Polymorphism of human telomeric quadruplex structure controlled by DNA concentration: a Raman study

Abstract

DNA concentration has been recently suggested to be the reason why different arrangements are revealed for K(+)-stabilized human telomere quadruplexes by experimental methods requiring DNA concentrations differing by orders of magnitude. As Raman spectroscopy can be applied to DNA samples ranging from those accessible by absorption and CD spectroscopies up to extremely concentrated solutions, gels and even crystals; it has been used here to clarify polymorphism of a core human telomeric sequence G(3)(TTAG(3))(3) in the presence of K(+) and Na(+) ions throughout wide range of DNA concentrations. We demonstrate that the K(+)-structure of G(3)(TTAG(3))(3) at low DNA concentration is close to the antiparallel fold of Na(+)-stabilized quadruplex. On the increase of G(3)(TTAG(3))(3) concentration, a gradual transition from antiparallel to intramolecular parallel arrangement was observed, but only for thermodynamically equilibrated K(+)-stabilized samples. The transition is synergically supported by increased K(+) concentration. However, even for extremely high G(3)(TTAG(3))(3) and K(+) concentrations, an intramolecular antiparallel quadruplex is spontaneously formed from desalted non-quadruplex single-strand after addition of K(+) ions. Thermal destabilization or long dwell time are necessary to induce interquadruplex transition. On the contrary, Na(+)-stabilized G(3)(TTAG(3))(3) retains its antiparallel folding regardless of the extremely high DNA and/or Na(+) concentrations, thermal destabilization or annealing.

Figure 1.

Raman spectra of G₃(TTAG₃)₃ in 200 mM K⁺ (30 mM of PBS, pH 6.8, t = 5°C) at the nucleoside concentrations of 8 mM (bottom trace) and 200 mM (top trace). Intermediate traces show the differences between the spectra at indicated concentration and that of the lowest one.

Figure 2.

Dependence of CD spectra of G₃(TTAG₃)₃ on DNA concentration at 23°C. Each sample was slowly annealed after dilution to the final DNA concentration. Concentration of K⁺ in all samples was 300 mM. Bottom: native PAGE of K⁺-stabilized G₃(TTAG₃)₃ at indicated DNA concentrations. The PAGE was performed at 20°C in 30 mM of PBS containing 240 mM of K⁺, pH 6.7.

Figure 3.

Dependence of normalized Raman spectra of G₃(TTAG₃)₃ on DNA concentration in the presence of 250 mM of K⁺ or Na⁺ (150 mM of PBS, pH 6.8, 5°C). Spectral differences between K⁺- and Na⁺-stabilized quadruplex structures and changes induced by increasing DNA concentration are expressed as abstract orthonormal factors obtained by SVD analysis (44). For better insight to real spectral differences and their extent, refer to Figure 4 and Supplementary Figure S3. Right top corner: scheme of a G-tetrad.

Figure 4.

Detail comparison of the spectral differences between K⁺- and Na⁺-stabilized G₃(TTAG₃)₃ quadruplexes at low (8 mM, top) and high (240 mM, bottom) DNA concentration. Normalized Raman spectra correspond to lower and upper concentration limits of the series analysed by SVD in Figure 3.

Figure 5.

Raman spectra of 50 mM G₃(TTAG₃)₃ in the presence of 100 and 500 mM of Na⁺ (top) or K⁺ (bottom) at 10°C. Difference spectra between the high- and low-salt structures are shown to highlight cation-specific spectral variance. Differential Raman features are labelled according to their apparent maxima/minima that can differ from the maxima/minima of the respective Raman bands.

Figure 6.

Top: CD spectra of 50 mM G₃(TTAG₃)₃ in the presence of 100 and 500 mM of Na⁺ or K⁺. Positive bands at ∼245 and ∼293 nm and negative band at ∼265 nm affirm antiparallel foldback of the Na⁺-stabilized G₃(TTAG₃)₃, regardless of the Na⁺ concentration. Even though without negative band at ∼265 nm, the CD spectrum of 100 mM K⁺-stabilized G₃(TTAG₃)₃ was recently proven to be indicative for essentially the same antiparallel topology as adopted by Na⁺-stabilized quadruplex (30), contradicting the previous interpretation attributing CD spectra of this shape to hybrid (3 + 1) form (18,19,22). Transition to the parallel structure via hybrid (3 + 1) forms is accompanied by appearance of a strong positive band at ∼263 nm, decrease of the positive band at ∼290 nm and the presence of negative band at ∼240 nm (30), the CD features visible in the presence of 500 mM K⁺. Bottom: native PAGE of 50 mM G₃(TTAG₃)₃ in 100–500 mM of K⁺ before heating (N) and after slow annealing (A). The PAGE was performed at 20°C in 30 mM PBS containing 300 mM K⁺ in total, pH 6.7.

Figure 7.

Spectral differences between non-quadruplex G₃(TTAG₃)₃ structure at extremely high DNA concentration (520 mM; no alkali cations present) and initial quadruplex structures adopted by highly concentrated G₃(TTAG₃)₃ (330 mM) after adjustment of K⁺ or Na⁺ concentrations to 230 mM, but before annealing.

Figure 8.

Effect of the annealing mode and K⁺ concentration on G₃(TTAG₃)₃ quadruplex structure at extremely high DNA concentration. Raman spectra of 330 and 315 mM G₃(TTAG₃)₃ in the presence 230 and 500 mM of K⁺, respectively, before thermal denaturation (No) and after fast (Fast) and slow (Slow) annealing. Differential spectra between thermally destabilized, thermodynamically equilibrated and initially adopted K⁺-stabilized G₃(TTAG₃)₃ structures are shown to depict differences.

Figure 9.

Heating and cooling Raman profiles of selected Raman bands sensitive (581, 611 and 1485 cm⁻¹) and insensitive (837, 1581 and 1719 cm⁻¹) to antiparallel-to-parallel switching of the K⁺-stabilized G₃(TTAG₃)₃ quadruplex. Initially, antiparallel K⁺-quadruplex (330 mM), spontaneously formed on addition of 230 mM K⁺, was heated from 10°C to 95°C and then cooled down at the same rate of ∼1.5°C/min.