Investigating G-quadruplex Formation and its Structural Perturbation by and Interactions with N-methyl mesoporphyrin IX

G-quadruplexes (GQs) are noncauonical secondary structures of DNA that can adopt a multitude of different topologies. Here, we explore the factors that drive the formation of these structures and favor the specific topologies they adopt. Our discussion of this topic begins with an investigation of the interactions of N-methyl mesoporphyrin IX (NMM), a small aromatic molecule, with Tel22, a 22-nucleotide DNA sequence that serves as a model for the putative quadruplex formed by the human telomeric repeat sequence. NMM is exceptionally selective for GQ DNA over all other types of nucleic acid secondary structure and specifically prefers the parallel GQ topology, inducing an isomerization in Tel22 from the mixed hybrid structure to the all-parallel form. Thus, we were interested in understanding the molecular basis of this specificity. Our recently obtained crystal structure of the NMM-Tel22 complex indicates that the N-methyl group plays a large role in this selectivity by distorting the porphyrin core of NMM, thereby optimizing its interactions with GQ DNA and precluding its association with double stranded B-form sequences. NMM\u27s peripheral side chains are largely unresolved in the crystal structure while the N-methyl group has well-defined electron density, suggesting either a large degree of thermal motion or the binding of 4 distinct NMM regioisomers. Here, we detail the separation and spectroscopic characterization of these isomers. Our preliminary data suggest that all four isomers are able to interact with Tel22, although there appears to be significant contamination of these samples. In the next chapter of this work, we explore the bimolecular quadruplex formation between a strand of DNA and its complement. Using a variety of different tactics, we attempted to induce GQ formation in engineered sequences endowed with what is known as duplex derived interstrand quadruplex forming potential (ddiQFP). Our efforts were mostly unsuccessful likely due to the extremely high thermal stability of the competing duplex structure and the difficulty of interpreting our spectroscopic data. In the final chapter of this work, we investigate quadruplex formation at mitochondrial DNA (mtDNA) sites associated with DNA deletions. Three of these sequences showed stable quadruplex formation under physiological conditions, indicating that GQs may be responsible for these breakpoints in vivo. In low salt conditions, the addition of lead (Pb²⁺) to two of these sequences appears to select for a small population of stable GQs and even induce GQ formation in one sequence. This suggests an additional mechanism for the toxicological effects of lead on mitochondrial structure and function. Because of the disparate nature of these topics, the conclusions and future directions will be discussed at the end of each chapter rather than together

Optimized End-Stacking Provides Specificity Of N-Methyl Mesoporphyrin IX For Human Telomeric G-Quadruplex DNA

N-Methyl mesoporphyrin IX (NMM) is exceptionally selective for G-quadruplexes (GQ) relative to duplex DNA and, as such, has found a wide range of applications in biology and chemistry. In addition, NMM is selective for parallel versus antiparallel GQ folds, as was recently demonstrated in our laboratory. Here, we present the X-ray crystal structure of a complex between NMM and human telomeric DNA dAGGG(TTAGGG)(3), Tel22, determined in two space groups, P2(1)2(1)2 and P6, at 1.65 and 2.15 angstrom resolution, respectively. The former is the highest resolution structure of the human telomeric GQ DNA reported to date. The biological unit contains a Tel22 dimer of 5\u27-5\u27 stacked parallel-stranded quadruplexes capped on both ends with NMM, supporting the spectroscopically determined 1:1 stoichiometry. NMM is capable of adjusting its macrocycle geometry to closely match that of the terminal G-tetrad required for efficient pi-pi stacking. The out-of-plane N-methyl group of NMM fits perfectly into the center of the parallel GQ core where it aligns with potassium ions. In contrast, the interaction of the N-methyl group with duplex DNA or antiparallel GQ would lead to steric clashes that prevent NMM from binding to these structures, thus explaining its unique selectivity. On the basis of the biochemical data, binding of NMM to Te122 does not rely on relatively nonspecific electrostatic interactions, which characterize most canonical GQ ligands, but rather it is hydrophobic in nature. The structural features observed in the NMM-Tel22 complex described here will serve as guidelines for developing new quadruplex ligands that have excellent affinity and precisely defined selectivity

G-Quadruplexes: A Role In The Mitochondrial Genome Stability

Single-stranded DNA or RNA regions rich in guanine (G) sequences can adopt non-canonical G-quadruplexes (G4) structures through the formation of Hoogsteen hydrogen bonds. Several studies report the existence of G4 structure formation both in vitro and in vivo, and have established their biological importance in nuclear DNA replication, transcriptional regulation and genome stability [1,2]. Genomic events, such as replication, lead to single-strand DNA formation and increase the probability of G4 formation, which could contribute to genome instability both in nuclear and mitochondrial DNA (mtDNA). The mitochondrial genome is present in thousands of copies per cell as a double-stranded circular molecule of 16 kb, encoding 13 proteins essential to oxidative phosphorylation (OXPHOS) and the RNAs necessary for their translation. Our recent in vitro study established that mtDNA has the potential to form G4 structures [3]. The same study demonstrated a tight correlation between G4 motifs and mtDNA deletion breakpoints, supporting a role for the G-quadruplexes in genome instability. To better understand the biological function of G-quadruplexes in mitochondria, we screened G4 stabilizing ligands for effects on mtDNA abundance. Here we report the activity a specific mitochondrial GQ ligand on mtDNA stability, gene expression and mitochondrial respiration