Thioflavin T fluorescence and NMR spectroscopy 1 suggesting a non-G-quadruplex structure for a 2 sodium binding aptamer embedded in DNAzymes

Recently, a Na+-binding aptamer was reported to be embedded in a few RNA-cleaving DNAzymes including NaA43, Ce13d and NaH1. These DNAzymes require Na+ for activity but show no activity in the presence of K+ or other metal ions. Given that DNA can selectively bind K+ by forming a G-quadruplex structure, this work aims to answer whether this Na+ aptamer also uses a G-quadruplex to bind Na+. The Na+ aptamer embedded in Ce13d consists of multiple GG sequences, which is also a pre-requisite for the formation of G4 structures. To delineate the structural differences and similarities between Ce13d and G-quadruplex in terms of metal binding, thioflavin T (ThT) fluorescence spectroscopy, NMR spectroscopy and CD spectroscopy were used. Through comparative ThT fluorescence spectrometry studies, we deciphered that while a control G-quadruplex DNA exhibited notable fluorescence enhancement up to 5 mM K+ with a Kd of 0.52 mM, the Ce13d DNAzyme fluorescence was negligibly perturbed with similar concentrations of K+. Opposed to this, Ce13d displayed specific remarkable fluorescence decrease with low millimolar concentrations of Na+. NMR experiments at two different pH values suggest that Ce13d adopts a significantly different conformation or equilibrium of conformations in the presence of Na+ versus K+ and has a more stable structure in the presence of Na+. Additionally, absence of characteristic peaks expected for a G-quadruplex structure in 1D 1H NMR suggest that G4 is not responsible for the Na+ binding. This theory is confirmed by absence of characteristic peaks in the CD spectra of this sequence. Therefore, we concluded that the aptamer must be selective for Na+ and binds using a structural element that does not contain G4.Natural Sciences and Engineering Research Council of Canada, Discovery Grant 303454

Quadruplex DNA: sequence, topology and structure.

G-quadruplexes are higher-order DNA and RNA structures formed from G-rich sequences that are built around tetrads of hydrogen-bonded guanine bases. Potential quadruplex sequences have been identified in G-rich eukaryotic telomeres, and more recently in non-telomeric genomic DNA, e.g. in nuclease-hypersensitive promoter regions. The natural role and biological validation of these structures is starting to be explored, and there is particular interest in them as targets for therapeutic intervention. This survey focuses on the folding and structural features on quadruplexes formed from telomeric and non-telomeric DNA sequences, and examines fundamental aspects of topology and the emerging relationships with sequence. Emphasis is placed on information from the high-resolution methods of X-ray crystallography and NMR, and their scope and current limitations are discussed. Such information, together with biological insights, will be important for the discovery of drugs targeting quadruplexes from particular genes

The structures of protonated and alkali metal cationized nucleobase self-assemblies and base pairs by infrared multiphoton dissociation spectroscopy and computational methods

