Stacking Interactions and Flexibility of Human Telomeric Multimers

G-quadruplexes (G4s) are helical four-stranded structures forming from guanine-rich nucleic acid sequences, which are thought to play a role in cancer development and malignant transformation. Most current studies focus on G4 monomers, yet under suitable and biologically relevant conditions, G4s undergo multimerization. Here, we investigate the stacking interactions and structural features of telomeric G4 multimers by means of a novel low-resolution structural approach that combines small-angle X-ray scattering (SAXS) with extremely coarse-grained (ECG) simulations. The degree of multimerization and the strength of the stacking interaction are quantitatively determined in G4 self-assembled multimers. We show that self-assembly induces a significant polydispersity of the G4 multimers with an exponential distribution of contour lengths, consistent with a step-growth polymerization. On increasing DNA concentration, the strength of the stacking interaction between G4 monomers increases, as well as the average number of units in the aggregates. We utilized the same approach to explore the conformational flexibility of a model single-stranded long telomeric sequence. Our findings indicate that its G4 units frequently adopt a beads-on-a-string configuration. We also observe that the interaction between G4 units can be significantly affected by complexation with benchmark ligands. The proposed methodology, which identifies the determinants that govern the formation and structural flexibility of G4 multimers, may be an affordable tool aiding in the selection and design of drugs that target G4s under physiological conditions.


Size exclusion chromatography experiments
Size exclusion chromatography (SEC) experiments were performed with an AZURA Fast Protein Liquid Chromatography system (Knauer) using a Superdex 200 Increase 10/300 GL column. The column was equilibrated with K + buffer (same used for the sample preparation for CD and SAXS). Samples were eluted at a flow rate of 0.9 mL/min. Although SEC is unable to predict the molecular weights of quadruplex scaffolds (and chain-length distribution), since no quadruplex calibration standards exist, it can be used to separate quadruplex monomeric and multimeric species and to evaluate their relative content in a sample, revealing changes in equilibrium due to e.g. annealing conditions. 1 Figure S1 shows the comparison between the elution traces of G4 samples at similar DNA concentration but obtained by using different annealing protocols. It is apparent that both samples exhibit a prominent peak at an elution volume of V l ≈ 18 mL, which is likely attributable to G4 monomers. In the multimeric sample, additional peaks are present at smaller elution volumes. The peaks are well separated from each other, indicating stable multimeric species. To attain more quantitative information, we determined the percentage * The authors L.C., C. D. M., and A. P. equally contributed to the manuscript. of monomers in the multimeric sample by calculating the ratio of the area under the monomer peak to the total area of the size exclusion chromatography (SEC) trace. The calculated value (∼ 60%) is in good agreement with that obtained from simulations by using the exponential law distribution (52%).

Form factor of the Tel22 monomer
The SAXS profile of the Tel22 monomeric solution is well reproduced by a cylinder form factor. By fitting the experimental data through the SasView package (https://www.sasview.org/), we obtained for the diameter and the length values equal to D 0 =2.12 ±0.02 nm and L 0 =3.10 ±0.05 nm, respectively (see Figure S2). Figure S2. SAXS pattern of Tel22 sample (C=0.5 mM) in K + buffer solution (circle). The form factor was reproduced using a cylindrical shape by means of the SasView Software Package (blue solid line).

Multimer chain length distribution
From ECG simulations we derived the distribution of multimer chain lengths for all the investigated concentrations. The corresponding results are reported in Figure S3 along with the best fit using the normalized exponential distribution:  Figure S4. Panel a: SAXS curves of G4 samples (C=0.5 mM) in K + (blue) and Na + (red) buffer solutions. In the inset, the CD spectra of the same samples are reported. Panel b and c show the best agreement between the experimental SAXS data and the simulated intensities respectively for the K + and Na + sample.

Comparison between G4 monomers in different buffer solutions
Tel22 monomeric solutions were measured by SAXS and CD techniques in both K + and Na + buffer at the same C=0.

Angular distribution of self-assembled dimers and trimers
In order to investigate the structural flexibility of the self-assembled G4 multimers, we derived from the ECG simulations the distributions of the angles formed by adjacent hard cylinders within dimers ( Figure S8) and trimers ( Figure S9) for all the investigated concentrations.
All the distributions are centered around θ 0 ≃ 20, suggesting that multimers preferentially stack into a coaxial arrangement. This trend is found to be concentration-independent, in accordance with the similar values of T * obtained for the best-fit simulations.

Structure factor
The static structure factor S(Q) was calculated from the ECG simulations according to the procedure described in Pal et al. 2 The resulting profiles are reported in Figure S10. We notice that the value of S(Q) in the low-Q region decreases with increasing the concentration. Such a behaviour reveals how the structure factor affects in a non trivial way the low-Q signal of the measured SAXS intensities.  value decomposition was used to quantify the fraction of the major G4 topologies. 4 In Figure   S11 the fit of experimental data obtained from the deconvolution into the three main folded topologies (parallel, antiparallel and hybrid), is reported, with the corresponding results represented in the pie charts. A population transfer is evident, as the hybrid component halves at the highest concentration, while the parallel component grows from 10% to 40%.

Circular dichroism of high-concentrated samples
In addition, in order to quantify the secondary structural element steps mainly involved in the multimerization process, a decomposition of the CD spectra is reported in terms of the glycosidic angle of the base steps, associated with anti-anti, syn-anti or anti-syn conformations, in diagonal or lateral loops, or in other sub-unities (see Figure S11, panel d-f).
Based on these results, we propose that the secondary structure components associated with the syn-anti and diagonal/lateral loop segments are correlated with the average number of G4 units in the multimer M obtained from ECG simulations (see Figure S12). This suggests a potential secondary/quaternary structural connection through multimerization.     Table SI1. The intensity curves are well described by a Porod behaviour for the G4 monomer (P = 4), whereas the multimers approach the fractal behaviour of a polymer in good solvent conditions (P mul ≈ 1.5). 6 In Figure S13, the experimental data together with the corresponding fitting functions are reported. It is interesting to compare the values of R mon and R mul obtained by fitting the SAXS data with those evaluated by the simulations. Specifically, R mul was directly