In Saccharomyces cerevisiae, the telomeres and rDNA repeats are repetitive silent chromatin domains that are tightly regulated to maintain silencing and genome stability. Disruption of the Cohibin complex, which maintains rDNA silencing and stability, also abrogates telomere localization and silencing. Cohibin-deficient cells have decreased Sir2 localization at telomeres, and restoring telomeric Sir2 concentrations rescues the telomeric defects observed in Cohibin-deficient cells. Genetic and molecular interactions suggest that Cohibin clusters telomeres to the nuclear envelope by binding inner nuclear membrane proteins. Futhermore, telomeric and rDNA sequences can form G-quadruplex structures. G-quadruplexes are non-canonical DNA structures that have been linked to processes affecting chromosome stability. Disruption of the G-quadruplex stabilizing protein Stm1, which also interacts with Cohibin, increases rDNA stability without affecting silent chromatin formation. In all, our findings have led to the discovery of new processes involved in the maintenance of repetitive silent chromatin domains that may be conserved across eukaryotes.

Small molecule mediated chemical intervention of biological processes using nucleic acid targets has proven extremely successful and is continually providing exciting new avenues for the development of anti-cancer agents and molecular probes. Among the alternative DNA confrormations, G-quadruplexes has certainly garnered much recognition due to increase in evidences supporting their involvement in diverse biological process. The grooves of the quadruplexes offer an alternate recognition site for ligand interactions with potentially higher selectivity than the traditional terminal stacking sites. DB832, a bifuryl-phenyl diamidine, was recently reported to selectively recognize human telomeric G-quadruplex, as a stacked species, with significant selectivity over duplex sequences. A series of biophysical studies were conducted to test the groove-binding mode of DB832, along with the selectivity for diverse quadruplex forming sequences. To gain better understanding of quadruplex groove-recognition by DB832, a series of structurally similar heterocyclic diamidines were also evaluated. The unique binding mode of DB832 may allow it to serve as a paradigm for the design of new class of highly selective quadruplex groove-binding molecules. Beyond the alternative secondary structures, it is also becoming increasingly apparent that the structure and dynamics of the canonical Watson–Crick DNA double helix play pivotal roles in diverse biological functions. DB1878, a phenyl-furan-indole diamidine, was shown to recognize a mixed GC/AT motif as a stacked antiparallel dimer, and a detailed structural analysis is reported here. Interestingly, the DNA recognition is completely different from the reported molecules in literature, and represents an entirely new motif for DNA minor groove recognition.

Oligonucleotides are short fragments of DNA (10-100nt) which are of great interest because their applications in molecular biology, biomedicine and nanotechnology. As a result of their ability to base pairing, oligonucleotides can be used as primers, hybridization probes in biosensors, agents for controlling gene expression, structural material in nanotechnology or as substrates for a variety of biochemical and biophysical studies. Chemical modification of oligonucleotides as well as conjugation to different functional molecules allows for modulation of both therapeutical and biotechnological properties. This thesis is focused in the nucleic acid chemistry field and the main objective is the synthesis of modified oligonucleotides for obtaining structures with therapeutical and/or biotechnological interest. Oligonucleotides capable to form structures other than the canonical DNA double helix have received considerable attention in the last years. The ability of triplex forming oligonucleotides (TFOs) to bind specifically to certain duplex DNA regions provides a strategy for site-directed modification of genomic DNA. Besides, G-quadruplexes are four-stranded DNA structures stabilized by stacking of guanine tetrads which have been found in telomeres and some promoters and play a role in regulation of transcription and translation. In addition, they are also interesting for nanotechnological devices. In this context, the first part of the research work was addressed to synthesize parallel stranded oligonucleotide clamps carrying LNA (locked nucleic acid) residues and study the stability of the triplex formed with DNA and RNA target sequences. Secondly, a novel strategy to obtain parallel clamps using the non-templated chemical ligation of two oligonucleotides by 5’-5’ linkages was developed. For this purpose, several protocols for introduce azido and alkyne moieties in the 5’-end of different sequences were developed so that the modified DNA strands could form a parallel hairpin after their chemical ligation by click chemistry. Thirdly, a system composed of four DNA strands whose 5’ ends are covalently attached was designed to form a monomolecular parallel G-quadruplex, which was used to study the effects of some nucleobase modifications in quadruplex structure. Finally, oligonucleotide conjugates carrying Cu(II) complexes were synthesized to construct arrays of electrochemical oscillators for nanotechnology applications.

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.