Nucleobases are the bricks of nucleic acids such as deoxyribonucleic acid (DNA)/ribonucleic acid (RNA) molecules. In the current research, ionic nucleobase self-assemblies and base pairs were studied using gas-phase ion techniques in either a Fourier transform ion cyclotron resonance (FTICR) or ‘paul-type’ quadrupole ion-trap (QIT) mass spectrometers. Sustained off-resonance irradiation collision-induced dissociation (SORI-CID) and infrared multiphoton dissociation (IRMPD) were used to fragment the target ions. IRMPD spectroscopy was employed to collect spectra by using tunable IR lasers, either an optical parametric oscillator or amplifier (OPO/A) or a free-electron laser (FEL). Density functional theory (DFT) was mainly used to study the structural information, calculate thermodynamic results, and perform IR frequency calculations for isomers. Besides, computed IR intensities were compared to experimental IRMPD spectra to explore their consistency. The study of uracil with Ca²⁺ clusters was presented in Chapter 3. There were agreements between the global minima IR spectra and the experimental results. Uracil tetramer, pentamer and hexamer with Ca²⁺ are composed of both tautomerized and canonical uracils, which were not proposed by previous work. Further research on discovering the structures of 1-methylcytosine dimers with alkali metal cations has revealed two possible structures; a new one which is in planar geometry containing the interbase hydrogen bonding as well as being bound by the metal cation (Chapter 4). There was a conclusion that, as alkali metal cations’ radii increased, the ion-dipole interaction weakened. In Chapter 5 the research dug into the effects of alkali metal cations and proton on guanine:cytosine (G:C) base pair. The heavier metal cations were found less likely to interrupt the hydrogen bonds between Watson-Crick G:C base pairs. Moreover, an unexpected great abundance of protonated cytosine for the dissociation of protonated G:C molecule, termed an anomaly by previous works, was explained because of the high proton transfer barrier but not the proposed thermochemistries. Guanine-involved base pair mismatches with protons were discussed in Chapters 6. For (9eG:1mT)H⁺ and (9eG:9eG)H⁺, the lowest energy structures were sufficient to explain their IRMPD spectra while the global minimum of (9eG:9eG)H⁺ presented only one classical hydrogen bond. For (9eG:9mA)H⁺ the lowest energy structures’ weighted-average spectrum was substantially consistent with its IRMPD spectrum

Structures and energetics of guanine tetrads and quadruplexes isolated in the gas phase

Structures, thermochemistries, reactivities, and kinetics of chemical systems in the gas phase can be determined by a variety of ion activation techniques. The internal energy of ions can be increased by colliding the accelerated ions with a target gas, collision-induced dissociation (CID). This technique can lead to a full or partial structural identification, and also to the relative stabilities of the ion clusters. Ions in the gas phase may reach a dissociation threshold by absorbing multiple photons from the laser radiation if the laser frequency is resonant with a vibrational mode which is called infrared multiphoton dissociation (IRMPD). This technique turns into IRMPD spectroscopy by varying the laser frequency and recording the resultant fragments as a function of laser frequency. Blackbody infrared radiative dissociation (BIRD) is another photodissociation technique used in this work that is well suited to slowly activate weakly-bound non-covalent interactions in order to determine their thermochemistries and also gain kinetic insights. Theoretical techniques have been used along with these activation techniques as a powerful complementary tool. This work provides physical chemistry insights about guanine tetrads and quadruplexes in the gas phase. The first phase of this research focuses on the alkali metal G-tetrads, M(9-eG)₄⁺ (M=Li, Na, K, Rb, Cs; 9eG = 9-ethylguanine). After their gas phase formation, their structures, stabilities, and energetics were examined by a combination of SORI-CID, IRMPD spectroscopy, and computational chemistry. The role of hydrogen bonding, the size of cation, and electrostatic interactions in the stability and structures were also examined. The formation of alkali metal G-quadruplexes, (M(9eG)₈⁺ (M = Na, K, Rb, Cs), was pursued in the next phase. The BIRD technique, in conjunction with master equation modeling, was applied to determine the binding energies and transition state nature of these G-quadruplexes. IRMPD spectroscopy, along with theoretical techniques, was used for structural elucidation. In the final step, gas phase formation of larger G-quadruplexes was assessed. The K₂(9-ethylguanine)₁₂⁺ quadruplex was successfully generated and isolated in the gas phase. BIRD and SORI-CID techniques were used to determine the quadruplex binding energies and gas phase stability, respectively. The structure of this quadruplex was determined by IRMPD spectroscopy and computational chemistry

Effect of DNA Sequence Context, DNA structure, and Excitation Method on Cyclobutane Pyrimidine Dimer Formation

