Homopurine guanine-rich sequences in complex with N-methyl mesoporphyrin IX form parallel G-quadruplex dimers and display a unique symmetry tetrad.

DNA can fold into G-quadruplexes (GQs), non-canonical secondary structures formed by π-π stacking of G-tetrads. GQs are important in many biological processes, which makes them promising therapeutic targets. We identified a 42-nucleotide long, purine-only G-rich sequence from human genome, which contains eight G-stretches connected by A and AAAA loops. We divided this sequence into five unique segments, four guanine stretches each, named GA1-5. In order to investigate the role of adenines in GQ structure formation, we performed biophysical and X-ray crystallographic studies of GA1-5 and their complexes with a highly selective GQ ligand, N-methyl mesoporphyrin IX (NMM). Our data indicate that all variants form parallel GQs whose stability depends on the number of flexible AAAA loops. GA1-3 bind NMM with 1:1 stoichiometry. The Ka for GA1 and GA3 is modest, ∼0.3 µM -1, and that for GA2 is significantly higher, ∼1.2 µM -1. NMM stabilizes GA1-3 by 14.6, 13.1, and 7.0 °C, respectively, at 2 equivalents. We determined X-ray crystal structures of GA1-NMM (1.98 Å resolution) and GA3-NMM (2.01 Å). The structures confirm the parallel topology of GQs with all adenines forming loops and display NMM binding at the 3' G-tetrad. Both complexes dimerize through the 5' interface. We observe two novel structural features: 1) a 'symmetry tetrad' at the dimer interface, which is formed by two guanines from each GQ monomer and 2) a NMM dimer in GA1-NMM. Our structural work confirms great flexibility of adenines as structural elements in GQ formation and contributes greatly to our understanding of the structural diversity of GQs and their modes of interaction with small molecule ligands.

The first crystal structures of hybrid and parallel four-tetrad intramolecular G-quadruplexes.

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.