Guanine-rich DNA and RNA sequences can fold, in the presence of alkali metal ions such as Na+ and K+, into G-quadruplex structures. These alkali metal ions are necessary for the stabilization of G-quadruplex structures. However, little is known about the ion dynamics in G-quadruplex structures. In this thesis, we used molecular dynamics (MD) simulations to study the energetics of ion transport in G-quadruplex DNA channels. In particular, we applied, for the first time, adaptive biasing force (ABF) and umbrella sampling (US) methods to obtain potential of mean force (PMF) profiles for Na+, K+, and NH4+ ion movement along [d(TG4T)]4 and [d(G3T4G4)]2 channels. We found that the ABF and US methods produce very similar PMF profiles, in qualitative agreement with the very limited experimental data in the literature. We found that, within a G-quadruplex channel, K+ and NH4+ ions experience significant energy barriers (13-17 kcal/mol) to cross a G-quartet, whereas the Na+ movement encounters minimal resistance (5-7 kcal/mol). All ions are nearly fully dehydrated inside the channel but quickly become hydrated after exiting the channel. Our simulations suggested that the free energy landscapes for ion movement between the channel exit points and bulk solution are quite flat (ca. 2-4 kcal/mol) regardless of the loop topology in the region. We discovered that the directional symmetry of the ion movement within any G-quadruplex channel depends critically on both the DNA sequence and the folding of the G-quadrupelx structure. While the ion movement inside the [d(TG4T)]4 channel shows the same free energy barrier in either direction, the [d(G3T4G4)]2 channel exhibits a free energy difference of 3-4 kcal/mol for NH4+ ions exiting from the two ends. We hypothesized that the mode of base-stacking is the determining factor for the G-quartet stiffness and this stiffness then contributes to the free energy barrier for any ion to across it. This hypothesis appears to be consistent with all currently available experimental observations. When a G-quadruplex channel contains multiple ions, we found that the ion-ion repulsion is an important factor that must be considered in order to have a complete understanding of the ion movement within G-quadruplex DNA channels. / Thesis (Ph.D, Chemistry) -- Queen's University, 2013-01-25 17:38:07.489

Structure-specific recognition of nucleic acids is a promising method to reduce the size of the recognition unit required to achieve the necessary selectivity and binding affinity for small molecules. It has been demonstrated recently that G-quadruplex DNA structures can be targeted by organic cations in a structure-specific manner. Structural targets of quadruplexes include the planar end surfaces of the G-tetrad stacked columns as well as four grooves. The significant structural differences between quadruplex DNA and duplex DNA make quadruplex DNA a very attractive target for highly selective, structure-specific drug design. We have used a variety of biophysical techniques including circular dichroism, surface plasmon resonance, thermal melting and absorbance spectroscopy to investigate small molecules that can selectively bind to the ends of human telomeric DNA as well as the ends of the G-quadruplex structure formed by the purine-rich promoter region of the c-MYC oncogene. We have also screened a library of heterocyclic diamidines, and identified one that binds selectively in the grooves of human telomeric quadruplex DNA. This compound is an excellent starting point for the design of new anti-cancer and anti-parasitic compounds with high affinity and selectivity for human telomeric DNA.

