Molecular models for intrastrand DNA G-quadruplexes

<p>Abstract</p> <p>Background</p> <p>Independent surveys of human gene promoter regions have demonstrated an overrepresentation of G₃X_<it>n</it>1G3X_<it>n</it>2G₃X_<it>n</it>3G₃motifs which are known to be capable of forming intrastrand quadruple helix structures. In spite of the widely recognized importance of G-quadruplex structures in gene regulation and growing interest around this unusual DNA structure, there are at present only few such structures available in the Nucleic Acid Database. In the present work we generate by molecular modeling feasible G-quadruplex structures which may be useful for interpretation of experimental data.</p> <p>Results</p> <p>We have used all quadruplex DNA structures deposited in the Nucleic Acid Database in order to select a list of fragments entailing a strand of three adjacent G's paired with another strand of three adjacent G's separated by a loop of one to four residues. These fragments were further clustered and representative fragments were finally selected. Further fragments were generated by assemblying the two strands of each fragment with loops from different fragments whenever the anchor G's were superimposable. The fragments were used to assemble G quadruplex based on a superimposability criterion.</p> <p>Conclusion</p> <p>Molecular models have been generated for a large number of G₃X_<it>n</it>1G₃X_<it>n</it>2G3X_<it>n</it>3G₃sequences. For a given sequence not all topologies are possible with the available repertoire of fragments due to steric hindrance and low superimposability. Since all molecular models are generated by fragments coming from observed quadruplex structures, molecular models are in principle reliable and may be used for interpretation of experimental data. Some examples of applications are given.</p

Biophysical and Structural Studies on Telomeric G-Quadruplexes from Tetrahymena thermophila

G-quadruplexes (GQs) are non-canonical DNA structures composed of stacks of stabilized guanine tetrads (G-tetrads). GQs are highly diverse structures that can be categorized by their strand directionality, number of G-tetrads, and loop types among other parameters. Due to their high guanine content, GQs are expected to fold in regions such as telomers or oncogene promoters. They are thus firmly established as biologically viable targets for the development of anticancer therapeutics. In order to harvest GQs’ therapeutic potential, extensive structural studies are required to elucidate the structures’ diverse topologies alone and in complex with selective ligands. Towards this goal, this thesis studies nine variants of the telomeric repeat (TTGGGG)ₙ from Tetrahymena thermophila (designated as TET) alone and in complex with the highly selective GQ ligand, N-Methyl Mesoporphyrin IX (NMM). Our biophysical characterization shows that almost all TET GQs are highly heterogenous and can form multiple conformations. The addition of NMM, however, converts all the sequences to a parallel conformation and increases their thermal stability. To gain further insight into the molecular structures of the variants, we sought out to solve the crystal structures of TET26-2, TET22-NMM, TET24A-NMM, and TET25-NMM. TET26-2, solved to 1.97 Å, shows a parallel GQ with a four G-tetrad core and three TT propeller loops. Preliminary solutions of TET22-NMM, TET24A-NMM, and TET25-NMM complexes show parallel GQs with NMM π − π stacking on top of the 3’- terminal G-tetrad. Significantly, the first example of an NMM-NMM dimer was observed in these preliminary solutions which contributes to the understanding of GQ folding and the structures’ interactions with small molecule ligands. Overall, T. thermophila telomeric variants display unusual structural diversity by forming very distinct GQ structures. Our results provide insight into the many GQ topologies available to telomeric repeats and how they interact with the selective ligand NMM, which is important for the design of optimized GQ-selective anticancer therapeutics

