3.2uC and 1.9?.6uC, respectively. Thus, the small-sized pyrimidines opposite the AP site allow for an effective stacking interaction, which is predicted to result in the observed greater emission enhancements for DNAn-C and -T than DNAn-G and A (n = 1, 2), and higher emission quenchings for DNAn-C and -T than DNAn-G and -A (n = 3, 4) (Figure 3 and S1). However, SG slightly stabilizes the FM-DNAs with the Tm increasing only of 0.5?.8uC. Therefore, it is reasonably concluded that SG can enter into the hydrophobic helix interior in the presence of the AP site and the AP site is believed to play an important role for the occurrence of the stacking interaction, implying a binding mode different from that for the FM-DNAs. This effective p 
stacking interaction should efficiently prevent the converted SG from contacting with water and induce a pronounced fluorescence alteration, and thus favor the formation of the emissive iminium form when the AP site neighbors are bases other than guanines. Finally, a comparison was made between SG binding to the AP site and to the mismatch site. With DNA1-C as an example, although we observed that the presence of a mismatch site (DNA1, X = T, Y = C, one of the most unstable mismatch sites [47]) also enhanced SG fluorescence, the enhancement arising from the AP site binding was still overwhelming (Figure S4), indicating the high selectivity of SG binding to the AP site. Additionally, a noticeable increase in the fluorescence response could be distinguished even with DNA1-C concentration.
