Biophysical and Structural Studies on Telomeric G-Quadruplexes from Tetrahymena thermophila

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 π − π 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

N-Methyl Mesoporphyrin IX As A Highly Selective Light-Up Probe For G-Quadruplex DNA

N-methyl mesoporphyrin IX (NMM) is a water-soluble, non-symmetric porphyrin with excellent optical properties and unparalleled selectivity for G-quadruplex (GQ) DNA. G-quadruplexes are non-canonical DNA structures formed by guanine-rich sequences. They are implicated in genomic stability, longevity, and cancer. The ability of NMM to selectively recognize GQ structures makes it a valuable scaffold for designing novel GQ binders. In this review, we survey the literature describing the GQ-binding properties of NMM as well as its wide utility in chemistry and biology. We start with the discovery of the GQ-binding properties of NMM and the development of NMM-binding aptamers. We then discuss the optical properties of NMM, focusing on the light-switch effect — high fluorescence of NMM induced upon its binding to GQ DNA. Additionally, we examine the affinity and selectivity of NMM for GQs, as well as its ability to stabilize GQ structures and favor parallel GQ conformations. Furthermore, a portion of the review is dedicated to the applications of NMM-GQ complexes as biosensors for heavy metals, small molecules (e.g. ATP and pesticides), DNA, and proteins. Finally and importantly, we discuss the utility of NMM as a probe to investigate the roles of GQs in biological processes

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

Interactions Of Ruthenium(II) Polypyridyl Complexes With Human Telomeric DNA

Eight [Ru(bpy)₂L]²⁺ and three [Ru(phen)₂L]²⁺ complexes (where bpy = 2,2′-bipyridine and phen = 1,10-phenanthroline are ancillary ligands, and L = a polypyridyl experimental ligand) were investigated for their G-quadruplex binding abilities. Fluorescence resonance energy transfer melting assays were used to screen these complexes for their ability to selectively stabilize human telomeric DNA variant, Tel22. The best G-quadruplex stabilizers were further characterized for their binding properties (binding constant and stoichiometry) using UV–vis, fluorescence spectroscopy, and mass spectrometry. The ligands\u27 ability to alter the structure of Tel22 was determined via circular dichroism and PAGE studies. We identified me₂allox as the experimental ligand capable of conferring excellent stabilizing ability and good selectivity to polypyridyl Ru(II) complexes. Replacing bpy by phen did not significantly impact interactions with Tel22, suggesting that binding involves mostly the experimental ligand. However, using a particular ancillary ligand can help fine-tune G-quadruplex-binding properties of Ru(II) complexes. Finally, the fluorescence “light switch” behavior of all Ru(II) complexes in the presence of Tel22 G-quadruplex was explored. All Ru(II) complexes displayed “light switch” properties, especially [Ru(bpy)₂(diamino)]²⁺, [Ru(bpy)₂(dppz)]²⁺, and [Ru(bpy)₂(aap)]2²⁺ Current work sheds light on how Ru(II) polypyridyl complexes interact with human telomeric DNA with possible application in cancer therapy or G-quadruplex sensing