The cis-syn cyclobutane pyrimdine dimer (CPD) is the major photoproduct resulting from UV irradiation of duplex DNA that results in C to T mutations found in human skin. CPDs with the anti stereochemistry were recently discovered to be formed in human telomeric DNA that adopts a quadruplex structure in vitro and may also play a role in the effects of sunlight in vivo. In this thesis, the effect of telomeric DNA structure on the formation of the anti-CPDs is investigated, as well as the effect of sequence context on cis-syn CPD formation which could help explain the origin of DNA mutation hot spots and cold spots. Originally it was hypothesized that anti CPDs formed from G-quadruplex structures, but I now show that anti CPDs can also form in human telomeric DNA sequences when complexed with lithium ions that are known to disfavor G-quadruplex formation. I also show that anti CPDs can also form in the presence of potassium ion when selected guanine bases are changed to inosine to interfere with G-quartet formation. Most significantly weI show that anti-CPDs form in sequences containing A’s in place of G’s that cannot form Hoogsteen hairpins, but can form reverse Hoogsteen hairpins. These results suggest that reverse Hoogsteen hairpins may play a hitherto unrecognized role in the biology and photoreactivity of DNA in telomeres, and possibly in other purine-rich sequences found in regulatory regions. To study sequence context effects, we designed 129-mer sequences containing all 64 possible NPyPyN tetrads (where PyPy is CC, CT, TC, or TT, and N is A, C, G or T), and used a T4 endonuclease gel electrophoresis assay to determine the relative yields of photoproduct formation at the different sites. The results show that CPD yields for different tetrads varied by as much as an order of magnitude. The yield of CPDs under UVC irradiation at a given site decrease in the order TT \u3e TC \u3e CT \u3e CC, whereas the yield of CPDs under UVB irradiation decreased in the order TT \u3e TC \u3e CC \u3e CT. The yield of CPD formation was lowest with a 5’-G and highest with a 5’-T, whereas the yield was lowest with either a 3’-C, G or T, and highest with a 3’-flanking A. We also studied the sequence context effect on photosensitized CPD formation in the presence of acetone and norfloxacin. The results not only show that the efficiency of photosensitized CPD formation depends on the flanking bases, but also indicates that the efficiency depends on the structure and properties of the photosensitizer. These results suggest that the photosensitizers may result in unique mutation spectra that can be used to identify endogenous photosensitizers such as those implicated in chemi-excitation pathways in melanoma induction

Synthetic Ion Channels From Lipophilic Guanosine Derivatives

Synthetic ion channels and pores not only represent models of natural transmembrane ion channels, but also demonstrate their potential applications in the areas of drug delivery, biosensors, antimicrobial agents and other molecular devices. In this thesis, lipophilic guanosine derivatives that combine both "molecular recognition" and "membrane soluble" features are utilized for the development of the self-assembled synthetic ion channels. The potential of lipophilic G-quadruplexes to function as synthetic ion channels has been investigated by tracing the cation exchange process between free cations and G-quadruplex bound cations. Cation exchange between bulk cations (K+, NH4+) in solution and the bound cations in G-quadruplexes (G 1)16*4Na+*4DNP- was investigated by electrospray ionization mass spectrometry and by 1H , 15N NMR spectroscopy. The ESI-MS and 1H NMR data showed that G-quadruplexes containing "mixed cations" formed through a sequential ion exchange process. The use of NMR-"visible" 15NH4+ cations in the NMR titration experiments allowed the determination of two "mixed-cation" intermediates by 15N-filtered 1H NMR and selective NOE spectroscopy. A "central insertion" pathway was proposed for the cation exchange process from (G 1)16* 4Na+* 4DNP- to (G 1)16* 4NH4+* 4DNP-. In the lipophilic G-quadruplex, the "central" Na+, bound between the 2 symmetry related G8-Na+ octamers, is bound less strongly than are the 2 "outer" Na+ ions sandwiched within the G8-octamers. These results demonstrated the dynamic nature of lipophilic G-quadruplex in solution and directed the design of a ditopic guanosine-sterol conjugate as an approach toward making synthetic ion channels. Guanosine-sterol conjugate 3-1 was prepared by coupling 2', 3'-bis-TBDMS, 5'-amino guanosine with a bis-lithocholic acid derivative. Voltage clamp experiments demonstrated a series of stable, single ion channel conductances when compound 3-1 was incorporated into a planar phospholipid membrane. These channels are large; with nanoSiemens conductance values and they last for seconds of "open" time. This feature distinguishes them from most synthetic channels, which typically conduct in the picosiemens range with millisecond lifetimes. The structural studies using the bis-lithocholamide linker demonstrated that the guanosine moiety plays an essential role in the self-assembly of the transmembrane ion channels. The sizes of the most prevalent single channels calculated by Hille's equation are much larger than the diameter of a G-quartet, which suggested that the ion transport proceeded through larger pore(s) that form upon self-assembly of lipophilic guanosine-lithocholate 3-1 within the phospholipid membrane. The large transmembrane pore(s) could be envisioned as a supramolecular structure with hydrophobic walls of bis-lithocholate linker and a central pillar of a cation-filled G-quadruplex. The use of a bis-urea functionality in the bis-lithocholic acid linker generated guanosine-sterol conjugate 4-1. The ion channel activity of 4-1 was demonstrated by voltage clamp experiment. Large ion channels formed from 4-1 had longer life-times than those formed from compound 3-1. The extra stabilization of self-assembled ion channels attributed to the bisurea hydrogen bonding is consistent with the structural hypothesis of ion channels. The stable large transmembrane ion channels self-assembled by lipophilic guanosine derivatives have potential for delivery of drugs or biomolecules

