Rubén Ferreira

Mass spectrometry and ion mobility spectrometry of G-quadruplexes. A study of solvent effects on dimer formation and structural transitions in the telomeric DNA sequence d(TAGGGTTAGGGT).

We survey here state of the art mass spectrometry methodologies for investigating G-quadruplexes, and will illustrate them with a new study on a simple model system: the dimeric G-quadruplex of the 12-mer telomeric DNA sequence d(TAGGGTTAGGGT), which can adopt either a parallel or an antiparallel structure. We will discuss the solution conditions compatible with electrospray ionisation, the quantification of complexes using ESI-MS, the interpretation of ammonium ion preservation in the complexes in the gas phase, and the use of ion mobility spectrometry to resolve ambiguities regarding the strand stoichiometry, or separate and characterise different structural isomers. We also describe that adding electrospray-compatible organic co-solvents (methanol, ethanol, isopropanol or acetonitrile) to aqueous ammonium acetate increases the stability and rate of formation of dimeric G-quadruplexes, and causes structural transitions to parallel structures. Structural changes were probed by circular dichroism and ion mobility spectrometry, and the excellent correlation between the two techniques validates the use of ion mobility to investigate G-quadruplex folding. We also demonstrate that parallel G-quadruplex structures are easier to preserve in the gas phase than antiparallel structures.

Specific loop modifications of the thrombin‐binding aptamer trigger the formation of parallel structures

Guanine‐rich sequences show large structural variability, with folds ranging from duplex to triplex and quadruplex helices. Quadruplexes are polymorphic, and can show multiple stoichiometries, parallel and antiparallel strand alignments, and different topological arrangements. We analyze here the equilibrium between intramolecular antiparallel and intermolecular parallel G‐quadruplexes in the thrombin‐binding aptamer (TBA) sequence. Our theoretical and experimental studies demonstrate that an apparently simple modification at the loops of TBA induces a large change in the monomeric antiparallel structure of TBA to yield a parallel G‐quadruplex showing a novel T‐tetrad. The present results illustrate the extreme polymorphism of G‐quadruplexes and the ease with which their conformation in solution can be manipulated by nucleotide modification.

Solution equilibria of cytosine- and guanine-rich sequences near the promoter region of the n-myc gene that contain stable hairpins within lateral loops.

BACKGROUND Cytosine- and guanine-rich regions of DNA are capable of forming complex structures named i-motifs and G-quadruplexes, respectively. In the present study the solution equilibria at nearly physiological conditions of a 34-base long cytosine-rich sequence and its complementary guanine-rich strand corresponding to the first intron of the n-myc gene were studied. Both sequences, not yet studied, contain a 12-base tract capable of forming stable hairpins inside the i-motif and G-quadruplex structures, respectively. METHODS Spectroscopic, mass spectrometry and separation techniques, as well as multivariate data analysis methods, were used to unravel the species and conformations present. RESULTS The cytosine-rich sequence forms two i-motifs that differ in the protonation of bases located in the loops. A stable Watson-Crick hairpin is formed by the bases in the first loop, stabilizing the i-motif structure. The guanine-rich sequence adopts a parallel G-quadruplex structure that is stable throughout the pH range 3-7, despite the protonation of cytosine and adenine bases at lower pH values. The presence of G-quadruplex aggregates was confirmed using separation techniques. When mixed, G-quadruplex and i-motif coexist with the Watson-Crick duplex across a pH range from approximately 3.0 to 6.5. CONCLUSIONS Two cytosine- and guanine-rich sequences in n-myc gene may form stable i-motif and G-quadruplex structures even in the presence of long loops. pH modulates the equilibria involving the intramolecular structures and the intermolecular Watson-Crick duplex. GENERAL SIGNIFICANCE Watson-Crick hairpins located in the intramolecular G-quadruplexes and i-motifs in the promoter regions of oncogenes could play a role in stabilizing these structures.

Structure and stability of human telomeric G-quadruplex with preclinical 9-amino acridines.

G-quadruplexes are higher-order DNA structures formed from guanine-rich sequences, and have been identified as attractive anticancer drug targets. Elucidating the three-dimensional structure of G-quadruplex with 9-amino acridines and the specific interactions involved in binding selectivity are the key to understanding their mechanism of action. Fluorescence titration assays, competitive dialysis and NMR studies have been used to study the binding specificity of 9-amino acridines to DNA. Structural models of the complexes with the telomeric DNA G-quadruplex based on NMR measurements were developed and further examined by molecular dynamics simulations and free energy calculations. Selective binding of 9-amino acridines for G-quadruplex sequences were observed. These compounds bind between A and G-tetrads, involving significant π-π interactions and several strong hydrogen bonds. The specific interactions between different moieties of the 9-amino acridines to the DNA were examined and shown to play a significant role in governing the overall stabilities of DNA G-quadruplex complexes. Both 9-amino acridines, with similar binding affinities to the G-quadruplex, were shown to induce different level of structural stabilization through intercalation. This unique property of altering structural stability is likely a contributing factor for affecting telomerase function and, subsequently, the observed differences in the anticancer activities between the two 9-amino acridines.

