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. 2021 Apr 26;22(9):4532.
doi: 10.3390/ijms22094532.

Supramolecular Polymorphism of (G4C2)n Repeats Associated with ALS and FTD

Melani Potrč  1   2 , Nerea Sebastián  2 , Miha Škarabot  3 , Irena Drevenšek-Olenik  2   4 , Lea Spindler  2   5
Affiliations

Supramolecular Polymorphism of (G4C2)n Repeats Associated with ALS and FTD

Melani Potrč et al. Int J Mol Sci. .

Abstract

Guanine-rich DNA sequences self-assemble into highly stable fourfold structures known as DNA-quadruplexes (or G-quadruplexes). G-quadruplexes have furthermore the tendency to associate into one-dimensional supramolecular aggregates termed G-wires. We studied the formation of G-wires in solutions of the sequences d(G4C2)n with n = 1, 2, and 4. The d(G4C2)n repeats, which are associated with some fatal neurological disorders, especially amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), represent a challenging research topic due to their extensive structural polymorphism. We used dynamic light scattering (DLS) to measure translational diffusion coefficients and consequently resolve the length of the larger aggregates formed in solution. We found that all three sequences assemble into longer structures than previously reported. The d(G4C2) formed extremely long G-wires with lengths beyond 80 nm. The d(G4C2)2 formed a relatively short stacked dimeric quadruplex, while d(G4C2)4 formed multimers corresponding to seven stacked intramolecular quadruplexes. Profound differences between the multimerization properties of the investigated sequences were also confirmed by the AFM imaging of surface films. We propose that π-π stacking of the basic G-quadruplex units plays a vital role in the multimerization mechanism, which might be relevant for transformation from the regular medium-length to disease-related long d(G4C2)n repeats.

Keywords: AFM; DNA-quadruplex; G-wires; d(GGGGCC) repeats; dynamic light scattering; self-assembly.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
d(G4C2)n sequences form quadruplex structures in which four guanines (gray squares) associate via Hoogsteen-type hydrogen bonding. (a) The d(G4C2) sequence forms a tetrameric symmetric quadruplex with 3′-CC overhangs (blue squares) [4]. (b) The d(G4C2)2 sequence shows a high polymorphism with the exact structures still being unknown [5]. Two possible dimeric folds are depicted. (c) The d(G4C2)4 sequence is believed to form a unimolecular antiparallel quadruplex with edgewise loops [11,12,13].
Figure 2
Figure 2
DLS measurements: (a) Autocorrelation curves taken at ϑ = 90° clearly reveal very slow dynamics of the fast mode in solution of the d(G4C2) sequence. (b) The measured dependencies of the inverse relaxation time of the fast mode 1/τf versus q2 were used to calculate the translational diffusion coefficients D (Equation (2)). Quite notably, there is no evident correlation between the obtained values of D and the number of G4C2 repeats in the investigated sequence, which indicates that these sequences exhibit very diverse self-assembly patterns.
Figure 3
Figure 3
Time dependences of the diffusion coefficient after thermal denaturation (performed at t = 0 h). The highest values of D observed in solution of d(G4C2)2 indicate the presence of small structures, most probably two stacked bimolecular quadruplexes. The intermediate values of D observed in solution of d(G4C2)4 are probably associated with higher-order quadruplexes. The lowest values of D observed in solution of d(G4C2) signify a formation of extremely long G-wires created by stacking of individual parallel quadruplex units.
Figure 4
Figure 4
AFM images of small globular islands formed in drop-cast surface films of (a) d(G4C2)2 and (b) d(G4C2)4. The height distribution of the structures is given in the right panels and shows an average height of around 2.4 nm. The lines are guides to the eye. The scale bars in the AFM images correspond to 500 nm.
Figure 5
Figure 5
(a) AFM image of a drop cast film of d(G4C2). The scale bar corresponds to 500 nm. (b) The height profiles along three single G-wires (see colored lines in (a)). (c) The length and (d) the height distribution of G-wires. The lines are guides to the eye. Length analysis was limited to objects above 1 nm and elongated in shape.
Figure 6
Figure 6
The model for the largest diffusing structures in the solutions [38] as deduced from the DLS data. The quadruplexes from d(G4C2), d(G4C2)2, and d(G4C2)4 show very different stacking ability with d(G4C2) forming the longest (a), d(G4C2)2 the shortest (b) and d(G4C2)4 intermediate sized G-wires (c). All aggregates determined by DLS are longer than previously reported with other methods.
See this image and copyright information in PMC

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