G-Quadruplex Aptamer Beacon for Detection of Prostate Cancer Biomarker

The prostate is the major male reproductive gland involved in male fertility and plays an important role in triggering of molecular pathways relevant to fertility success. Unfortunately, in Portugal prostate cancer is the most common cancer type among men, being asymptomatic in earlier stages. Thus, is important early detection of disease. NCL is a multifunctional protein involved in multiple biological processes under both physiological and pathological processes and can have several cellular localizations. Cell surface protein overexpression was found restricted to cancer cells, namely in prostate cancer cells. Thus, we can consider NCL as a potential biomarker for cancer diagnosis and a target for cancer treatment. The AS1411 is an aptamer capable to recognise and binds specifically NCL and have a therapeutic effect on cancer cells through of induction of antiproliferative activity. Beyond its therapeutic use, AS1411 can be used in imaging and diagnostic, particularly on aptasensors development. One of the most relevant characteristics of this aptamer is the ability to fold in a G4 conformation, a secondary structure of nucleic acids. G4 structure confers stabilization to sequence and availability to bind NCL. Thus, in this work is presented the first approach of use AS1411 aptamer to prostate cancer diagnosis, namely through the design of molecular beacon (MB) designated by AS1411N5. Initially, biophysical characterization of AS1411-N5 was done by circular dichroism, nuclear magnetic resonance or fluorometric spectroscopies. Additionally, it was performed microfluidic experiments, to detect NCL using AS1411-N5 in biological samples. The results demonstrated that the proposed AS1411-N5 adopt a G4 structure and it is capable to bind with specificity and selectivity NCL, even in plasma of human patients with prostate cancer.A próstata é a maior glândula reprodutiva masculina e tem um papel importante nas vias moleculares relevantes para o sucesso da fertilização. Infelizmente, em Portugal o cancro da próstata é o cancro mais comum entre os homens, sendo assintomático em estadios iniciais. Assim é imperativo a deteção precoce da doença. A nucleolina (NCL) é uma proteína multifuncional envolvida em múltiplos processos biológicos sob condições fisiológicas e patológicas, podendo ter várias localizações celulares. A sobre-expressão da proteína na superfície das células é apenas encontrada em células cancerosas, nomeadamente as do cancro da próstata. Assim a NCL pode ser considerada como um potencial biomarcador para o diagnóstico e tratamento do cancro da próstata. O AS411 é um aptamero capaz de reconhecer e ligar especificamente a esta proteína, e de ter um efeito terapêutico nas células cancerosas ao induzir atividade antiproliferativa. Além do uso terapêutico, a sequência pode ser utilizada na imagiologia e diagnóstico, particularmente através do desenvolvimento de aptasensores. Uma das características mais relevantes do aptamero AS1411 é a capacidade de adotar a configuração de G-quadruplex (G4), uma estrutura secundária dos ácidos nucleicos. As estruturas G4 conferem estabilização à sequência e capacidade de ligar à NCL quando adota esta estrutura. Assim, neste trabalho é apresentada uma primeira abordagem do uso do AS1411 no diagnóstico do cancro da próstata, nomeadamente através da construção de uma sonda a partir da sequência deste aptamero designado por AS1411N5. Inicialmente foi efetuada a caracterização biofísica do AS1411-N5 a nível da estrutura e interação com o alvo, recorrendo às espectroscopias dicroísmo circular e ressonância magnética nuclear, e ensaios fluorométricos. Adicionalmente foram efetuadas experiências de microfluídica, para o uso do AS1411N5 como sonda de deteção da NCL. Estes resultados demonstraram, que o AS1411-N5adota a estrutura G4 e é capaz de ligar especificamente e com seletividade com a NCL, mesmo em amostras biológicas

Beyond small molecules: Targeting G-quadruplex structures with oligonucleotides and their analogues

G-Quadruplexes (G4s) are widely studied secondary DNA/RNA structures, naturally occurring when G-rich sequences are present. The strategic localization of G4s in genome areas of crucial importance, such as proto-oncogenes and telomeres, entails fundamental implications in terms of gene expression regulation and other important biological processes. Although thousands of small molecules capable to induce G4 stabilization have been reported over the past 20 years, approaches based on the hybridization of a synthetic probe, allowing sequence-specific G4-recognition and targeting are still rather limited. In this review, after introducing important general notions about G4s, we aim to list, explain and critically analyse in more detail the principal approaches available to target G4s by using oligonucleotides and synthetic analogues such as Locked Nucleic Acids (LNAs) and Peptide Nucleic Acids (PNAs), reporting on the most relevant examples described in literature to date

Monomolecular G-quadruplex structures with inversion of polarity sites: new topologies and potentiality

In this paper, we report investigations, based on circular dichroism, nuclear magnetic resonance spectroscopy and electrophoresis methods, on three oligonucleotide sequences, each containing one 3- 3 and two 5-5 inversion of polarity sites, and four G-runs with a variable number of residues, namely two, three and four (mTG2T, mTG3T andmTG4T with sequence 3-TGnT-5-5-TGnT-3-3-TGnT-5-5-TGnT-3 in which n = 2, 3 and 4, respectively), in comparison with their canonical counterparts (TGnT)4 (n = 2, 3 and 4). Oligonucleotides mTG3T and mTG4 T have been proven to form very stable unprecedented monomolecular parallel G-quadruplex structures, characterized by three side loops containing the inversion of polarity sites. Both G-quadruplexes have shown an all-syn G-tetrad, while the other guanosines adopt anti glycosidic conformations. All oligonucleotides investigated have shown a noteworthy antiproliferative activity against lung cancer cell line Calu 6 and colorectal cancer cell line HCT-116 p53−/−. Interestingly, mTG3T andmTG4T have proven to be mostly resistant to nucleases in a fetal bovine serum assay. The whole of the data suggest the involvement of specific pathways and targets for the biological activity

