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

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

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.)

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The thesis entitled “New DNA-Targeting Small Molecules as Potential Anticancer Agents and for in vivo Specificity toward Enhanced Silk Production” encompasses design, computational calculations, and syntheses of diverse small molecular scaffolds to explicitly target duplex and higher order DNA morphologies (G-quadruplex DNA). Some of these molecules have a potential as anticancer agents. Besides, an attempt has been made elucidate the importance of novel oligopyrrole carboxamides in the enhancement of silk yield, hence proving to a boon in the field of sericulture. The work has been divided into six chapters. Chapter 1. DNA Binding Small Molecules as Anticancer Agents Figure 1. DNA targeting by small molecules. Cancer has always been a dreadful disease and continues to attract extensive research investigations. Various targets have been identified to restrain cancer. Among these DNA happens to be the most explored one. A wide variety of small molecules, often referred to as “ligands”, has been synthesized to target numerous structural features of DNA (Figure 1). The sole purpose of such molecular design has been to interfere with the transcriptional machinery in order to drive the cancer cell toward apoptosis. The mode of action of the DNA targeting ligands focuses either on the sequence-specificity by groove binding and strand cleavage, or by identifying the morphologically distinct higher order structures like that of the G-quadruplex DNA. Chapter 2. Ligand 5, 10, 15, 20-tetra(N-methyl-4-pyridyl)porphine (TMPyP4) Prefers the Parallel Propeller-type Human G-Quadruplex DNA over its other Polymorphs The binding of ligand 5, 10, 15, 20-tetra(N-methyl-4-pyridyl)porphine (TMPyP4) with telomeric and genomic G-quadruplex DNA has been extensively studied. However, a comparative study of interactions of TMPyP4 with different conformations of human telomeric G-quadruplex DNA, namely parallel propeller-type (PP), antiparallel basket-type (AB), and mixed hybrid-type (MH) G-quadruplex DNA has not been done. We considered all the possible binding sites in each of the G-quadruplex DNA structures and docked TMPyP4 to each one of them. The resultant most potent sites for binding were analyzed from the mean binding free energy of the complexes. Molecular dynamics simulations were then carried out and analysis of the binding free energy of the TMPyP4-G-quadruplex complex showed that the binding of TMPyP4 with parallel propeller-type G-quadruplex DNA is preferred over the other two G-quadruplex DNA conformations. The results obtained from the change in solvent excluded surface area (SESA) and solvent accessible surface area (SASA) also support the more pronounced binding of the ligand with the parallel propeller-type G-quadruplex DNA (Figure 2). Figure 2. Ligand TMPyP4 prefers parallel propeller-type G-quadruplex DNA morphology. Chapter 3. A Theoretical Analysis on the Selective Stabilization of Intermolecular G-quadruplex RNA with a bis-Benzimidazole Ligand EtBzEt over TMPyP4 in K+ Environment Ever since the discovery of G-quadruplex RNA, a constant urge exists to target these higher order RNA conformations. These structures play a significant role in the transcriptional and translational mechanism. Herein we have determined the mode and extent of association of certain G-quadruplex DNA binding bisbenzimidazole ligand (EtBzEt) in comparison to a known porphyrin ligand (TMPyP4). We have performed docking studies of the known G-quadruplex DNA binding ligands with the parallel propeller G-quadruplex RNA (PPR) to determine the most potent binding conformation which showed EtBzEt to be a better RNA binder than others. Furthermore, a molecular dynamics (MD) simulation (6 ns) was performed for the most stable docked complex in explicit solvent environment. The role of K+ ions, Hoogsteen hydrogen bond formation and backbone dihedral angle between the tetrads were carefully analyzed during the entire simulation run to determine the stability of each ligand associated PPR complex. All the analyses conclusively showed that while TMPyP4 merely stabilized the PPR, the ligand EtBzEt stabilized PPR very efficiently (Figure 3). Figure 3. Stabilzation and destabilization by EtBzEt and TMPyP4, repectively. Red and green ovals represent EtBzEt and TMPyP4, repectively. Chapter 4A. Design and Synthesis of New DNA Binding Fe(III) and Co(II) Salen Complexes with Pendant Oligopyrrole Carboxamides Extensive research on these oligopyrrole carboxamides has shown their specificity toward AT-rich sequences with high binding affinity. Here we have designed and synthesized Fe (III)-and Co (II)-based salen complexes attached with minor groove targeting oligopyrrole carboxamide side-chains (Figure 4). While the ligands showed excellent activity toward DNA damage, they also exhibited high affinity toward the minor grooves of the ds-DNA. This was also reflected in the high efficiency of the ligands toward cancer cell cytotoxicity. Further studies revealed that the ligands resulted in prominent nuclear condensation and fragmentation thereby driving the cells toward apoptosis. The presence of metal coordinated salen moiety conjugated with positively charged pendants ending with minor groove binding oligopyrrole carboxamides might have resulted in the increased activity of the ligands toward DNA targeting and cancer cell death. Figure 4. Chemical structures of the ligands used in this study. Chapter 4B. Design and synthesis of novel oligopyrrole based salen metal complexes and their efficiency toward stabilization of G-quadruplex DNA DNA targeting has been the key strategy toward the restriction of cancer cell proliferation. In a similar effort, we have designed and synthesized novel salen based Ni(II) and Pd(II) metal complexes with positively charged flanking side-chains comprising attached N-methylpyrrole carboxamides of varying lengths (Figure 5). The ligands showed efficient stabilization of the G-quadruplex DNA morphologies, with specificity over the duplex DNA. Sufficient inhibition of the telomerase activity was observed by the TRAP-LIG assay which was ascertained by the prominent restriction of cancer cell proliferation in the long-term cell viability assay. The ligands exhibited condensation and fragmentation of the nucleus when observed under confocal microscopy which is indicative of the cells undergoing apoptosis. Further annexin V-FITC and PI dual staining showed apoptosis to be the mechanistic pathway underlying the cancer cell cytotoxicity by the ligands. Modeling studies clearly showed the stacking of the salen moiety over the G-tetrads with the association of the pendant oligopyrrole carboxamide units to the grooves. Figure 5. Chemical structures of the ligands used in this study. Chapter 5A. Role of Metal Ions in Novel Fluorescein based Salen and Salphen Complexes toward Efficient DNA Damage and their Effect on Cancer Cells Metal ions play an important role toward DNA damage and numerous ligands have been synthesized for their use in anticancer therapy. Herein, we have designed and synthesized Fe(III) and Co(II) based salen/salphens by bridging two fluorescein moieties with varying spacers (Figure 6). Although the ligands exhibit dual binding mode, the more flexible salen ligands prefer to associate to the minor groove of the DNA while the relatively rigid salphen ligands show greater intercalation. The biophysical experiments reveal better binding affinity of the salphens toward duplex DNA as compared to the salen ligands. The metal coordination resulted in efficient DNA cleavage of plasmid at low ligand concentrations. The ligands also showed cancer cell cytotoxicity, cellular internalization with apoptosis as the proposed mechanism for cell death. Figure 6. Chemical structures of the salen and salphen ligands used in this study. Chapter 5B. Fluorescein based Salen and salphen Complexes as stabilizers of the Human G-quadruplex DNA and Promising Telomerase Inhibitors Metal based salen complexes have been considered as an important scaffold toward targeting of DNA structures. In the present work we have designed and synthesized nickel(II)-and palladium(II)-salen and salphen ligands by using fluorescein as the backbone to provide an extended aromatic surface (Figure 7). The ligands exhibit sufficient affinity toward the human telomeric G-quadruplex (G4) DNA in preference to the duplex DNA and also exhibit promising inhibition of telomerase activity. This is ascertained by their potency in the long-term cell viability assay which shows significant cancer cell cytotoxicity in presence of the ligands. Confocal microscopy showed cellular internalization followed by nuclear localization. Considerable population at the sub-G1 phase of the cell cycle showed cell death via apoptotic pathway. Figure 7. Chemical structures of the ligands used in this study. Chapter 6. Knockdown of Broad-Complex Gene Expression of Bombyx mori by Oligopyrrole carboxamides Enhances Silk Production Bombyx mori (B. mori) is important due to its major role in the silk production. Though DNA binding ligands often influence gene expression, no attempt has been made to exploit their use in sericulture. The telomeric heterochromatin of B. mori is enriched with 5′-TTAGG-3′ sequences. These sequences were also found to be present in several genes in the euchromatic regions. We examined three synthetic oligopyrrole carboxamides that target 5′-TTAGG-3′ sequences in controlling the gene expression in B. mori (Figure 8). The ligands did not show any defect or feeding difference in the larval stage, crucial for silk production. The compounds caused silencing of various isoforms of the broad-complex transcription factor and cuticle proteins which resulted in late pupal developmental defects. This study shows for the first time use of oligopyrrole carboxamide drugs in controlling gene expression in B. mori and their long term use in enhancing silk production. Figure 8. Chemical structures of the ligands used in this study (top) and increased cocoon size on ligand treatment.