MYC is overexpressed in most types of tumors, but a means to selectively decrease its expression is yet to be found. Our recent findings on modulation of BCL2 gene expression through protein interactions with the BCL2 i-motif have provided a basis for further investigation of MYC gene control. It is proposed that the MYC i-motif could function by a similar molecular switch mechanism as in BCL2.Binding sites for heterogeneous nuclear ribonucleoprotein K (hnRNP K) within the MYC promoter also exist in the i-motif-forming sequence. Circular dichroism and bromine footprinting confirmed that this DNA sequence is able to form an i-motif, and systematic mutation of the cytosine residues in this sequence has revealed a 5:5:5 loop configuration. Indeed, all loops of the i-motif, when folded into a 5:5:5 loop configuration, contain the hnRNP K consensus sequence (CCCT). Previous studies show that hnRNP K binds to this i-motif-forming sequence, but it was assumed to be single-stranded. Binding studies revealed that hnRNP K has more binding affinity to its consensus sequence in the i-motif compared to a mutant sequence where the i-motif cannot form. Further investigation of the MYC promoter revealed an additional two runs of cytosine seven bases downstream of the MYC i-motif. Biophysical studies showed that the additional two runs were not involved in i-motif formation, however recent studies describe their importance for transcriptional activation. We found that hnRNP K preferred the longer 5CT sequence compared to the i-motif forming 4CT sequence when using a competitive binding assay. Utilizing luciferase reporters containing either the 4CT or 5CT sequence validated that hnRNP K required both the i-motif and 5th CT element for maximum transcriptional activation. Competition binding studies and bromine footprinting showed that hnRNP K bound to the downstream 5th CT element and the central and lateral loops of the i-motif.Additionally, we found that co-overexpression of Sp1 and hnRNP K induced a 10-fold increase in luciferase activity in the 5CT reporter only. We hypothesize that Sp1 continuously primes the promoter to initiate transcription inducing more negative superhelicity and increasing the melting of duplex DNA. This increased melting grants hnRNP K’s three KH domains access to the i-motif loops and the 5Th CT element. Confirmation by ChIP analysis validated that Sp1 overexpression causes an increase in hnRNP K occupancy at the MYC promoter. These findings provide new insight into the mechanisms of MYC transcriptional control by the i-motif and G-quadruplex.Recently, our group has demonstrated that two small molecules IMC-48 and IMC-76 can interact with the i-motif and can be an effective means to modulate BCL2 expression. Based on these results with the BCL2 i-motif, we employed a similar strategy and screened and identified small drug-like molecules that interact with MYC i-motif, using a FRET high-throughput assay. We then further validated that IMC-16 stabilizes the MYC i-motif through the interactions with the loops of the i-motif. No stabilization by IMC-16 treatment was observed with the MYC G-quadruplex and the BCL2 and PDGFRβi-motifs demonstrating selectivity for the MYC i-motif.Finally, we investigated the effects of IMC-16 on MYC expression in three lymphoma cell lines all expressing different levels of MYC. In the case of both Daudi and RAJI Burkitt’s lymphoma cell lines we demonstrated that selectively stabilizing the i-motif by IMC-16 could increase MYC expression. Furthermore, we demonstrated that the MYC G-quadruplex stabilizing compound GQC-05 and IMC-16, which stabilizes the MYC i-motif, have antagonistic effects on MYC expression, providing further evidence of a molecular switch mechanism in the NHEIII1. Directly targeting MYC expression through the i-motif offers advantages over targeting the G-quadruplex, because of the reduced stability and dynamic nature of the i-motif, additionally the i-motif is only found in DNA. The use of such i-motif interactive compounds is the first step into the development of new innovative approaches to treat cancers.

Dysregulation of cellular metabolism is associated with multiple diseases including cancer. Polyamines are organic cations shown to control gene expression at the transcriptional, post-transcriptional, and translational level. The activity of ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, is associated with normal and neoplastic growth. A single nucleotide polymorphism (SNP, rs2302615, SNP +316 nucleotides 3' of the transcriptional start site) in the ODC1 gene has been found to be both functional and prognostic for risk of colorectal carcinogenesis. A comprehensive investigation of genetic variability in ODC1 gene was performed. We confirmed frequencies of 12 SNPs occurring in participants of a clinical cancer prevention trial. We identified haplotypes accounting for over 90% of the genetic diversity in the ODC1 gene. Mechanistically, we addressed two of them, which account for more than half of the participants in the clinical trial. Two ODC1 intron 1 SNPs, rs2302616 (SNP +263 nucleotides 3' of the transcriptional start site) and rs2302615, were found to be associated with disease processes. Both of them predicted metachronous adenoma and response to agents targeting the polyamine pathway in participants of the clinical trial. The rs2302616 functionally modulate a DNA G-quadruplex structure and predicted the ODC1 rate-limiting product putrescine by genotype. Both SNPs cooperate to modulate ODC1 transcriptional activity involving both a G-quadruplex structure and Sp1 binding site at rs2302616, and rs2302615 flanked MYC-binding E-boxes. Haplotype analysis, using both these SNPs, might provide better discrimination of both disease prognosis and treatment prediction in cancer chemoprevention clinical trials.

Herein we describe the regulatory significance and molecular targeting of novel non-B-DNA secondary structures formed from the PDGFR-Beta core promoter nuclease hypersensitivity element.