In vitro, G-rich sequences form highly stable secondary structures known as G-Quadruplexes. These structures have been characterized by circular dichroism nuclear magnetic resonance and X-ray crystallography; although their detection in vivo has remained elusive. Due to the biological implication of a transisent and polymorphic secondary structure forming within the hypothetical G-Quadruplex forming regions, there is growing interest to understand their in vivo molecular dynamics. / text

Chapter 1 provides a general introduction to organofluorine chemistry and focuses on recent developments in fluorination techniques. It also details how the C–F bond influences conformational and physiochemical properties of organic molecules. Chapter 2 highlights the biological role of the telomere, telomerase and quadruplex DNA in cells. It discusses the inhibition of telomerase with small molecules that stabilise quadruplex DNA as a treatment for cancer. An overview of the development of structurally related telomerase inhibitors and recent X-ray crystallographic structural data with BSU6039 and BRACO-19 telomeric DNA is presented. Chapter 3 discusses the synthesis of fluorinated BSU6039 analogues for the investigation of the conformational effects of fluorine in 5-membered rings and its influence on binding with quadruplex DNA. These compounds have been successfully co-crystallised with telomeric DNA and their relative stabilisation of telomeric DNA has been assessed. The latter half of this chapter focuses on the co-crystal structures between (S,S)- and (R,R)-144 with Oxytricha nova telomeric DNA, discussing the key differences between the two stereoisomers. Chapter 4 details the synthesis of fluorinated BRACO-19 analogues. The syntheses of such fluorinated analogues were achieved through a base mediated coupling between 3,6-diaminoacridone and an α-fluorinated-β-amino ester. The α-fluorinated-β-amino ester was synthesised through a deoxyfluorination-mediated approach, using the stereochemistry of natural amino acids. Chapter 5 describes the stereo- and regio- selectivity of deoxyfluorination reactions with dipeptides bearing the β-amino alcohol functionality. Understanding this selectivity enabled the development of a method towards α-fluorination of tertiary amides. The application of this fluorination method with an orthogonally protected tertiary amide is described.

The G-quadruplex DNA is a four-stranded DNA structure that is highly susceptible to oxidation due to its G-rich sequence and its structure. Oxidative DNA base damages can be mutagenic or lethal to cells if they are left unrepaired. The base excision repair (BER) pathway is the predominant pathway for repair of oxidized DNA bases. DNA glycosylases are the first enzymes in BER and are responsible for removing base lesions from DNA. How DNA glycosylases remove base lesions from duplex and single-stranded DNA has been intensively studied, while how they act on G-quadruplex DNA remains to be explored. In Chapter II of this dissertation, we studied the glycosylase activity of the five mammalian DNA glycosylases (OGG1, NTH1, NEIL1, NEIL2 and mouse Neil3) on G-quadruplex DNA formed by telomere sequences that contain a single base lesion. We found that telomeric sequences that contain thymine glycol (Tg), 8-oxo-7,8-dihydroguanine (8-oxoG), guanidinohydantoin (Gh) or spiroiminodihydantoin (Sp) all formed the basket form of an antiparallel G-quadruplex DNA structure in Na+ solution. We also showed that no glycosylase was able to remove 8-oxoG from quadruplex DNA, while its further oxidation products, Sp and Gh, were good substrates for mNeil3 and NEIL1 in quadruplex DNA. In addition, mNeil3 is the only enzyme that removes Tg from quadruplex DNA and the glycosylase strongly prefers Tg in the telomere sequence context in both single-stranded and double-stranded DNA. In Chapter III, we extended our study to telomeric G-quadruplex DNA in K+ solution and we also studied quadruplex DNA formed by promoter sequences. We found that 8-oxoG, Gh and Sp reduce the thermostability and alter the folding of telomeric quadruplex DNA in a location-dependent manner. Also, the NEIL1 and NEIL3 DNA glycosylases are able to remove hydantoin lesions but none of the glycosylases, including OGG1, are able to remove 8-oxoG from telomeric quadruplex DNA in K+ solution. Interestingly, NEIL1 or NEIL3 do not efficiently remove hydantoin lesions at the site that is most prone to oxidation in quadruplex DNA. However, hydantoin lesions at the same site in quadruplex DNA are removed much more rapidly by NEIL1, NEIL2 and NEIL3, when an extra telomere TTAGGG repeat is added to the commonly studied four-repeat quadruplex DNA to make it a five-repeat telomere quadruplex DNA. We also show that APE1 cleaves furan in selected positions in Na+-coordinated telomeric quadruplex DNA structures. We use promoter sequences of the VEGF and c-MYC genes as models to study promoter G-quadruplex DNA structures, and show that the NEIL glycosylases primarily remove Gh from Na+-coordinated antiparallel quadruplex DNA but not from K+-coordinated parallel quadruplex DNA containing VEGF or c-MYC promoter sequences. Taken together, our data show that the NEIL DNA glycosylases may be involved in both telomere maintenance and gene regulation.