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

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

Investigating Noncanonical DNA Structures as Anti-Cancer Drug Targets

This work focuses on the structure and interactions with ligands of noncanonical DNA structures. These noncanonical structures include guanine quadruplexes (GQs), as well as a novel type of quadruple helix. Sequences capable of forming these structures are overrepresented at telomeres and replication perturbed loci, respectively. Better understanding of these structures and ligand interactions will provide an improved platform upon which mechanistic understanding and therapeutic treatments can be designed. Our discussion begins with the development and biophysical characterization of a model for the smallest stable structure formed by the repeat sequence d(CAGAGG)ₙ. This sequence was one of many short purine rich tandem repeats recently identified by the Brown lab to be associated with intrinsic replication stress and increased likelihood of replication fork stalling. The resulting model is a monomolecular tetraplex containing two stacked GCGC tetrads and three 4-nucleotide loops that connect the tetrads in an antiparallel manner. This work describes the extension of the repeat to 3-nucleotide overhangs on the 5\u27 and 3\u27 ends to improve the structure\u27s stability, as well as the results of systematic mutations in the core tetrad and loop regions which further support the model. Additional work to extend the model to longer, more biologically relevant repeat lengths is discussed. We sought further validation of the biophysical model, as well as elucidation of its atomic details, through x-ray crystallography. A variety of constructs were screened for crystal results, with varying degrees of success. The best crystals contain short mutations to the 5\u27 and 3\u27 ends, and diffracted to l.92 Å. However, due to a lack of a suitable model for molecular replacement, poor anomalous redundancy, and striking nonisomorphism, solution of the phase problem has been a major impediment. Results for all phasing approaches are discussed below. The final chapter addresses the potential for small molecule ligands, in particular the highly cationic porphyrin TMPyP4, to stabilize the human telomere GQ structure (Tel22) selectively as an anti-cancer therapeutic precursor. Five TMPyP4 derivatives with varying cationic charge were assayed for binding strength and stoichiometry, stabilization of Te122, and selectivity for Tel22 over duplex DNA. A small decrease in cationic charge, exemplified in porphyrin 4P3, resulted in both improved stabilization and selectivity for Tel22 over TMPyP4

G-quadruplexes in Prague: a Bohemian Rhapsody

The Sixth International Meeting on Quadruplex Nucleic Acids was held at the Hotel Internationale in Prague, Czech Republic from 31 May – 3 June 2017. A vibrant interdisciplinary community of almost over 300 scientists gathered to share their newest results in this exciting field and exchange ideas for further investigations

Modeling the influence of DNA lesion on the regulation of gene expression

Nucleic acids are organic macromolecules that result from the polymerization of nucleotides. These molecules are generally considered as the support of the genetic information. Two families of nucleic acids are currently known: DNA and RNA. From a structural point of view, the most popular form is the double helix of DNA. However, other forms exist and among them are the G-quadruplex. This is a folding of the DNA, or RNA, in an area rich in guanines. These form quadruplex of guanines, which are stacked on top of each other and are stabilized by a central cation. G-quadruplex structures are increasingly studied. This is not surprising since their biological role involves the regulation of genetic mechanisms. They are notably involved in the regulation of the cell cycle, but they also play a role in cancer, certain neurological or viral diseases. The aim of this PhD thesis is to study G-quadruplex using theoretical chemistry tools. The three years of work raise very important points for the research on G-quadruplex. First, the modeling of a theoretical G-quadruplex structure can be achieved by sequence homology and validated by calculations of a theoretical circular dichroism spectrum. Consequently, it is possible to use these tools to propose and use a G-quadruplex structure if it is not yet experimentally solved. Then, the work done shows that G-quadruplex form a very stable folding since they are globally conserved even when 8-oxo-guanine or strand breaks lesions are introduced at the quartets. Then, the paper focuses on the interaction between G-quadruplex and proteins. It highlights the important role of G-quadruplex RNA in the infection of the viral pathogen SARS-CoV-2. This RNA promotes the dimerization of the SUD protein of the virus, which in turn is responsible for the disruption of the immune system. Finally, this thesis provides a structural explanation for the specific interaction between the DARPin 2E4 protein and the G-quadruplex of the c-Myc promoter