Biophysical and Structural Characterization of Non-Canonical DNA Structures

Non-canonical DNA structures known as G-quadruplexes (GQs) and i-motifs can form from G-rich and C-rich sequences within the genome, respectively, which are prominent within telomeres and oncogene promoters. These non-canonical quadruplexes likely play roles in regulating gene expression and in DNA replication and repair. However, much remains to be understood about their structural features and diversity. In this work, we investigate the interaction of a GQ-forming sequence with a water-soluble porphyrin, N-methyl mesoporphyrin IX (NMM). Biophysical studies revealed an impressively tight, thermodynamically favorable binding interaction. We then solved the GQ-NMM crystal structure at 2.39 Å, which showed that the DNA forms a dimer of parallel GQs with NMM bound at both ends via end-stacking interactions. Furthermore, we demonstrate that an unusually large, four-tetrad GQ adopts the same structure in solution as it does in crystalline form, thereby validating the crystal structure. We characterize its loop mutants to show that loop interactions fine tune GQ stability, but do not affect GQ folding. Finally, we present ongoing work towards solving the crystal structures of a different GQ-ligand complex and of a monomolecular i-motif. The work in this thesis advances our understanding of quadruplex structural diversity and ligand binding interactions by contributing to the limited number of solved GQ-ligand crystal structures, as well as by characterizing an atypically large GQ. Non-canonical quadruplexes likely function in vivo as regulatory elements. Accordingly, our improved understanding of their structural features informs the design and in silico screening of novel drugs, which can selectively recognize these features and subsequently modulate quadruplex stability for therapeutic purposes, particularly against cancer

Quadruplexes In ‘Dicty’: Crystal Structure Of A Four-Quartet G-Quadruplex Formed By G-Rich Motif Found In The Dictyostelium Discoideum Genome

Guanine-rich DNA has the potential to fold into non-canonical G-quadruplex (G4) structures. Analysis of the genome of the social amoeba Dictyostelium discoideum indicates a low number of sequences with G4-forming potential (249–1055). Therefore, D. discoideum is a perfect model organism to investigate the relationship between the presence of G4s and their biological functions. As a first step in this investigation, we crystallized the dGGGGGAGGGGTACAGGGGTACAGGGG sequence from the putative promoter region of two divergent genes in D. discoideum. According to the crystal structure, this sequence folds into a four-quartet intramolecular antiparallel G4 with two lateral and one diagonal loops. The G-quadruplex core is further stabilized by a G-C Watson–Crick base pair and a A–T–A triad and displays high thermal stability (Tm \u3e 90°C at 100 mM KCl). Biophysical characterization of the native sequence and loop mutants suggests that the DNA adopts the same structure in solution and in crystalline form, and that loop interactions are important for the G4 stability but not for its folding. Four-tetrad G4 structures are sparse. Thus, our work advances understanding of the structural diversity of G-quadruplexes and yields coordinates for in silico drug screening programs and G4 predictive tools

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

Biophysical And X-Ray Structural Studies Of The (GGGTT)3GGG G-Quadruplex In Complex With N-Methyl Mesoporphyrin IX

The G-quadruplex (GQ) is a well-studied non-canonical DNA structure formed by G-rich sequences found at telomeres and gene promoters. Biological studies suggest that GQs may play roles in regulating gene expression, DNA replication, and DNA repair. Small molecule ligands were shown to alter GQ structure and stability and thereby serve as novel therapies, particularly against cancer. In this work, we investigate the interaction of a G-rich sequence, 5’-GGGTTGGGTTGGGTTGGG-3’ (T1), with a water-soluble porphyrin, N-methyl mesoporphyrin IX (NMM) via biophysical and X-ray crystallographic studies. UV-vis and fluorescence titrations, as well as a Job plot, revealed a 1:1 binding stoichiometry with an impressively tight binding constant of 30–50 μM-1 and ΔG298 of -10.3 kcal/mol. Eight extended variants of T1 (named T2 –T9) were fully characterized and T7 was identified as a suitable candidate for crystallographic studies. We solved the crystal structures of the T1- and T7-NMM complexes at 2.39 and 2.34 Å resolution, respectively. Both complexes form a 5’-5’ dimer of parallel GQs capped by NMM at the 3’ G-quartet, supporting the 1:1 binding stoichiometry. Our work provides invaluable details about GQ-ligand binding interactions and informs the design of novel anticancer drugs that selectively recognize specific GQs and modulate their stability for therapeutic purposes

Identification And Characterization Of A B-Raf Kinase α-Helix Critical For The Activity Of MEK Kinase In MAPK Signaling

In the MAPK pathway, an oncogenic V600E mutation in B-Raf kinase causes the enzyme to be constitutively active, leading to aberrantly high phosphorylation levels of its downstream effectors, MEK and ERK kinases. The V600E mutation in B-Raf accounts for more than half of all melanomas and ∼3% of all cancers, and many drugs target the ATP binding site of the enzyme for its inhibition. Because B-Raf can develop resistance against these drugs and such drugs can induce paradoxical activation, drugs that target allosteric sites are needed. To identify other potential drug targets, we generated and kinetically characterized an active form of B-RafV600E expressed using a bacterial expression system. In doing so, we identified an α-helix on B-Raf, found at the B-Raf–MEK interface, that is critical for their interaction and the oncogenic activity of B-RafV600E. We assessed the binding between B-Raf mutants and MEK using pull downs and biolayer interferometry and assessed phosphorylation levels of MEK in vitro and in cells as well as its downstream target ERK to show that mutating certain residues on this α-helix is detrimental to binding and downstream activity. Our results suggest that this B-Raf α-helix binding site on MEK could be a site to target for drug development to treat B-RafV600E-induced melanomas

Identification And Characterization Of A B-Raf Kinase Alpha Helix Critical For The Activity Of MEK Kinase In MAPK Signaling

In the mitogen-activated protein kinase (MAPK) pathway, an oncogenic V600E mutation in B-Raf kinase causes the enzyme to be constitutively active, leading to aberrantly high phosphorylation levels of its downstream effectors, MEK and ERK kinases. The V600E mutation in B-Raf accounts for more than half of all melanomas and ~3% of all cancers and many drugs target the ATP-binding site of the enzyme for its inhibition. Since B-Raf can develop resistance against these drugs and such drugs can induce paradoxical activation, drugs that target allosteric sites are needed. To identify other potential drug targets, we used information from the available B-Raf-MEK crystal structure to generate an active form of B-RafV600E that can be expressed using a bacterial expression system. In doing so, we identified an alpha helix on B-Raf, found at the B-Raf-MEK interface, that is critical for their interaction and the oncogenic activity of B-RafV600E. We introduced mutations along this alpha helix to pinpoint regions that are important for the B-Raf-MEK interaction and tested their effects on binding and phosphorylation. We performed binding experiments between B-Raf mutants and MEK using pull downs and biolayer interferometry. We also assessed phosphorylation levels of MEK, as well as its downstream target ERK, in vitro and in cells. These studies showed that mutating certain residues on this alpha helix is detrimental to binding and downstream activity. This result suggests that this B-Raf alpha helix binding site on MEK could be a site to target for drug development to treat B-RafV600E-induced melanomas. Our cell-based data with a point mutation in B-Raf further suggests that combination therapies with ATP-competitive inhibitors would be useful to further reduce B-Raf activity and prevent the development of resistanc