STUDIES ON THE SIZE AND NON-PLANARITY OF AROMATIC STACKING MOIETY ON CONFORMATION SELECTIVITY AND THERMAL STABILIZATION OF G-QUADRUPLEXES

Targeting DNA has the advantage over proteins for cancer remediation because of the fewer copies of the ligands required for the desired therapeutic effect. Traditionally, covalent DNA binders like alkylating agents have been used to induce genetic instability through the formation of DNA lesions and strand breaks, leading to cellular apoptosis. The primary drawback of this treatment is the non-specific binding that affects both cancerous and non-cancerous cells. G-quadruplexes are the DNA secondary structures that are present in abundance near the promoter regions of the oncogenes and are involved in the regulation of their activities. A ligand-mediated stabilization of G-quadruplexes in the promoter regions and down-regulation of the associated oncogenes have been validated. In contrast to alkylating agents, G-quadruplex ligands induce genetic stabilization through non-covalent interactions. They can be designed to interact specifically with G-quadruplex DNA over duplex DNA, which reduce side effects arising from the off-targeting. G-quadruplex ligands invariably have the large planar aromatic moiety to interact with G-quadruplexes through π- π stacking interactions. For determining the size effect of the aromatic moiety on stabilization of G-quadruplexes, a series of ligands were synthesized by conjugating nucleobases or 1,10-phenanthroline with an aminoglycoside, neomycin. The resulting conjugates increased the binding affinity synergistically and enabled us to study the effect of the stacking moiety required for G-quadruplex stabilization. Nucleobase-neomycin conjugates did not show stabilization stabilize of human telomeric G-quadruplex. 1,10-Phenanthroline-neomycin conjugate (7b) on the other hand binds to human telomeric G-quadruplex with a Ka of (8.92.4)×108 M-1 and inhibits telomerase activity at 1.56 µM probably through G-quadruplex stabilization. Moving forward, we further enlarged the aromatic moiety by tethering two 1,10-phenantholine molecules together through a five-atom linker. The resulting molecule (2-Clip-phen) was conjugated with various amino-containing side chains. 2-Clip-phen derivatives showed at least 30 times weaker binding to duplex DNA over G-quadruplex DNA. In addition, compounds showed a preference for the antiparallel G-quadruplex conformation over parallel and hybrid G-quadruplex conformations, as shown in the CD spectroscopy studies. Ligands 11 and 13 induced the formation of an antiparallel G-quadruplex from random coils and stabilize it to 60 oC (Tm) in a salt-free condition. Mass spectrometry study showed the formation of a two-tetrad G-quadruplex with the 2-Clip-phen ligand. Docking study showed that the ligand interacts most favorably with antiparallel G-quadruplex conformation, which is supported further by the larger thermal stabilization effect on antiparallel G-quadruplex compared with other G-quadruplex conformations. Our study suggests that 2-Clip-phen can be used as a scaffold for designing G-quadruplex binding ligands that preferentially bind to antiparallel G-quadruplexes, which has never been reported before