Small molecules interacting with DNA is an emerging theme in scientific research due to its specificity and minimal side-effect. Moreover, a large amount of research has been done on finding compounds that can stabilize G-quadruplex DNA, a non-canonical secondary DNA structure, to inhibit cancerous cell proliferation. G-quadruplex DNA is found in the guanine-rich region of the chromosome that has an important role in protecting chromosomes from unwinding, participate in gene expression, contribute in the control replication of cells and more. In this research, rationally designed, synthetic cyanine dye derivatives, which were tested under physiologically relevant conditions, were found to selectively bind to G-quadruplex over duplex DNA and are favored to one structure over another. The interactions were observed using UV-Vis thermal melting, fluorescence titration, circular dichroism titration, and surface plasmon resonance analysis. For fluorescence and selectivity properties, cyanine dyes, therefore, have the potential to become the detections and/or therapeutic drugs to target cancers and many other fatal diseases.

The thesis entitled “Benzimidazole based Novel Ligands for Specific Recognition of Duplex and G-Quadruplex DNA” deals with the design, synthesis and modeling of several benzimidazole based molecules and their interaction with duplex and G-quadruplex DNA structures. It also elucidates the inhibition effect of the ligands on the activity of Topoisomerase I and Telomerase. The work has been divided into six chapters. Chapter 1. DNA Interacting Small Organic Molecules: Target for Cancer Therapy This first chapter presents an overview on the various types of small molecules that interact with duplex and G-quadruplex structures of DNA or interfere with the activity of DNA targeted enzymes like topoisomerase and telomerase. The importance of such molecules as chemotherapeutic agents is highlighted. Chapter 2. DNA Recognition: Conformational Switching of Duplex DNA by Mg2+ ion Binding to Ligand Bis-benzimidazoles like Hoechst 33258 are well known ligands that bind to duplex DNA (ds-DNA) minor grooves. Here a series of dimeric bisbenzimidazole based ligands in which two Hoechst units are connected via oxyethylene based hydrophilic [Ho-4ox-Ho (1), Ho-3ox-Ho (2)] or via hydrophobic oligomethylene [Ho-(CH2)8-Ho (3)](Figure 1) spacers have been synthesized. The aim of this investigation is to examine the binding property of these dimers on the ds-DNA to explore whether the variation in the length of the spacer has any effect on DNA binding properties particularly in presence of selected metal ions. The changes of individual dimers in DNA binding efficiency was studied in detail by fluorescence, circular dichroism spectral titrations and thermal denaturation experiment with selected duplex DNA formed from appropriate oligonucleotides. We have also examined the changes that occur in geometry of the molecules from linear to hairpin motif in presence of Mg2+ ion. A large difference was observed in [ligand]/ [DNA] ratio and binding efficiency with ds-DNA upon change in the ligand geometry from linear to hairpin motif. The experimental results were then substantiated using docking and molecular dynamics simulations using a model ds-DNA scaffold. Both experimental and theoretical studies indicate that the DNA binding is highly dependent on the spacer type and length between the two monomeric Hoechst units. The spacer length actually helps to achieve shape complimentarity with the double-helical DNA axis. Figure1: Chemical structures of the dimeric ligands Ho-4ox-Ho, Ho-3ox-Ho, Ho-(CH2)8-Ho and Hoechst 33258 (Ho) used in this study. Chapter 3. DNA Binding and Topoisomerase I Inhibiting Properties of New Benzimidazole Substituted Polypyridyl Ruthenium (II) Mixed-Ligand Complexes In this study, we have synthesized and fully characterized three new Ru(II) based polypyridyl and benzimidazole mixed complexes: (1) [Ru(bpy)2(PMI)], 2+ (2) [Ru(bpy)2(PBI)]2+ and (3) [Ru(bpy)2(PTI)]2+ (Figure 