Exposure of DNA to reactive oxygen species (ROS) results in the modified nucleobases (lesions) as well as strand scissions under physiological conditions. Due to its lowest oxidation potential (1.29 eV), guanine is the most easily oxidisable nucleobase. Furthermore, it has been observed that the 5'-guanine in G-tracts (e.g. GGG) has even lower oxidation potential (1.00 V vs. NHE). One of the representative G-rich examples is telomeres that consist of repeating units of 5'-d [TTAGGG]-3' found at the ends of chromosomes. Telomeres play an important role in biological functions, serving as guardians of genome stability; however, their G-rich nature implies that they can be readily oxidized. So how does nature protect these biologically important regions from oxidation? We believe the formation of a secondary structure known as G-Quadruplex in telomeric regions can partly serve as a protective role. In the first part of this work, we investigated DNA G-Quadruplex damage under various oxidation conditions and compare the damage results with single-stranded telomeric sequences. Damage to G-Quadruplex is generally less than single strands and is condition dependent. Guanines are the primary damage sites, but damage of adenine and thymine is also possible. Based on our studies, telomeric DNA can be readily oxidized to produce DNA lesions. How do DNA lesions affect the conformation and the stability of telomeric G-Quadruplex DNA? In the second part, we sought to address this question using various biophysical methods. Several native (OxodG, OxodA, and abasic site) and non-native (8-NH 2 -dA and 8-Br-dA) lesions were tested. UV thermal denaturation and circular dichroism revealed that the conformation and the stability of G-Quadruplex DNA are dependent on the location and the type of lesion in the sequence. G-Quadruplex DNA containing OxodG maintains its conformation with a decreased stability. Abasic site in the TTA loop affects the conformation of G-Quadruplex DNA but shows little effect on its stability. An unexpected stabilization of telomeric G-Quadruplex DNA was observed when deoxyadenosine (dA) in the loops was replaced with its native oxidized form OxodA. This is the first example of native DNA lesion that increases the stability of G-Quadruplex DNA. Like OxodA lesion, 8-NH 2 -dA (a non native DNA lesion) increases the stability of G-Quadruplex DNA while 8-Br-dA only affects the stability in KCl but has no significant effect in NaCl. In addition, studies of the effect of OxodA lesion on the human telomerase activity using TRAP assay will be discussed.