Y charged iminium (in the pH rang 1.0?.0) and the neutral alkanolamine

Y charged TBHQ supplier iminium (in the pH rang 1.0?.0) and the neutral alkanolamine (in the pH rang 8.5?1.0) forms (Figure 1) [31,32]. The iminium form is unsaturated and completely planar, while the alkanolamine form has a buckled structure. SG can interact with polymorphic nucleic acid structures including DNA (B form [33], Z form, triplex [34], quadruplex [35]) and RNA (for example, poly(A) [36]). It is widely believed that the iminium form is mainly responsible for the DNA binding [37]. In addition, binding-induced fluorescence quenching and a strong GC base pair binding preference were observed [38?41]. In this work, we found that SG exhibits a sequence-dependent AP site binding behavior in the aspect of the enhanced emission for the iminium form that is converted from the alkanolamine form. Thus, targeting the AP site with a larger emission shift can be realized by thorough conversion of the alkanolamine emission band to the iminium emission band. The mechanism of the sequence-dependent fluorescence behavior is discussed.annealed in a thermocycler (first at 92uC, then cooled down to room temperature slowly) in 0.1 M phosphate buffer (pH 7.0) containing 1 mM EDTA. Sanguinarine (SG, Sigma Chemical Co., St. Louis, USA) was added to the duplex DNA solution to an appropriate molar ratio at 0.1 M phosphate buffer (pH 7.0) containing 1 mM EDTA. After mixing, the solution was incubated for 15 minutes with gentle stirring. The resulting solution was examined at room temperature within 2 h. Nanopure water (18.2 mV; Millipore Co., USA) was used in all experiments. Fluorescence RE640 supplier spectra were acquired with a FLSP920 spectrofluorometer (Edinburgh Instruments Ltd., UK) at 1861uC, equipped with a temperature- controlled circulator (Julabo, Germany). Time-resolved fluorescence decays were recorded on a time-correlated single photon counting FLSP920 system, with excitation at 375 nm. A ludox solution was used as the scatter for the instrument response. The data were fitted with a multiexponential decay and x2 was less than 1.15. UV/Vis absorption spectra and melting temperatures (Tm) were determined with a UV2550 spectrophotometer (Shimadzu Corp., Japan), equipped with an accessory of TMSPC-8 Tm analysis system which can simultaneously control the chamber temperature and detect up to 8 samples by a micro multi-cell.Results and Discussion Experimental sectionDNA species (Figure 1) were synthesized by TaKaRa Biotechnology Co., Ltd (Dalian, China) and purified by HPLC. The DNA concentrations were measured by UV absorbance at 260 nm using extinction coefficients calculated by the nearest neighbor analysis. Tetrahydrofuran residue was used as the chemically stable abasic site (AP site) for replacement of the naturally-occurred unstable deoxyribose structure. To prepare DNA duplex solutions, the probe and target strands were mixed in equimolar amounts and In aqueous solution, SG exists in the forms of iminium and alkanolamine and their population is dependent on pH (Figure 1). As shown in Figure 2, the 415 nm emission band increases with the solution pH increasing, while the 604 nm band simultaneously deceases under excitation at 336 nm. Thus, the iminium and alkanolamine forms emit at 604 and 415 nm, respectively [31]. The fitted equilibrium constant pKa is about 7.7, which is in good agreement with the previously reported value [32]. The alkanolamine form is not further deprotonated when the solution pH is lower than 11 [42]. According to the absorbance of the two f.Y charged iminium (in the pH rang 1.0?.0) and the neutral alkanolamine (in the pH rang 8.5?1.0) forms (Figure 1) [31,32]. The iminium form is unsaturated and completely planar, while the alkanolamine form has a buckled structure. SG can interact with polymorphic nucleic acid structures including DNA (B form [33], Z form, triplex [34], quadruplex [35]) and RNA (for example, poly(A) [36]). It is widely believed that the iminium form is mainly responsible for the DNA binding [37]. In addition, binding-induced fluorescence quenching and a strong GC base pair binding preference were observed [38?41]. In this work, we found that SG exhibits a sequence-dependent AP site binding behavior in the aspect of the enhanced emission for the iminium form that is converted from the alkanolamine form. Thus, targeting the AP site with a larger emission shift can be realized by thorough conversion of the alkanolamine emission band to the iminium emission band. The mechanism of the sequence-dependent fluorescence behavior is discussed.annealed in a thermocycler (first at 92uC, then cooled down to room temperature slowly) in 0.1 M phosphate buffer (pH 7.0) containing 1 mM EDTA. Sanguinarine (SG, Sigma Chemical Co., St. Louis, USA) was added to the duplex DNA solution to an appropriate molar ratio at 0.1 M phosphate buffer (pH 7.0) containing 1 mM EDTA. After mixing, the solution was incubated for 15 minutes with gentle stirring. The resulting solution was examined at room temperature within 2 h. Nanopure water (18.2 mV; Millipore Co., USA) was used in all experiments. Fluorescence spectra were acquired with a FLSP920 spectrofluorometer (Edinburgh Instruments Ltd., UK) at 1861uC, equipped with a temperature- controlled circulator (Julabo, Germany). Time-resolved fluorescence decays were recorded on a time-correlated single photon counting FLSP920 system, with excitation at 375 nm. A ludox solution was used as the scatter for the instrument response. The data were fitted with a multiexponential decay and x2 was less than 1.15. UV/Vis absorption spectra and melting temperatures (Tm) were determined with a UV2550 spectrophotometer (Shimadzu Corp., Japan), equipped with an accessory of TMSPC-8 Tm analysis system which can simultaneously control the chamber temperature and detect up to 8 samples by a micro multi-cell.Results and Discussion Experimental sectionDNA species (Figure 1) were synthesized by TaKaRa Biotechnology Co., Ltd (Dalian, China) and purified by HPLC. The DNA concentrations were measured by UV absorbance at 260 nm using extinction coefficients calculated by the nearest neighbor analysis. Tetrahydrofuran residue was used as the chemically stable abasic site (AP site) for replacement of the naturally-occurred unstable deoxyribose structure. To prepare DNA duplex solutions, the probe and target strands were mixed in equimolar amounts and In aqueous solution, SG exists in the forms of iminium and alkanolamine and their population is dependent on pH (Figure 1). As shown in Figure 2, the 415 nm emission band increases with the solution pH increasing, while the 604 nm band simultaneously deceases under excitation at 336 nm. Thus, the iminium and alkanolamine forms emit at 604 and 415 nm, respectively [31]. The fitted equilibrium constant pKa is about 7.7, which is in good agreement with the previously reported value [32]. The alkanolamine form is not further deprotonated when the solution pH is lower than 11 [42]. According to the absorbance of the two f.