2) . The affinities of these complexes toward duplex DNA were investigated. In addition, the photocleavage reaction of DNA and topoisomerase I inhibition properties of these metal complexes were also studied. The DNA binding efficiency of individual complexes was studied in detail by absorbance, fluorescence spectral titrations and thermal denaturation experiment using natural calf-thymus DNA. Upon irradiation at 365 nm, all three Ru(II) complexes were found to promote the cleavage of plasmid DNA from negatively supercoiled to nicked circular and subsequently to linear DNA. The inhibition of topoisomerase I mediated by these Ru(II) complexes was also examined. These experiments demonstrate that each complex serves as an efficient inhibitor toward topoisomerase I and such inhibition activity is consistent with interference with the DNA religation step catalyzed by topoisomerase. Figure 2. Chemical structures of the metal complexes used in this present study. Chapter 4. Synthesis and Evaluation of a Novel Class of G-Quadruplex-Stabilizing small molecules based on the 1,3-Phenylene-bis (piperazinyl benzimidazole) syatem Achieving stabilization of telomeric DNA in the G-quadruplex conformation by various organic compounds is an important goal for the medicinal chemists seeking to develop new anticancer agents. Several compounds are known to stabilize the G-quadruplexes. However, relatively few are known to induce their formation and/or alter the topology of the pre-formed G-quadruplex DNA. Herein, four compounds having the 1,3-phenylene-bis(piperazinyl benzimidazole) (Figure 3) unit as a basic skeleton have been synthesized, and their interactions with the 24-mer telomeric DNA sequences from Tetrahymena thermophilia d(T2G4)4 have been investigated using high-resolution techniques such as circular dichroism (CD) spectropolarimetry, CD melting, emission spectroscopy, and polyacrylamide gel electrophoresis. The data obtained, in the presence of one of three ions (Li+, Na+ or K+), indicate that all the new compounds have a high affinity for G-quadruplexDNA, and the strength of the binding with G-quadruplex depends on (i) phenyl ring substitution, (ii) the piperazinyl side chain, and (iii) the type of monovalent cation present in the buffer. Results further suggest that these compounds are able to abet the conversion of the intramolecular G-quadruplex DNA into parallel stranded intermolecular G-quadruplex DNA. Notably, these compounds are also capable of inducing and stabilizing the parallel stranded G-quadruplex DNA from randomly structured DNA in the absence of any stabilizing cation. The kinetics of the structural changes induced by these compounds could be followed by recording the changes in the CD signal as a function of time. Figure 3. Chemical structures of the ligands used in this study. Chapter 5A. The Spacer Segment in the Dimeric 1,3-phenylene-bis (piperazinyl benzimidazole) has a Dramatic Influence on the Binding and Stabilization of Human Telomeric G-Quadruplex DNA Ligand-induced stabilization of G-quadruplex structures formed by human telomeric DNA is an active area of basic and clinical research. The compounds which stabilize the G-quadruplex structures lead to suppression of telomerase activity. Herein, we present the interaction of a series of monomeric and dimeric compounds having 1,3-phenylene-bis(piperazinyl benzimidazole) (Figure 4) as basic pharmacophore unit with G-quadruplex DNA formed by human telomeric repeat d[(G3T2A)3G3]. These new compounds provide an excellent stabilization property to the pre-formed G-quadruplex DNA in the presence of one of three ions (100 mM Li+, Na+ or K+ ions). Also the G-quadruplex DNA formed in the presence of low concentrations of ligands in 100 mM K+, adopts a parallel-stranded conformation which attains an unusual thermal stability. The dimeric ligands having oxyethylene based spacer provide much higher stability to the pre-formed G-quadruplex DNA and the G-quadruplexes formed in presence of the dimeric compounds than the corresponding