Biophysical and biological properties of quadruplex oligodeoxyribonucleotides

Abstract

Single-stranded guanosine-rich oligodeoxyribonucleotides (GROs) have a propensity to form quadruplex structures that are stabilized by G-quartets. In addition to intense speculation about the role of G-quartet formation in vivo, there is considerable interest in the therapeutic potential of quadruplex oligonucleotides as aptamers or non-antisense antiproliferative agents. We previously have described several GROs that inhibit proliferation and induce apoptosis in cancer cell lines. The activity of these GROs was related to their ability to bind to a specific cellular protein (GRO-binding protein, which has been tentatively identified as nucleolin). In this report, we describe the physical properties and biological activity of a group of 12 quadruplex oligonucleotides whose structures have been characterized previously. This group includes the thrombin-binding aptamer, an anti-HIV oligonucleotide, and several quadruplexes derived from telomere sequences. Thermal denaturation and circular dichroism (CD) spectropolarimetry were utilized to investigate the stability, reversibility and ion dependence of G-quartet formation. The ability of each oligonucleotide to inhibit the proliferation of cancer cells and to compete for binding to the GRO-binding protein was also examined. Our results confirm that G-quartet formation is essential for biological activity of GROs and show that, in some cases, quadruplex structures formed in the presence of potassium ions are significantly more active than those formed in the presence of sodium ions. However, not all quadruplex structures exhibit antiproliferative effects, and the most accurate factor in predicting biological activity was the ability to bind to the GRO-binding protein. Our data also indicate that the CD spectra of quadruplex oligonucleotides may be more complex than previously thought.

Figure 1

Schematic presentation of G-quartet structures. (A) G-quartet. (B) Molecularity and loop orientation of quadruplexes

Figure 2

Cation dependence of antiproliferative activity. HeLa cervical carcinoma cells were either treated with a single dose of oligonucleotide (10 µM final concentration) annealed in buffer containing either 50 mM KCl or 50 mM NaCl, or received no oligonucleotide (None). Cell viability was assayed 7 days after addition of oligonucleotide using the MTT assay. All experiments were performed in triplicate, and bars represent the standard error of the data. In this figure, GRO20A, GRO23A, GRO29A, GRO15B and T30695 are abbreviated to 20A, 23A, 29A, 15B and T30 respectively. Dark bars represent oligonucleotides annealed in KCl and light bars represent oligonucleotides annealed in NaCl.

Figure 3

UV thermal denaturation–renaturation studies. Profiles were classified as NT, R or H, as shown. Oligonucleotides (2–10 µM final concentration) were annealed in T_m buffer (20 mM Tris–HCl pH 8.0, 140 mM KCl, and 2.5 mM MgCl₂) and absorbance was measured at 295 nm with a 1 cm path length. The melting curve is the lighter line; the annealing curve is the darker line. Melting and annealing temperatures are reported in Table 2.

Figure 4

CD spectroscopy studies. CD spectra of oligonucleotides (5 µM final concentration) were obtained in the presence of either 0.1 M KCl (dark lines) or 0.1 M NaCl (light lines) at 25°C with a path length of 4 mm.

Figure 5

Protein-binding assay. (A) Competitive mobility shift assay showing the ability of unlabeled oligonucleotides to compete with radiolabeled TEL for binding to nuclear proteins. The band marked by an asterisk indicates the specific GRO-binding protein (thought to be nucleolin). The names of the oligonucleotides are abbreviated without the KS or GRO prefixes. (B) Plot of the relative intensity of the protein band in (A) versus the relative number of viable cells remaining after treatment with oligonucleotide annealed in KCl buffer. The squared correlation coefficient (R²) for the relationship between these variables is 0.82. The names of the oligonucleotides are abbreviated without the KS or GRO prefixes.

Figure 6

Structural characteristics of GRO29A. (A) UV thermal denaturation–renaturation profile (295 nm) of GRO29A (2 µM concentration, 1 cm path length). (B) CD spectra of GRO29A (5 µM concentration, 4 mm path length) in buffers containing KCl (dark line) or NaCl (light line). (C) Non-denaturing polyacrylamide electrophoresis of GRO29A (150 µM) annealed in 50 mM KCl, 50 mM NaCl or no salt.