Extended Data Figure 1. Spectroscopic characterization of DNA/RNA structural polymorphism.

a) The decanucleotide GGGGCCGGGG from the C9orf72 HRE adopts a parallel G-quadruplex conformation indicated by the CD spectra in the presence of KCl (left). The proposed intermolecular parallel G-quadruplex topology (right) of two decanucleotide sequences that form three stacks of G-quartets 5´ to 3´ with three or four nucleotides, GCCG, forming a loop region. b) The complementary sequence strand for the C9orf72 HRE, (CCCCGG)₄, shows no apparent structural differences ± KCl. c) The variability in CD spectra increases with the number of repeats (dotted lines), but the CD spectra can be recapitulated (lines) by fitting a linear combination of three component spectra for hairpin, antiparallel Gquadruplex, and parallel G-quadruplexes from the spectra corresponding to 0 mM KCl (GGGGCC)₄, 100 mM KCl (GGGGCC)₄, and 100 mM KCl GGGGCCGGGG, respectively. The residuals of the fit are plotted below. d) CD spectral analysis of the thermal stability of the DNA antiparallel G-quadruplex. CD spectra were obtained as described in Methods with temperatures ranging from 25–95°C in 10 mM Tris-HCl pH 7.4 with 50 mM KCl. This two-state transition was evaluated by singular value decomposition (SVD) of the CD melting spectra (Matlab). The two basis component spectra, shown as left coefficients, represent an antiparallel G-quadruplex and an unfolded single-stranded DNA oligo. The residuals show the difference between the original CD spectrum and the basis component spectra fit. e) Increasing KCl concentrations increase the abundance and stability of antiparallel DNA G-quadruplexes, as shown by UV-VIS melting curves. CD absorbance was normalized to the equation (Abs_t – min)*(max – min)⁻¹, where Abs_t is the absorbance at a given temperature, max is the observed maximum absorbance at 295 nm at that [KCl], and min is the minimum value obtained for all [KCl]s. Data was fit to a Boltzmann distribution (Prism) with a lower boundary constraint set to min. f) The decamer HRE sequence forms a parallel G-quadruplex that is less stable than the antiparallel conformation. The data obtained were normalized as described above, except the CD absorptivity was measured at 260 nm. g) CD melting spectrum shows a two-state transition from a folded parallel RNA G-quadruplex state to an unfolded linear state. The CD absorptivity data measured at 260 nm were normalized as described above and fit to a Boltzmann distribution without constraints. h) The (GGGGCC)₄ forms a G-quadruplex that is unperturbed by a 3ʹ biotin label. CD spectra were obtained ± KCl as in Figure 1 to identify the stabilization of K⁺ in the formation of a G-quadruplex that had been chemically modified with the biotin tag. All calculated melting temperatures are provide in Extended Data Table 1 and results detailed further in Supplementary Results.