G-Quadruplex Aptamer-Ligand Characterization

Abstract

In this work we explore the structure of a G-rich DNA aptamer termed AT11-L2 (TGGTGGTGGTTGTTGTTGGTGGTGGTGGT; derivative of AT11) by evaluating the formation and stability of G-quadruplex (G4) conformation under different experimental conditions such as KCl concentration, temperature, and upon binding with a variety of G4 ligands (360A, BRACO-19, PDS, PhenDC3, TMPyP4). We also determined whether nucleolin (NCL) can be a target of AT11-L2 G4. Firstly, we assessed by circular dichroism, UV and NMR spectroscopies the formation of G4 by AT11-L2. We observed that, for KCl concentrations of 65 mM or less, AT11-L2 adopts hybrid or multiple topologies. In contrast, a parallel topology predominates for buffer containing 100 mM of KCl. The T_m of AT11-L2 in 100 mM of KCl is 38.9 °C, proving the weak stability of this sequence. We also found that upon titration with two molar equivalents of 360A, BRACO-19 and PhenDC3, the G4 is strongly stabilized and its topology is maintained, while the addition of 3.5 molar equivalents of TMPyP4 promotes the disruption of G4. The K_D values between AT11-L2 G4, ligands and NCL were obtained by fluorescence titrations and are in the range of µM for ligand complexes and nM when adding NCL. In silico studies suggest that four ligands bind to the AT11-L2 G4 structure by stacking interactions, while the RBD1,2 domains of NCL interact preferentially with the thymines of AT11-L2 G4. Finally, AT11-L2 G4 co-localized with NCL in NCL-positive tongue squamous cell carcinoma cell line.

Keywords: G-quadruplex aptamer; aptamer–ligand interactions; biophysical techniques; ligands.

Figure 1

AS1411 derivatives and chemical structure of G4 ligands.

Figure 2

Evaluation of G4 formation using distinct spectroscopic techniques. (A) CD spectra of AT11-L2 (10 μΜ) in 10 mM of lithium cacodylate buffer with increasing concentrations of KCl in the range of 220–340 nm. (B) IDS spectrum resultant from the difference of UV spectra in the presence and absence of KCl. (C) TDS spectrum of AT11-L2 in lithium cacodylate containing 100 mM of KCl, obtained by subtracting the spectrum in folded and unfolded states and then normalized relative to the maximum absorbance (D) Effect of KCl salt on the structure formation of AT11-L2 G4 (50 μΜ) was monitored by ¹H NMR spectroscopy recorded in lithium cacodylate supplemented with 10% D₂O and increased concentrations of KCl (20 to 100 mM). All measurements were performed at 20 °C.

Figure 3

CD spectra of AT11-L2 in the absence and presence of increased molar equivalents of ligands (A) 360 A, (B) BRACO-19, (C) PhenDC3, (D) TMPyP4 and (E) PDS. CD spectra were acquired in a buffer containing 10 mM of lithium cacodylate and 100 mM of KCl.

Figure 4

CD melting spectra of AT11-L2 in the absence and presence of increased molar equivalents of ligands (A) 360A, (B) BRACO-19, (C) PhenDC3, (D) TMPyP4 and (E) PDS. CD spectra were acquired in a buffer containing 10 mM of lithium cacodylate (pH 7.2) and 100 mM of KCl.

Figure 5

T_m radar plot of AT11-L2 in the presence of different molar equivalents of tested ligands obtained by CD-melting experiments.

Figure 6

Fluorescence titration spectra of pre-folded 5′-Cy5-AT11-L2 G4 with increasing concentrations of (A) 360A, (B) TMPyP4, (C) PhenDC3, (D) BRACO-19 and (E) PDS. The experiments were performed in buffer containing 10 mM of lithium cacodylate and 100 mM of KCl with excitation set at 647 nm, and emission was recorded ranging from 655 to 800 nm.

Figure 7

Final snapshots of 200 ns MD simulations of the complex between AT11-L2 G4 and PhenDC3. (A) Side view and (B) top view. The backbone is highlighted in light grey, while nucleotides are depicted in orange. K⁺ is shown in purple. The ligand is depicted in blue. The first and last nucleotides of AT11-L2 are also shown. (C) RMSD plot of the 200 ns simulation of AT11-L2 G4/PhenDC3 complex.

Figure 8

Final snapshot of 200 ns MD simulations of the complex AT11-L2 G4/NCL RBD1,2. G4 backbone is depicted in light grey, while nucleotides are highlighted in orange. K⁺ is shown in purple. NCL residues that interact with AT11-L2 G4 by hydrogen bonding are highlighted in cornflower blue.

Figure 9

Non-denaturing gel electrophoresis of AT11-L2 G4 (2 μM) in (A) presence of ligands (4 μM) (Lanes: M—Marker; 1—ultrapure water; 2—annealing buffer; 3—360A; 4—PhenDC3; 5—TMPyP4; 6—BRACO-19 and 7—PDS) and (B) Ligands (4 μM) and NCL (2 μM) (Lanes: M—Marker; 1–NCL; 2—360A; 3—PhenDC3; 4—TMPyP4; 5—BRACO-19 and 6-PDS).

Figure 10

NCL immunofluorescence in UPCI-SCC-154 cells with 5´-FAM-AT11-L2-3´-TAMRA (green) conjugate with PhenDC3. The anti-NCL primary antibody was conjugated with an Alexa Fluor 647 secondary antibody (red) and nuclei were stained with Hoechst (blue). (A) Original images obtained with a 63× objective (scale bar: 20 µm). (B) Magnification from the original image (scale bar: 5 µm).