2'-Fluoroarabinonucleic acid modification traps G-quadruplex and i-motif structures in human telomeric DNA

Abstract

Human telomeres and promoter regions of genes fulfill a significant role in cellular aging and cancer. These regions comprise of guanine and cytosine-rich repeats, which under certain conditions can fold into G-quadruplex (G4) and i-motif structures, respectively. Herein, we use UV, circular dichroism and NMR spectroscopy to study several human telomeric sequences and demonstrate that G4/i-motif-duplex interconversion kinetics are slowed down dramatically by 2'-β-fluorination and the presence of G4/i-motif-duplex junctions. NMR-monitored kinetic experiments on 1:1 mixtures of native and modified C- and G-rich human telomeric sequences reveal that strand hybridization kinetics are controlled by G4 or i-motif unfolding. Furthermore, we provide NMR evidence for the formation of a hybrid complex containing G4 and i-motif structures proximal to a duplex DNA segment at neutral pH. While the presence of i-motif and G4 folds may be mutually exclusive in promoter genome sequences, our results suggest that they may co-exist transiently as intermediates in telomeric sequences.

Figure 1.

(A) A:T and C:G Watson-Crick base pairs (top) and schematic representation of an antiparallel duplex (bottom). The G-rich strand is represented in blue and the C-rich strand in red. (B) G-quartet (left) and schematic representation of an intramolecular G-quadruplex (right). (C) C:CH⁺ base pair (left) and schematic representation of an intramolecular i-motif (right).

Figure 2.

Structures of 2′-deoxy-2′-fluoro-arabinocytidine (2′F-araC, left), 2′-deoxy-2′-fluoro-arabinoguanosine (2′F-araG, middle) and 2′-deoxy-2′-fluoro-guanosine (2′F-riboG, right).

Figure 3.

Models representing G4/i-motif-duplex interconversion of (I) 22mer human telomeric repeat forming G4, (II) 22mer human telomeric repeat forming G4 and i-motif secondary structures due to 2′F-ANA modifications, (III) 35mer human telomeric repeat forming a duplex and G4 and (IV) 35mer human telomeric repeat forming a duplex, a G4, and an i-motif within the same complex.

Figure 4.

(A) UV-melting curves of 22mer sample D5 (2 μM) recorded at 260 nm and 0.5 °C / min. (B) CD spectra of D5 (15 μM) recorded at 5 °C. (C) UV-melting curves of DQ (2 μM) recorded at 260 nm and 0.5 °C / min, pH 7.0. (D) CD spectra of DQ (15 μM) recorded at 5 °C, pH 7.0. Buffer conditions: 20 mM KPi and 70 mM KCl.

Figure 5.

¹H-NMR monitored kinetic experiments for (A) D1 and (B) D6 at 5 °C and 25 °C in a 1:1 mixture at pH 7.1 in 20 mM KPi and 70 mM KCl.

Figure 6.

¹H-NMR melting experiments at pH 7.1 in 20 mM KPi and 70 mM KCl. G4 imino signals show at 10–12 ppm and i-motif imino signals at 15–16 ppm.

Figure 7.

¹H-NMR spectra acquired at different times and at 5 °C for a 1:1 mixture of (A) DQ1 (35G0:35C0), (B) DQ2 (35G0:35C2), (C) DQ3 (35G3:35C0) and (D) DQ4 (35G3:35C2). Buffer conditions: 20 mM KPi and 70 mM KCl, pH 7.1.

Figure 8.

Schematic representation of the proposed scenarios: Scenario (A) where a duplex moiety co-exists in the same complex with G4 and i-motif. Scenario (B) where a duplex moiety co-exists with a C-rich ssDNA and a G4 (B1), a G-rich ssDNA and an i-motif (B2). (C) Imino region of the ¹H-NMR spectrum of 35G3:35C2 recorded at T = 5 °C after 30 min from mixing the two strands and showing the values resulting from the integration of i-motif, duplex, and G4 imino signals.