How long is too long? Effects of loop size on G-quadruplex stability - PubMed (original) (raw)

Comparative Study

. 2010 Nov;38(21):7858-68.

doi: 10.1093/nar/gkq639. Epub 2010 Jul 26.

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Comparative Study

How long is too long? Effects of loop size on G-quadruplex stability

Aurore Guédin et al. Nucleic Acids Res. 2010 Nov.

Abstract

We compared here 80 different sequences containing four tracts of three guanines with loops of variable length (between 1 and 15 bases for unmodified sequences, up to 30 for fluorescently labeled oligonucleotides). All sequences were capable of forming stable quadruplexes, with T(m) above physiological temperature in most cases. Unsurprisingly, the melting temperature was systematically lower in sodium than in potassium but the difference between both ionic conditions varied between 1 and >39°C (average difference: 18.3°C). Depending on the sequence context, and especially for G4 sequences involving two very short loops, the third one may be very long without compromising the stability of the quadruplex. A strong inverse correlation between total loop length and T(m) was found in K(+): each added base leads to a 2°C drop in T(m) or ∼0.3 kcal/mol loss in ΔG°. The trend was less clear in Na(+), with a longer than expected optimal loop length (up to 5 nt). This study will therefore extend the sequence repertoire of quadruplex-prone sequences, arguing for a modification of the widely used consensus (maximal loop size of 7 bases).

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Figures

Figure 1.

Figure 1.

TDS, CD and UV melting experiments. Oligonucleotides from the var-TTT-var series are shown for illustration. Results obtained with the other series are shown as

supplementary information

(

Supplementary Figures S1–S8

). The sequences were tested at 4 µM strand concentration. Left panels in 100 mM KCl; Right panels in 100 mM NaCl. Both buffers contained 10 mM lithium cacodylate at pH 7.2. (A–B) Examples of Thermal difference spectra. Thermal difference spectra result from the difference between the absorbance recorded at 88 ± 2°C and at 04 ± 2°C. They were recorded over the 220–335 nm wavelength range. (C–D) Examples of Circular dichroism spectra. CD spectra were recorded at 10−25°C (in K+) or 4–25°C in Na+ (to maximize quadruplex formation in the case of relatively unstable quadruplexes) on a JASCO-810 spectropolarimeter using 1 cm path length quartz cuvettes. Oligonucleotides were prepared as a 4 µM and annealed by heating to 90°C for 2 min, followed by slow cooling. (E–F) Examples of UV melting profiles. Absorbance at 295 nm is plotted as a function of temperature for a selection of sequences.

Figure 2.

Figure 2.

Effect of concentration on _T_m. _T_m (in °C) is shown versus strand concentration (log scale) for a few examples. For most sequences, the effect was negligible or modest, in agreement with intramolecular folding. Notable exceptions are 121 (in sodium) or 313 (in potassium) (solid lines; filled circles and triangles, respectively).

Figure 3.

Figure 3.

Behavior of the G-quadruplex forming oligonucleotides on a non-denaturing gel. Sequences were compared by non-denaturing electrophoresis and revealed by UV-shadow with 30 µM of oligonucleotide. Samples were incubated in 10 mM Tris–HCl pH 7.5 buffer with 100 mM NaCl (A–B) or 100 mM KCl (C–D). The gel was prepared at 12% acrylamide supplemented with 20 mM of the corresponding salt and run at 23°C. Migration markers are oligothymidylate markers (dT15, dT21 or dT30) or double-stranded markers (dx9: 5′-d-GCGATACGG + 5′-d-CCGATACGC dx12: 5′-d-GCGTGACTTCGG + 5′-d-CCGAAGTCACGC). Oligonucleotide length (in nucleotide) is indicated above each band for comparison purposes.

Figure 4.

Figure 4.

Sequence effects on _T_m. _T_m (deduced from UV-melting experiments) is shown as a function of loop size (in nucleotides) both in potassium (left panels) and sodium (right panels).

Figure 5.

Figure 5.

Sodium–Potassium difference. (A) Correlation between _T_m values (deduced from UV-melting experiments) found in K+ and Na+. (B) Δ_T_m (K+ - Na+) is shown as a function of loop size (in nucleotides) for each family of sequences.

Figure 6.

Figure 6.

FRET melting experiments. _T_m (deduced from FRET melting experiments) is shown as a function of loop size (in nucleotides) both in potassium (full lines) and sodium (dotted lines) for the T-var-T and TTT-var-TTT families.

Figure 7.

Figure 7.

_T_m as a function of total loop size. _T_m (deduced from UV melting experiments) is plotted versus total (L1 + L2 + L3) loop size in K+ (A) and Na+ (B).

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