Hänsel R , Löhr F , Foldynová-Trantírková S , Bamberg E , Trantírek L , Dötsch V .

	Figure 1. Schematic structures of intramolecular G-quadruplexes formed by four-repeat human telomeric sequences. Loops are colored magenta; anti and syn guanines are colored grey and white, respectively.
	Figure 2. CD spectra and imino regions of 1D 1H 11-echo NMR spectra for [TA-core], [A-core-TT] and [core-T] constructs in intra-oocyte buffer (A–C), and in intra-oocyte buffer supplemented with 40% PEG 200 (D–F) are shown, accompanied by schematic representations of the assumed G-quadruplex conformations. Loops are colored magenta; anti and syn guanines are colored grey and white, respectively.
	Figure 3. Overlays of CD spectra and imino regions of 1D 1H 11-echo NMR spectra for [TA-core], [A-core-TT] and [core-T] constructs in intra-oocyte buffer, and in intra-oocyte buffer supplemented with 40% Ficoll are shown, accompanied by schematic representations of the assumed G-quadruplex conformations (A–C). Loops are colored magenta; anti and syn guanines are colored grey and white, respectively.
	Figure 4. Overlays of CD spectra of 50 µM [TA-core], [A-core-TT] and [core-T] constructs in intra-oocyte buffer (A–C black lines), supplemented with either 40% PEG 200 (A–C red lines) or 40% Ficoll 70 (A–C green lines) and after the addition of 100 µM TMPyP4 and 12 h sample incubation at 4°C (D–F). Corresponding melting profiles were recorded by measuring the ellipticity at 290 nm, monitoring the stability of antiparallel G-quadruplex conformations of [TA-core], [A-core-TT] and [core-T] in the presence of TMPyP4 (G–I).
	Figure 5. CD spectra of 5 × 10−6 M [TA-core], [A-core-TT] and [core-T] constructs mixed with 40% PEG at different temperatures and incubated for various periods of time. In studies employing synthetic polymers as MC mimetics, re-annealing of DNA constructs in the presence of MC agents is typically used to accelerate the MC promoted DNA refolding. However, re-annealing of DNA constructs in X. laevis egg extracts cannot be performed as it would lead to thermal denaturation of proteins that are dominant contributing factors to the native MC environment. Here, we show that the CD spectra of telomeric constructs incubated in the presence of PEG for 2 h at 37°C are essentially the same as those obtained for the telomeric constructs re-annealed in the presence of PEG (cf. Figure 2D–F). To avoid any bias from differences in sample preparations, the telomeric repeat constructs were incubated in X. laevis egg extract and 40% PEG 200 for 2 h at 37°C prior to acquisition of NMR spectra (see Figure 6).
	Figure 6. Overlays of imino regions of 1D 1H 11-echo NMR spectra of the telomeric constructs in X. laevis egg extract (A–C blue lines) and in intra-oocyte buffer supplemented with 40% PEG 200 (A–C black lines). In the presence of PEG, several small and dispersed peaks can be observed between 11.5 and 12.4 ppm in the NMR spectra of [TA-core] and [A-core-TT] constructs (A and B black lines). These peaks can be attributed to low-level populated alternative conformation(s) co-existing in equilibrium with the dominant parallel high-order structures based on the analysis native PAGE data (Supplementary Figures S1 and S2).

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