Spectroscopic Properties of Two 5'-(4-Dimethylamino)Azobenzene Conjugated G-Quadruplex Forming Oligonucleotides

Abstract

The synthesis of two 5'-end (4-dimethylamino)azobenzene conjugated G-quadruplex forming aptamers, the thrombin binding aptamer (TBA) and the HIV-1 integrase aptamer (T30695), was performed. Their structural behavior was investigated by means of UV, CD, fluorescence spectroscopy, and gel electrophoresis techniques in K⁺-containing buffers and water-ethanol blends. Particularly, we observed that the presence of the 5'-(4-dimethylamino)azobenzene moiety leads TBA to form multimers instead of the typical monomolecular chair-like G-quadruplex and almost hampers T30695 G-quadruplex monomers to dimerize. Fluorescence studies evidenced that both the conjugated G-quadruplexes possess unique fluorescence features when excited at wavelengths corresponding to the UV absorption of the conjugated moiety. Furthermore, a preliminary investigation of the trans-cis conversion of the dye incorporated at the 5'-end of TBA and T30695 showed that, unlike the free dye, in K⁺-containing water-ethanol-triethylamine blend the trans-to-cis conversion was almost undetectable by means of a standard UV spectrophotometer.

Keywords: 5′-(4-dimethylamino)azobenzene phosphoramidite derivative; CD G-quadruplexes; G-quadruplexes; azobenzenes; conjugated aptamers; fluorescence G-quadruplexes; trans-cis conversion.

Figure 1

Schematic representation of T30695 (A) and TBA (B) Q structures. Red and blue balls containing the letters G and T represent G and T residues, respectively. Gray rectangles schematize the G-quartets, regardless the syn-anti orientation of each G residue.

Scheme 1

Synthesis of the DMAzo moiety. (a) 1, (1.0 g, 7.2 mmol), KOH (2 mg, 3.6 × 10⁻² mmol), R-glycidol 574 µL (0.640 g, 8.6 mmol) in dry toluene (4.0 mL) at 90 °C; (b) 2 (0. 560 g, 2.6 mmol), Pd/C (28 mg, 5% w/w with respect to 2) and EtOH (25 mL); (c) 3 (0.400 g, 2.2 mmol), 205 µL fluoroboric acid (water solution, 48%, 3.3 mmol), EtOH (4.0 mL), −15 °C, isoamyl nitrite (0.258 g, 2.2 mmol); (d) 4 (0.360 g, 1.8 mmol) acetic acid (10 mL, 14.0 g, 232 mmol), sodium acetate (4.0 g, 511 mmol) and N,N-dimethylaniline (254 µL, 0.270 g, 2.2 mmol). (e) 5 (0.400 g, 1.3 mmol), 4,4-dimethoxytrityl chloride (0.430 g, 1.3 mmol), 4-dimethylaminopyridine (0.0076 g, 0.062 mmol) dry Py (6.35 mL), 2.5 h and (f) 6 (0.350 g, 0.57 mmol), β-cyanoethyl-N,N’-diisopropyl-chloro-phosphoramidite (0.190 mL, 0.85 mmol), DIPEA (0.296 mL, 1.7 mmol), dry DCM (3 mL), 1.0 h.

Figure 2

(A) UV spectra of TBA (black line) and 5′-DMAzo-TBA (fuchsia line) in 100 K buffer (pH = 7.4) in the range 200–600 nm. (B) UV spectra of T30695 (black line) and 5′-DMAzo-T30695 (fuchsia line) in 10 K buffer (pH = 7.4), in the range 200–600 nm. The spectra were acquired at 10 °C using [ON] = 20 µM. cuvette o.l. 0.1 cm, V = 400 µL.

Figure 3

CD spectra of (A) TBA (black line) and 5′-DMAzo-TBA (fuchsia line), acquired in 100 K buffer (pH = 7.4) and of (B) T30695 (black line) and 5′-DMAzo-T30695 (fuchsia line), acquired in 10 K buffer (pH = 7.4). T = 10 °C; [ON] = 20 µM. Cuvette o.l. 1.0 cm, Vol = 1400 µL.

Figure 4

CD melting profiles of TBA or T30695 and of the corresponding conjugated sequences in different solvent conditions. (A) TBA (black line) and 5′-DMAzo-TBA (fuchsia line) in 100 K buffer (pH=7.4). (B) T30695 (black line) and 5′-DMAzo-T30695 (fuchsia line), in 10 K buffer (pH = 7.4). (C) TBA (black line) and 5′-DMAzo-TBA (fuchsia line) in 5KEW blend. (D) T30695 (black line) and 5′-DMAzo-T30695 (fuchsia line) in 5KEW blend. [ON] = 2.0 µM.

Figure 5

The normalized fluorescence spectra of (A) TBA (black line) and 5′-DMAzo-TBA (red line) in 100 K buffer excited at 255 nm and acquired in the range 300–460 nm. (B) T30695 (black line) and 5′-DMAzo-T30695 (red line) in 10 K buffer excited at 255 nm and acquired in the range 300–460 nm. (C) 5′-DMAzo-TBA (100 K buffer, red line), 5′-DMAzo-T30695 (10K buffer, black line) and free DMAzo (10 K buffer, blue line) excited at 450 nm and acquired in the range 480–650 nm.

Figure 6

(A,B) Fluorescence spectra at different temperatures (from 25 to 95 °C, ΔT = 10 °C) of T30695 and 5′-DMAzo-T30695 excited at 255 nm acquired in the region 300–460 nm. (C,D). Fluorescence spectra at different temperatures (from 25 to 95 °C, ΔT = 10 °C) of TBA and 5′-DMAzo-TBA excited at 255 nm acquired in the region 300–460 nm. In all cases 5KWE was used as a solvent.

Figure 7

(A) Fluorescence spectra of the free DMAzo (blue line), 5′-DMAzo-TBA (red line) and 5′-DMAzo-T30695 (black line) at 25 °C in the wavelength range 480–650 nm, after excitation at 450 nm. (B) Temperature-dependent fluorescence spectra of the 5′-DMAzo-T30695, in the wavelength range 480–650 nm, after excitation at 450 nm. In all cases 5KWE was used as solvent.

Figure 8

Electrophoresis mobility shift assay of the conjugated and unmodified sequences. Lane 1: TBA; lane 2: 5′-TT-T30695; lane 3: 5′-DMAzo-TBA; lane 4: 5′-DMAzo-T30695 and lane 5: T30695.

Figure 9

(A) UV spectra of the free DMAzo in the 5KEW containing 0.1 mM of TEA before (green line) and after (blue line) excitation with LED at 436 nm. (B) UV spectra of the free DMAzo in the 5KEW containing 0.5 mM of TEA before (green line) and after (blue line) excitation with LED at 436 nm.

Figure 10

(A) Time-dependent UV spectrum of the 5′-DMAzo-TBA in the 5KEW-TEA blend; after irradiation at 436 nm. (B) The enlargement of the UV-vis region between 320–600 nm. The band intensities around 440 nm attributable to the absorption of the conjugated 5′DMAB slightly decrease after irradiation (blue line), and consecutively increases over time to reach the values observed before irradiation (violet line) in dark. [ON] = 3.5 × 10⁻⁶ M.