Design and Properties of Ligand-Conjugated Guanine Oligonucleotides for Recovery of Mutated G-Quadruplexes

Abstract

The formation of a guanine quadruplex DNA structure (G4) is known to repress the expression of certain cancer-related genes. Consequently, a mutated G4 sequence can affect quadruplex formation and induce cancer progression. In this study, we developed an oligonucleotide derivative consisting of a ligand-containing guanine tract that replaces the mutated G4 guanine tract at the promoter of the vascular endothelial growth factor (VEGF) gene. A ligand moiety consisting of three types of polyaromatic hydrocarbons, pyrene, anthracene, and perylene, was attached to either the 3' or 5' end of the guanine tract. Each of the ligand-conjugated guanine tracts, with the exception of anthracene derivatives, combined with other intact guanine tracts to form an intermolecular G4 on the mutated VEGF promoter. This intermolecular G4, exhibiting parallel topology and high thermal stability, enabled VEGF G4 formation to be recovered from the mutated sequence. Stability of the intramolecular G4 increased with the size of the conjugated ligand. However, suppression of intermolecular G4 replication was uniquely dependent on whether the ligand was attached to the 3' or 5' end of the guanine tract. These results indicate that binding to either the top or bottom guanine quartet affects unfolding kinetics due to polarization in DNA polymerase processivity. Our findings provide a novel strategy for recovering G4 formation in case of damage, and fine-tuning processes such as replication and transcription.

Keywords: G-quadruplex; cancer; ligand; replication.

Figure 1

Schematic illustration of the recovery of mutated VEGF G4 by intermolecular G4 formation using a ligand-conjugated guanine tract.

Figure 2

Structures of ligand-containing guanine oligonucleotides used in this study. (A) Chemical structures of the ligand-containing linkers. (B) Structures of ligand-conjugated guanine tracts. The attachment of a ligand-containing linker was either to the 3′ or 5′ OH group of the respective deoxyguanosine residue via the phosphodiester linkage.

Figure 3

Circular dichroism (CD) spectra of 10 µM VEGF G2T with 10 µM (A) PySG₃, (B) G₃PyS, (C) PyLG₃, (D) G₃PyL, (E) PEPyG₃, and (F) G₃PEPy. All measurements were conducted in a buffer consisting of 10 mM Tris-HCl (pH 7.5), 8 mM MgCl₂, and 50 mM KCl.

Figure 4

CD spectra of 10 µM VEGF G2T with 10 µM (A) BPEAG₃, (B) G₃BPEA, (C) PerG₃, and (D) G₃Per. All the experiments were conducted in a buffer consisting of 10 mM Tris-HCl (pH 7.5), 8 mM MgCl₂, and 50 mM KCl.

Figure 5

CD melting profiles of 10 µM VEGF G2T with (A) pyrene-conjugated guanin tracts including PySG₃ (black), G₃PyS (purple), PyLG₃ (light blue), G₃PyL (blue), PEPyG₃ (pale green), and G₃PEPy (green), and (B) anthracene or perylene derivatives including BPEAG₃ (black), G₃BPEA (purple), PerG₃ (light blue), and G₃Per (blue). CD melting data were collected by measuring CD intensity at 263 nm in a buffer consisting of 10 mM Tris-HCl (pH 7.5), 8 mM MgCl₂, and 50 mM KCl.

Figure 6

Schematic illustration of the replication assay.

Figure 7

Denaturing-PAGE images of replication products from the VEGF G2T template with each ligand-conjugated guanine tract. All assays were performed in a buffer consisting of 10 mM Tris-HCl (pH 7.5), 8 mM MgCl₂, and 50 mM KCl with 1 µM primer, 1 µM template, 10 µM ligand-conjugated guanine tract, 250 µM dNTPs, and 1 µM KF exo- at 37 °C.

Figure 8

Regulation of G4 replication by a guanine tract conjugated at its (A) 5′ end and (B) 3′ end. The boxes highlighted in red indicate the affected G-quartet with the ligand moiety to decrease the unfolding kinetics.