Session: Current Topics in Biological Chemistry:

G-quadruplexes (GQs) are non-canonical DNA structures composed of stacks of stabilized guanine tetrads (G-tetrads). GQs are firmly established as biologically viable targets for anticancer and antiviral therapeutics. Understanding the 3D structures of those targets is of the utmost importance for the design of appropriate ligands. To investigate the structural diversity of GQs formed by the Tetrahymena thermophila telomeric repeat, (TTGGGG)_n, nine variants designated as TET were designed and studied. Their interactions with the highly selective GQ ligand, N-Methyl Mesoporphyrin IX (NMM) were also thoroughly investigated. Biophysical characterization concluded that most TET sequences fold into heterogenous mixtures of GQ structures. All GQs were found to adopt a hybrid conformation with the exception of TET12 (parallel), TET22, and TET24A (both predominantly antiparallel). Moreover, all TET GQs are stable above 60 °C. As expected based on our previous work, NMM converts all the sequences to a parallel conformation and increases their stability by ~7-10 °C. We were successful in obtaining diffraction quality crystals of TET25, TET26, TET26+NMM, and TET22+NMM. TET25 was solved via Molecular Replacement (MR) which revealed a hybrid GQ structure with a four G-tetrad core, three lateral loops, and a propeller loop. Two TET26 GQs were also solved via MR in space groups P3₁21 and P2₁2₁2. Both structures show a parallel conformation with a four G-tetrad core and three TT propeller loops. An initial solution of TET22+NMM shows a parallel topology, consistent with biophysical data, with NMM stacking on top of a terminal G-tetrad. Efforts to solve TET26 in a third space group and TET26+NMM are currently underway. Overall, T. thermophila telomeric variants display unusual structural diversity by forming very distinct GQ structures. Our results can guide future efforts in the synthesis and identification of GQ-targeting anticancer small-molecule ligands.

Dana Beseiso

Swarthmore College

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