Effects of abasic sites on structural, thermodynamic and kinetic properties of quadruplex structures

Abstract

Abasic sites represent the most frequent lesion in DNA. Since several events generating abasic sites concern guanines, this damage is particularly important in quadruplex forming G-rich sequences, many of which are believed to be involved in several biological roles. However, the effects of abasic sites in sequences forming quadruplexes have been poorly studied. Here, we investigated the effects of abasic site mimics on structural, thermodynamic and kinetic properties of parallel quadruplexes. Investigation concerned five oligodeoxynucleotides based on the sequence d(TGGGGGT), in which all guanines have been replaced, one at a time, by an abasic site mimic (dS). All sequences preserve their ability to form quadruplexes; however, both spectroscopic and kinetic experiments point to sequence-dependent different effects on the structural flexibility and stability. Sequences d(TSGGGGT) and d(TGGGGST) form quite stable quadruplexes; however, for the other sequences, the introduction of the dS in proximity of the 3'-end decreases the stability more considerably than the 5'-end. Noteworthy, sequence d(TGSGGGT) forms a quadruplex where dS does not hamper the stacking between the G-tetrads adjacent to it. These results strongly argue for the central role of apurinic/apyrimidinic site damages and they encourage the production of further studies to better delineate the consequences of their presence in the biological relevant regions of the genome.

Figure 1.

Structure of the tetrahydrofuranyl analog, dSpacer (dS) introduced in ODNs in Table 1 as AP sites mimic.

Figure 2.

Aromatic and imino protons regions of the ¹H-NMR spectra of the ODNs (Table 1).

Figure 3.

2D NOESY (500 MHz) region correlating base and H2′/H2′′ sugar protons in AQ2 [d(T1G2S3G4G5G6T7)]₄. The arrow indicates a NOE between aromatic protons of G2 and G4.

Figure 4.

ODN AQ2. CD spectroscopy: (A) the panel reports the spectrum at 20°C before heating (solid line) and at 100°C (dashed line). Thermal analysis (B) showing the melting curve (solid line) and the annealing one (dashed line) recorded at a heating and cooling scanning rates of 1°C/min. Association/dissociation kinetic analysis (C and D) of the quadruplex structure following the change in the CD signal at 263 nm; the experimental points are reported as black squares and the interpolating curves as a grey solid line. The panels (E and F) show the Arrhenius plots for the association and the dissociation processes; the experimental values of the kinetic constants (black squares) have been interpolated with the Equation (5) in order to get the activation energies and the pre-exponential values.

Figure 5.

ODN AQ3. The panel (A) reports the spectra of the quadruplex structure at 90°C (solid line), at 5°C after annealing by cooling the sample from 90°C to 1°C/min (dashed line) and after 1 week storage at 5°C (dotted line). The difference between the last two spectra at 5°C suggests that a slow conformational rearrangement has occurred. In the panel (B) are represented the melting curves obtained at a heating rate of 1°C/min recorded after the annealing procedure (1) and after 1 week of equilibration at 5°C (3). In both cases, the annealing profiles (2, 4) share high similarities.

Figure 6.

ODN AQ4. CD spectroscopy: (A) the panel reports the spectra at 20°C after storage at 2°C and before heating (solid line), at 105°C (dash-dot-dot line) and at 20°C after annealing (dashed line). Thermal analysis (B) showing the melting curve (solid line) and the annealing one (dashed line) obtained with a heating/cooling rate of 1°C/min. The panels (C and D) show the Arrhenius plots for the association and the dissociation processes.

Figure 7.

Molecular models of the quadruplexes formed by ODNs TGGGGGT (left) and AQ2 (right). The structures are oriented with the 5′-end upward (carbons, green; nitrogens, blue; oxygens, red; hydrogens, white).