DNA abasic site-selective enhancement of sanguinarine fluorescence with a large emission shift

Abstract

Small molecules that can specifically bind to a DNA abasic site (AP site) have received much attention due to their importance in DNA lesion identification, drug discovery, and sensor design. Herein, the AP site binding behavior of sanguinarine (SG), a natural alkaloid, was investigated. In aqueous solution, SG has a short-wavelength alkanolamine emission band and a long-wavelength iminium emission band. At pH 8.3, SG experiences a fluorescence quenching for both bands upon binding to fully matched DNAs without the AP site, while the presence of the AP site induces a strong SG binding and the observed fluorescence enhancement for the iminium band are highly dependent on the nucleobases flanking the AP site, while the alkanolamine band is always quenched. The bases opposite the AP site also exert some modifications on the SG's emission behavior. It was found that the observed quenching for DNAs with Gs and Cs flanking the AP site is most likely caused by electron transfer between the AP site-bound excited-state SG and the nearby Gs. However, the flanking As and Ts that are not easily oxidized favor the enhanced emission. This AP site-selective enhancement of SG fluorescence accompanies a band conversion in the dominate emission from the alkanolamine to iminium band thus with a large emission shift of about 170 nm. Absorption spectra, steady-state and transient-state fluorescence, DNA melting, and electrolyte experiments confirm that the AP site binding of SG occurs and the stacking interaction with the nearby base pairs is likely to prevent the converted SG iminium form from contacting with water that is thus emissive when the AP site neighbors are bases other than guanines. We expect that this fluorophore would be developed as a promising AP site binder having a large emission shift.

Figure 1. Structures of SG and DNA sequences.

(A) SG at the variable forms and schematic representation of the AP site-targeted association of SG with a large emission shift. (B) For AP site-containing DNAs, X = AP site (dSpacer, tetrahydrofuran residue) that is opposed by base Y and flanked by base Fs. Fully matched DNAs (FM-DNA) with X/Y = A/T, C/G, G/C, and T/A were used as controls.

Figure 2. pH dependence of SG fluorescence.

(A) emission spectra and (B) the relative intensity alterations of SG (5 µM). λ_ex = 336 nm.

Figure 3. AP site-dependent fluorescence behaviors of SG.

Excitation (A and C, measured at 586 nm) and emission (B and D, excited at 336 nm) spectra of SG (5 µM) in the absence and presence of 5 µM DNA1-Ys (A and B) and DNA3-Ys (C and D). The corresponding fully matched DNAs (FM-DNA) were used as controls. Inset: the photographs of SG in the absence and presence of 5 µM FM, DNA1-A, DNA1-C, DNA1-G and DNA1-T (from left to right) under UV illumination.

Figure 4. Absorption spectra of SG.

(A) UV-Vis absorption spectra of SG (5 µM) in the absence and presence of 5 µM DNA1-Ys. The corresponding fully matched DNAs (FM-DNA) were used as controls. (B) UV-Vis absorption spectra of SG (5 µM) alone at pH 8.3 (a) and pH 6.0 (b).

Figure 5. Emission spectra of SG (1 µM) in pH 8.3 aqueous solution.

(a) SG alone; (b) in the presence of 5 µM FM-DNA; (c) after further addition of 1 µM DNA1-C with the SG solution pretreated with 5 µM FM-DNA.

Figure 6. Effect of added NaCl concentrations on SG fluorescence at 586 nm.

F₀ and F represent the fluorescence responses of the SG-DNA complexes in the absence and presence of NaCl, respectively.