monomeric counterparts. Consistent with the above observation, the dimeric compounds exert significantly higher telomerase inhibition activity than the monomeric compounds. The ligand induced G-quadruplex DNA complexes were further investigated by computational molecular modeling, which provide useful information on their structure-activity relationship. Figure 4. Chemical structures of the monomeric and dimeric ligands used in this study. Chapter 5B. Role of Spacer in Symmetrical Gemini bisbenzimidazole based Ligands on the Binding and Stabilization of Dimeric G-Quadruplex DNA derived from Human Telomeric Repeats The design and development of anticancer agents that act via stabilization of the telomeric G-quadruplex DNA is an active area of research because of its importance in the negative regulation of telomerase activity. Several classes of G-quadruplex DNA binding ligands have been developed so far, but they mainly act on the DNA sequences which are capable of forming a single Gquadruplex unit. In the present work, we have developed few new dimeric (Gemini) bisbenzimidazole ligands (Figure 5), in which the spacer joining the two bisbenzimidazole units have been varied using oligooxyethylene units of different length. Herein we show the interaction of each of these ligands, with the G-quadruplex DNA, derived from oligodeoxynucleotides d(T2AG3)4 and d(T2AG3)8, which fold into a monomeric and dimeric (having two folded G-tetrad units) G-quadruplex DNA, respectively. We also present evidence that the G-quadruplex DNA structure formed by these sequences in K+ solution in presence of the ligands is parallel, with unusual stability, and the spacer length between the two bisbenzimidazole units has critical role on the G-quadruplex stability, particularly on the G-quadruplex structures formed by the 48-mer sequence. The computational aspects of the ligand-G-quadruplex DNA association have also been analyzed. Interestingly, the gemini ligand having longer spacer was highly potent in the inhibition of telomerase activity than the corresponding gemini ligands having shorter spacer or the monomeric ligand. Also, the dimeric ligands are more cytotoxic toward the cancer cells than normal cells. Figure 5. Chemical structures of the monomeric and gemini ligands used in this study. Chapter 6. Stabilization and Structural Alteration of G-Quadruplex DNA made from Human Telomeric Repeat Mediated by Novel Benzimidazole Derivatives based on Tröger’s Base Ligand-induced stabilization of G-quadruplex formation by the telomeric DNA single stranded 3'-overhang is a nice strategy to inhibit telomerase from catalyzing telomeric DNA synthesis and form capping telomeric ends. Herein we present the first report of the interactions of two novel bisbenzimidazoles (TBBz1 and TBBz2)(Figure 6) based on the Tröger’s base skeleton with the G-quadruplex DNA. These molecules stabilize the G-quadruplex DNA derived from a human telomeric sequence. Significantly strong binding affinity of these molecules to G-quadruplex DNA relative to duplex DNA was observed by CD spectroscopy, thermal denaturation and UV-vis titration studies. The above results obtained are in excellent agreement with the biological activity, measured in vitro using a modified TRAP assay. Additionally exposure of cancer cells to these compounds showed a remarkable decrease in the population growth. Also, it has been observed that the ligands are selectively more cytotoxic toward the cancerous cells than the corresponding noncancerous cells. To understand further, the ligand-G-quadruplex DNA complexes were investigated by computational molecular modeling. This provided additional insights on the structure activity relationship. Computational studies suggest that the adaptive scaffold not only allows these ligands to occupy the G-quartet but also binds with the grooves of the G-quadruplex DNA. Figure 6. Chemical structures of the ligands, TBBz1 and TBBz2 used in this study, (For structural formula pl see the abstact.pdf file.)

No description available.

