Stability of telomeric G-quadruplexes

Abstract

In most eukaryotes, telomeric DNA consists of repeats of a short motif that includes consecutive guanines and may hence fold into G-quadruplexes. Budding yeasts have telomeres composed of longer repeats and show variation in the degree of repeat homogeneity. Although telomeric sequences from several organisms have been shown to fold into G-quadruplexes in vitro, surprisingly, no study has been dedicated to the comparison of G-quadruplex folding and stability of known telomeric sequences. Furthermore, to our knowledge, folding of yeast telomeric sequences into intramolecular G-quadruplexes has never been investigated. Using biophysical and biochemical methods, we studied sequences mimicking about four repetitions of telomeric motifs from a variety of organisms, including yeasts, with the aim of comparing the G-quadruplex folding potential of telomeric sequences among eukaryotes. G-quadruplex folding did not appear to be a conserved feature among yeast telomeric sequences. By contrast, all known telomeric sequences from eukaryotes other than yeasts folded into G-quadruplexes. Nevertheless, while G(3)T(1-4)A repeats (found in a variety of organisms) and G(4)T(2,4) repeats (found in ciliates) folded into stable G-quadruplexes, G-quadruplexes formed by repetitions of G(2)T(2)A and G(2)CT(2)A motifs (found in many insects and in nematodes, respectively) appeared to be in equilibrium with non-G-quadruplex structures (likely hairpin-duplexes).

Figure 1.

Schematic structures of intramolecular G-quadruplexes formed by about four repetitions of telomeric motifs from (A) vertebrates in Na⁺ (51), (B, E–G) vertebrates in K⁺ (52–57), (B and C) Giardia in K⁺ (60), (D) Bombyx in K⁺ (59), (F) Tetrahymena in Na⁺ (50) and (H) Oxytricha in Na⁺ (49). Anti and syn guanines are in white and grey, respectively. Structures of Giardia and Bombyx G-quadruplexes were obtained with modified sequences. The two G-quartets illustrate the possible donor-to-acceptor hydrogen-bond orientations (left: clockwise, right: anticlockwise).

Figure 2.

CD spectra at 4°C (right panels) and normalized TDS (left panels) of all the sequences listed in Table 1, in NaCl (circles) and KCl (triangles), at 3 µM oligonucleotide strand concentration. The CD axis scale is the same for all sequences (–17/+17 mdeg), with the exception of Oxy28 (–32/+32 mdeg). TDS and CD spectra did not depend on strand concentration (3, 10 and 30 µM), with the exception of Gia18, Tet22 and Scer21 CD spectra in KCl.

Figure 3.

Circular dichroism spectra at 4°C of (A) Gia18, (B) Tet22 and (C) Scer21 in KCl at 3, 10 and 30 µM oligonucleotide strand concentration (circles, crosses and triangles, respectively).

Figure 4.

(A and B) Thermal melting (cooling and heating) followed by absorbance at 295 nm of (A) Asc20 and (B) Bom17 in NaCl (circles) and KCl (triangles), at 3 µM oligonucleotide strand concentration. (C and D) CD spectra at 4°C of (C) FAsc20T and (D) FBom17T in NaCl (circles) and KCl (triangles), at 3 µM oligonucleotide strand concentration. (E and F) Thermal melting (heating) followed by FRET (excitation at 470 nm, emission at 520 nm) of (E) FAsc20T and (F) FBom17T in NaCl (circles) and KCl (triangles), at 0.2 µM oligonucleotide strand concentration.

Figure 5.

Non-denaturing PAGE in (A) NaCl and (B) KCl at 30 µM oligonucleotide strand concentration. dT₂₁ is an oligothymidylate marker; dx9 and dx12 are two double-stranded markers of 9 and 12 bp, respectively. Oligonucleotides were detected by UV-shadow.