In Saccharomyces cerevisiae, the telomeres and rDNA repeats are repetitive silent chromatin domains that are tightly regulated to maintain silencing and genome stability. Disruption of the Cohibin complex, which maintains rDNA silencing and stability, also abrogates telomere localization and silencing. Cohibin-deficient cells have decreased Sir2 localization at telomeres, and restoring telomeric Sir2 concentrations rescues the telomeric defects observed in Cohibin-deficient cells. Genetic and molecular interactions suggest that Cohibin clusters telomeres to the nuclear envelope by binding inner nuclear membrane proteins. Futhermore, telomeric and rDNA sequences can form G-quadruplex structures. G-quadruplexes are non-canonical DNA structures that have been linked to processes affecting chromosome stability. Disruption of the G-quadruplex stabilizing protein Stm1, which also interacts with Cohibin, increases rDNA stability without affecting silent chromatin formation. In all, our findings have led to the discovery of new processes involved in the maintenance of repetitive silent chromatin domains that may be conserved across eukaryotes.

This thesis consists of two major chapters, each with its own introduction, experimental section and discussion. The TG4T/daunomycin and G4/daunomycin complexes described in Chapter One are two out of only five crystallographic quadruplex/ligand structures reported to date. In both structures daunomycin molecules stack onto a terminal G quartet preventing the G4 quadruplex from destacking and unwinding. The number of interacting ligand molecules depends on the quadruplex structure itself. The G4 quadruplex can accommodate four daunomycin molecules within one layer, while the TG4T tetraplex only accommodates three. In both structures daunosamine moieties form hydrogen bonds with the quadruplex but only daunosamine moieties from the TG4T/daunomycin structure make slight incursions into the quadruplex grooves. Both structures are stabilised by π-π interactions, hydrogen bonds, Van der Waals contacts and electrostatic interactions. The daunomycin/TG4T complex is the first ever reported and the only structure where a ligand interacts directly with the quadruplex groove. Chapter Two describes nine crystal structures of Hoechst 33258 analogues with d(CGCAAATTTGCG)2 and d(CGCGAATTCGCG)2 oligonucleotides, and is divided into two sections. Section A includes seven structures with Halogenated Hoechst 33258 analogues that are potential agents in radiotherapy, phototherapy, radioimmunotherapy or photoimmunotherapy, and the structure of the precursor. In all of the examined complexes the ligand binds to the minor groove but not all halogen substituents refine to 100% occupancy. The refined occupancies of the halogen atoms reveal that the degree of carbon-halogen cleavage is highest for ortho and lowest for para substitution. Among meta substituents pointing outside the minor groove, bromine atoms had a higher occupancy than the larger iodines. The position of the halogen atom in the minor groove is influenced by additional substituents on the phenyl ring. In most cases the bulky halogen atom is facing outside of the minor groove. Only in the 3-iodo-5-isopropylHoechst complex is iodine positioned towards the floor of the groove allowing the big isopropyl group to face outside. Section B describes the structure of a carborane-containing ligand (JW-B) bound to the minor groove of d(CGCAAATTTGCG)2. The analysis shows that is possible to position boron-rich moieties close to the cell nucleus, and JW-B may have potential in Boron Neutron Capture Therapy. / Data file restricted at the request of the author, but available by individual request, use the feedback form to request access.

This thesis consists of two major chapters, each with its own introduction, experimental section and discussion. The TG4T/daunomycin and G4/daunomycin complexes described in Chapter One are two out of only five crystallographic quadruplex/ligand structures reported to date. In both structures daunomycin molecules stack onto a terminal G quartet preventing the G4 quadruplex from destacking and unwinding. The number of interacting ligand molecules depends on the quadruplex structure itself. The G4 quadruplex can accommodate four daunomycin molecules within one layer, while the TG4T tetraplex only accommodates three. In both structures daunosamine moieties form hydrogen bonds with the quadruplex but only daunosamine moieties from the TG4T/daunomycin structure make slight incursions into the quadruplex grooves. Both structures are stabilised by π-π interactions, hydrogen bonds, Van der Waals contacts and electrostatic interactions. The daunomycin/TG4T complex is the first ever reported and the only structure where a ligand interacts directly with the quadruplex groove. Chapter Two describes nine crystal structures of Hoechst 33258 analogues with d(CGCAAATTTGCG)2 and d(CGCGAATTCGCG)2 oligonucleotides, and is divided into two sections. Section A includes seven structures with Halogenated Hoechst 33258 analogues that are potential agents in radiotherapy, phototherapy, radioimmunotherapy or photoimmunotherapy, and the structure of the precursor. In all of the examined complexes the ligand binds to the minor groove but not all halogen substituents refine to 100% occupancy. The refined occupancies of the halogen atoms reveal that the degree of carbon-halogen cleavage is highest for ortho and lowest for para substitution. Among meta substituents pointing outside the minor groove, bromine atoms had a higher occupancy than the larger iodines. The position of the halogen atom in the minor groove is influenced by additional substituents on the phenyl ring. In most cases the bulky halogen atom is facing outside of the minor groove. Only in the 3-iodo-5-isopropylHoechst complex is iodine positioned towards the floor of the groove allowing the big isopropyl group to face outside. Section B describes the structure of a carborane-containing ligand (JW-B) bound to the minor groove of d(CGCAAATTTGCG)2. The analysis shows that is possible to position boron-rich moieties close to the cell nucleus, and JW-B may have potential in Boron Neutron Capture Therapy. / Data file restricted at the request of the author, but available by individual request, use the feedback form to request access.