Synthesis and structural properties of oligonucleotides covalently linked to acridine and quindoline derivatives through a threoninol linker

Oligonucleotide conjugates containing acridine and quindoline derivatives linked through a threoninol molecule were synthesized. We showed that these conjugates formed duplexes and quadruplexes with higher thermal stability than the corresponding unmodified oligonucleotides. When acridine is located in the middle of the sequence, DNA duplexes have a similar stability independently of the natural base present in front of acridine. Self-complementary oligonucleotides and thrombin binding aptamers (TBA) carrying the acridine and quindoline molecules are studied by NMR.

Synthesis, DNA-Binding and Antiproliferative Properties of Acridine and 5-Methylacridine Derivatives

Several acridine derivatives were synthesized and their anti-proliferative activity was determined. The most active molecules were derivatives of 5-methylacridine-4-carboxylic acid. The DNA binding properties of the synthesized acridines were analyzed by competitive dialysis and compared with the anti-proliferative activities. While inactive acridine derivatives showed high selectivity for G-quadruplex structures, the most active 5-methylacridine-4-carboxamide derivatives had high affinity for DNA but showed poor specificity. An NMR titration study was performed with the most active 5-methylacridine-4-carboxamide, confirming the high affinity of this compound for both duplex and quadruplex DNAs.

Synthesis and structural characterization of stable branched DNA g-quadruplexes using the trebler phosphoramidite.

Guanine (G)-rich sequences can form a noncanonical four-stranded structure known as the G-quadruplex. G-quadruplex structures are interesting because of their potential biological properties and use in nanosciences. Here, we describe a method to prepare highly stable G-quadruplexes by linking four G-rich DNA strands to form a monomolecular G-quadruplex. In this method, one strand is synthesized first, and then a trebler molecule is added to simultaneously assemble the remaining three strands. This approach allows the introduction of specific modifications in only one of the strands. As a proof of concept, we prepared a quadruplex where one of the chains includes a change in polarity. A hybrid quadruplex is observed in ammonium acetate solutions, whereas in the presence of sodium or potassium, a parallel G-quadruplex structure is formed. In addition to the expected monomolecular quadruplexes, we observed the presence of dimeric G-quadruplex structures. We also applied the method to prepare G-quadruplexes containing a single 8-aminoguanine substitution and found that this single base stabilizes the G-quadruplex structure when located at an internal position.

Acridine and quindoline oligomers linked through a 4-aminoproline backbone prefer G-quadruplex structures

BACKGROUND DNA-intercalating drugs are planar molecules with several fused aromatic rings that form stacks between DNA base pairs, reducing the opening and unwinding of the double helix. Recently, interest on intercalating agents has moved in the search for new ligands to G-quadruplex structures. METHODS The DNA binding properties of 4-aminoproline oligomers functionalized with one, two or three units of acridine and/or quindoline have been analyzed by competitive dialysis. A NMR/molecular dynamics study was performed on G-quadruplex telomeric sequence and the 4-aminoproline dimer carrying two quindolines. A model of the complex with the telomeric DNA quadruplex is described. RESULTS AND CONCLUSIONS A selectivity of quindoline 4-aminoproline oligomers for G-quadruplex and triplex structures was observed, especially for those quadruplex sequences found in telomeres and in the promoter regions of c-myc and bcl-2 oncogenes. In this model the quindoline dimer is stabilized by π-π stacking interactions between the aromatic rings of the ligand and the nucleobases of the telomeric sequence that are located above and below the molecule. GENERAL SIGNIFICANCE The results of this work can be used for the design of new molecules with high affinity to telomeres which may have anticancer properties.

Synthesis and G-Quadruplex-Binding Properties of Defined Acridine Oligomers

The synthesis of oligomers containing two or three acridine units linked through 2-aminoethylglycine using solid-phase methodology is described. Subsequent studies on cell viability showed that these compounds are not cytotoxic. Binding to several DNA structures was studied by competitive dialysis, which showed a clear affinity for DNA sequences that form G-quadruplexes and parallel triplexes. The fluorescence spectra of acridine oligomers were affected strongly upon binding to DNA. These spectral changes were used to calculate the binding constants (K). Log K were found to be in the order of 4–6.

Receptor-Based Virtual Screening and Biological Characterization of Human Apurinic/Apyrimidinic Endonuclease (Ape1) Inhibitors

The endonucleolytic activity of human apurinic/apyrimidinic endonuclease (AP endo, Ape1) is a major factor in maintaining the integrity of the genome. Conversely, as an undesired effect, Ape1 overexpression has been linked to resistance to radio‐ and chemotherapeutic treatments in several human tumors. Inhibition of Ape1 using siRNA or the expression of a dominant negative form of the protein has been shown to sensitize cells to DNA‐damaging agents, including various chemotherapeutic agents. Therefore, inhibition of the enzymatic activity of Ape1 might result in a potent antitumor therapy. A number of small molecules have been described as Ape1 inhibitors; however, those compounds are in the early stages of development. Herein we report the identification of new compounds as potential Ape1 inhibitors through a docking‐based virtual screening technique. Some of the compounds identified have in vitro activities in the low‐to‐medium micromolar range. Interaction of these compounds with the Ape1 protein was observed by mass spectrometry. These molecules also potentiate the cytotoxicity of the chemotherapeutic agent methyl methanesulfonate in fibrosarcoma cells. This study demonstrates the power of docking and virtual screening techniques as initial steps in the design of new drugs, and opens the door to the development of a new generation of Ape1 inhibitors.