Les acides nucléiques simple-Brins contenant des répétitions de guanines peuvent former des structures secondaires non canoniques dites G-Quadruplexes, composées de plusieurs couches de quartets de guanine. Malgré de nombreuses études in vivo, les preuves de présence de structures quadruplexes in vivo restent indirectes. L’objectif de ce travail était la recherche de sondes fluorescentes capables de signaler la présence d'ADN quadruplex et détecter sa structure (topologie).Deux séries de sondes fluorescentes ont été envisagées et préparées : les colorants styryles (majoritairement distyryles) et les dérivés PDC-Coumarines. La conception de ces deux séries est basée sur l’échafaudage bisquinolinium pyrido¬dicarboxamide (PDC-360A), un ligand sélectif ayant une bonne affinité vis-À-Vis des structures d’ADN quadruplexes, mais qui est non-Fluorescent. En s’inspirant de cette molécule et du motif styryle, connu pour ses propriétés spectroscopiques, nous avons préparé une librairie de colorants distyryles. Une deuxième série, les dérivés PDC-Coumarine, est synthétisée afin d’introduire la propriété fluorescente de la coumarine dans le PDC par une liaison covalente.Les propriétés de colorant de ces deux librairies (65 composés) ont été étudiées en présence de nombreuses structures d’ADN (quadruplex et duplex) en utilisant un criblage par fluorescence sur microplaques et des méthodes de titration. Nos résultats montrent que certains colorants synthétisés possèdent une haute réponse fluorimétrique (facteur d’augmentation de fluorescence de 200 à 600) vis-À-Vis de différentes structures d’ADN et d’ARN quadruplex, ayant une très faible réponse fluorimétrique vis-À-Vis de l’ADN duplex. Cela permet de marquer sélectivement l’ADN quadruplex dans la solution ou sur les gels d’électrophorèse. Ces résultats représentent une première étape vers l’utilisation de ces sondes dans un contexte biologique, par exemple dans l’imagerie de fluorescence. / Single-Stranded nucleic acids containing guanine repeats can form non-Canonical secondary structures called G-Quadruplexes. These structures are composed of several guanine quartets, maintained by hydrogen bonds and metal cations (K+ or Na+) coordinated between G-Quartets. In spite of being well-Studied in vitro, the evidence for the presence of quadruplex DNA structures in vivo remains mainly indirect. The objective of this work was research of fluorescent probes that can signal the presence of quadruplex DNA and detect its structure (topology).Two series of fluorescent probes were considered and prepared: styryls dyes (mostly distyryls) and PDC-Coumarin derivatives. The design of these two series is based on the molecular scaffold of bisquinolinium pyridodicarboxamide (PDC-360A), a selective ligand with good affinity for quadruplex DNA structures but which is not fluorescent. Inspired by this molecule and the styryl motif, which is known for its spectroscopic properties, we considered a library of distyryles dyes. A second series, the PDC-Coumarin derivatives, was developed to introduce the fluorescence property of coumarin in the PDC by a covalent link. The properties of dyes of these two libraries (65 compounds) were studied in the presence of a number of DNA structures (quadruplex and duplex) by a fluorescent screening using microplate and titration methods. Our results show that some of synthesized dyes display high fluorescence response (i.e. fluorescence increase factor from 200 to 600) for different quadruplex DNA and RNA structures, while having a very low fluorimetric response for duplex DNA. This allows a selective visualization of quadruplex DNA in solution or in electrophoresis gel. These results represent the first steps towards the use of these probes in a biological context, for example in fluorescence imaging

G-quadruplex structures are found in important regions of the eukaryotic genome, such as telomeres and regulatory sequences of genes, and are likely to play important roles in regulation of biological events. The significant structural differences with duplex DNA make quadruplex DNA a very attractive target for anticancer drug design. The purpose of this study is to explore conformational space in a series of heterocyclic cations to discover novel structural motifs that can selectively bind and stabilize specific G-quadruplex arrangements. A variety of biophysical techniques such as thermal melting experiments, biosensor surface plasmon resonance, circular dichroism, fluorescence displacement assay and mass spectrometry were employed to evaluate the affinity of the compounds and their recognition properties. The screening of the molecules allowed the identification of not only selective G-quadruplex ligands but also potential quadruplex groove binders. These results can be useful for the development of new efficient telomerase inhibitors which are endowed with pharmacological activity.