Abstract:
G-quadruplexes (GQs) are non-canonical DNA structures composed of stacks of
stabilized guanine tetrads (G-tetrads). GQs are highly diverse structures that can be
categorized by their strand directionality, number of G-tetrads, and loop types among
other parameters. Due to their high guanine content, GQs are expected to fold in regions
such as telomers or oncogene promoters. They are thus firmly established as biologically
viable targets for the development of anticancer therapeutics. In order to harvest GQs’
therapeutic potential, extensive structural studies are required to elucidate the structures’
diverse topologies alone and in complex with selective ligands.
Towards this goal, this thesis studies nine variants of the telomeric repeat
(TTGGGG)n from Tetrahymena thermophila (designated as TET) alone and in complex
with the highly selective GQ ligand, N-Methyl Mesoporphyrin IX (NMM). Our
biophysical characterization shows that almost all TET GQs are highly heterogenous and
can form multiple conformations. The addition of NMM, however, converts all the
sequences to a parallel conformation and increases their thermal stability. To gain further
insight into the molecular structures of the variants, we sought out to solve the crystal
structures of TET26-2, TET22-NMM, TET24A-NMM, and TET25-NMM.
TET26-2, solved to 1.97 Å, shows a parallel GQ with a four G-tetrad core and
three TT propeller loops. Preliminary solutions of TET22-NMM, TET24A-NMM, and
TET25-NMM complexes show parallel GQs with NMM ! − ! stacking on top of the 3’-
terminal G-tetrad. Significantly, the first example of an NMM-NMM dimer was observed
in these preliminary solutions which contributes to the understanding of GQ folding and
the structures’ interactions with small molecule ligands.
Overall, T. thermophila telomeric variants display unusual structural diversity by
forming very distinct GQ structures. Our results provide insight into the many GQ
topologies available to telomeric repeats and how they interact with the selective ligand
NMM, which is important for the design of optimized GQ-selective anticancer
therapeutics.
