G-quadruplexes (GQs) are non-canonical DNA structures composed of stacks of stabilized G-tetrads. GQs play an important role in a variety of biological processes and may form at telomeres and oncogene promoters among other genomic locations. Here, we investigate nine variants of telomeric DNA from Tetrahymena thermophila with the repeat (TTGGGG)n. Biophysical data indicate that the sequences fold into stable four-tetrad GQs which adopt multiple conformations according to native PAGE. Excitingly, we solved the crystal structure of two variants, TET25 and TET26. The two variants differ by the presence of a 3′-T yet adopt different GQ conformations. TET25 forms a hybrid [3 + 1] GQ and exhibits a rare 5′-top snapback feature. Consequently, TET25 contains four loops: three lateral (TT, TT, and GTT) and one propeller (TT). TET26 folds into a parallel GQ with three TT propeller loops. To the best of our knowledge, TET25 and TET26 are the first reported hybrid and parallel four-tetrad unimolecular GQ structures. The results presented here expand the repertoire of available GQ structures and provide insight into the intricacy and plasticity of the 3D architecture adopted by telomeric repeats from T. thermophila and GQs in general.

The crystal structure of the G-rich human telomeric DNA Tel22 has been determined at 1.35 Å resolution in space group P6. Tel22 forms a non-canonical DNA structure called the G-quadruplex. The space group and unit-cell parameters are comparable to those in the crystal structures with PDB codes 6ip3 (1.40 Å resolution) and 1kf1 (2.15 Å resolution). The G-quadruplexes are highly similar in all of the structures. However, this structure of Tel22 displays clear density for polyethylene glycol and two potassium ions, which are located outside the ion channel in the G-quadruplex and play an important role in stabilizing the crystal contacts. In addition, 111 water molecules were identified (compared with 79 and 68 in PDB entries 6ip3 and 1kf1, respectively) that participate in intricate and extensive networks providing high stability to the G-quadruplex.

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