Dna Glycosylases Remove Oxidized Base Damages From G-Quadruplex Dna Structures

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

Targeting of noncanonical nucleic acid structures for therapeutic intervention and biological investigation

G-quadruplex (G4) structures fall among the most extensively studied noncanonical DNA/RNA conformations, with the current knowledge suggesting that they might play an integral part in many biological processes, including transcriptional regulation, telomeric maintenance, malignant transformation, and cancer development. In the last decade, mounting attention has been also focused on i-motif (iM) structures. Even though the structural aspects of iM formation, the factors leading to its stabilization, and its putative biological roles have been recently reviewed and discussed by the scientific community, there is still much to know about such noncanonical nucleic acid conformation. This PhD project aimed, on the one hand, at providing a more in-depth knowledge about the i-motif structures and, on the other hand, at identifying and developing new molecular tools able to target alternative nucleic acid secondary structures for either therapeutic intervention or biological investigation. Particularly, the thesis has been subdivided as follow: Chapter 1 provides a general description of nucleic acids and noncanonical DNA/RNA conformations, stressing on their structural features and biological roles. Chapter 2 offers an overview of the main methodologies employed in the herein presented studies. Chapter 3 includes two detailed investigations on iM structures. The first one is a systematic study to simultaneously analyze the effect of pH, cation type, and cation concentration (and their possible interactions) on the formation of an i-motif structure in vitro. The second one uses multivariate data analysis to bring out valuable structural information on i-motif DNA from circular dichroism and thermal difference spectra. Chapter 4 describes new G4-targeting compounds that we identified or rationally designed and synthesized as potential anti-cancer agents. In Chapter 5, new molecules are reported that might be used as tools to clarify the biological roles of G4 and iM structures and their intriguing relationship. The last section of this PhD thesis collects the general conclusions. Finally, the Appendix reports the published scientific articles as cited throughout the thesis. Paper IV is not included since the Manuscript is still in preparation and has not been submitted at the present time

Biomacromolecule-ligand interactions

The interactions and binding of various ligands to biomacromoleculcs e.g. DNA and proteins finds widespread application in the design and development of novel pharmaceuticals. DNA has been identified as the target molecule for a number of drugs and carcinogens and the supramolecular synthetic approach has led to the discovery of a range of bimetallo iron cylinders that bind to DNA inducing remarkable structural effects. The cylinders arc chiral and the enantiomers were separated on cellulose packed in paper or in a column. The optimum mobile phase for efficient separation was found to be 90% acctonitrilc: 10% 0.02 M NaCl. The (M)-enantiomers of the parent cylinder have been found to bind to DNA in the major groove. I Hydrophobic methyl groups were added at various positions on the ligand backbone. UV/visible absorbance, circular and linear dichroism were used to investigate any interactions of the metal complex with DNA with the aim of investigating any sequence preference or selectivity upon binding. Competitive binding studies and molecular dynamics simulations were used to probe the binding geometries of the enantiomers of the parent cylinder and two methylated cylinders to DNA as the exact site of interaction of the (P)-enantiomers of the parent cylinder was unclear. It was concluded that the methylated bimetallo iron cylinders bind to DNA and provide major groove recognition and may show some sequence preference. Circular dichroism was used to structurally characterise a range of buanosine-rich oligonucleotides (GRO's) and to investigate their interactions with a nucleolar protein - nucicolin. Biological/anti-proliferative activity has been related to the ability of the oligonucleotide to bind to this protein. It was found that nucleolin does bind to a biologically active GRO in the presence of K+ and induces a structural change in it