Mateus Webba da Silva

Topological Characterization of Nucleic Acid G‐Quadruplexes by UV Absorption and Circular Dichroism

The emergence of nucleic acid four-stranded architectures, denominated G-quadruplexes as a prolific area of research, has led to an interest in the development of inexpensive methods for the rapid assessment of their structural characterization in solution. Research in this area is motivated by their potential impact in regulation of biological mechanisms and technological applications. For many applications, light absorption techniques, such as circular dichroism (CD) and UV, have been sufficient to discriminate the quadruplex fold from other architectures. CD is also useful to discriminate a single quadruplex topology from all other 25 generic folding topologies. Here we demonstrate the use of these techniques for characterizing three different types of G-quadruplex topologies classified through the sequence of glycosidic bond angles (GBA) adopted by guanosines of the G-quadruplex stem.

DNA quadruplex folding formalism--a tutorial on quadruplex topologies.

Quadruplexes of DNA adopt a large variety of topologies that are dependent on their environment. We have been developing a formalism for quadruplex folding based on the relationship between base and its sugar--as defined by the glycosidic bond angle. By reducing the quadruplex stem to a description based on two finite states of the range of angles the glycosidic bond angle may adopt, the description of the relationships of type of loop and groove widths of a quadruplex stem are possible. In its current form this formalism has allowed for the prediction of some unimolecular quadruplex topologies. Its rules, whilst developed for unimolecular quadruplexes of three loops, are of general utility in understanding the interdependency of structural characteristics of multimolecular folds, as well as unimolecular quadruplexes of more than three loops. Here we describe current understanding of the interdependent structural features that define the quadruplex fold, and provide a tutorial for the use and application of this formalism.

Design of a G-Quadruplex Topology through Glycosidic Bond Angles†

To realize a programmed build up of DNA objects, devices,and materials, a systematization of the principles that formthe basis of control for the assembly process is necessary. Thusfar the discovery of four-stranded DNA architecturesdenominated G quadruplexes was either serendipitous orthrough coincidental emergence. Herein we utilize a rationalapproach for the design of G quadruplexes based on the twostatedisposition of the glycosidic bond angle.

Unique Structural Features of Interconverting Monomeric and Dimeric G-Quadruplexes Adopted by a Sequence from the Intron of the N-myc Gene

A multidimensional heteronuclear NMR study has demonstrated that a guanine-rich DNA oligonucleotide originating from the N-myc gene folds into G-quadruplex structures in the presence of K(+), NH(4)(+), and Na(+) ions. A monomeric G-quadruplex formed in K(+) ion containing solution exhibits three G-quartets and flexible propeller-type loops. The 3D structure with three single nucleotide loops represents a missing element in structures of parallel G-quadruplexes. The structural features together with the high temperature stability are suggestive of the specific biological role of G-quadruplex formation within the intron of the N-myc gene. An increase in K(+) ion and oligonucleotide concentrations resulted in transformation of the monomeric G-quadruplex into a dimeric form. The dimeric G-quadruplex exhibits six stacked G-quartets, parallel strand orientations, and propeller-type loops. A link between the third and the fourth G-quartets consists of two adenine residues that are flipped out to facilitate consecutive stacking of six G-quartets.

Kinetics of double-chain reversals bridging contiguous quartets in tetramolecular quadruplexes

Repetitive 5′GGXGG DNA segments abound in, or near, regulatory regions of the genome and may form unusual structures called G-quadruplexes. Using NMR spectroscopy, we demonstrate that a family of 5′GCGGXGGY sequences adopts a folding topology containing double-chain reversals. The topology is composed of two bistranded quadruplex monomeric units linked by formation of G:C:G:C tetrads. We provide a complete thermodynamic and kinetic analysis of 13 different sequences using absorbance spectroscopy and DSC, and compare their kinetics with a canonical tetrameric parallel-stranded quadruplex formed by TG4T. We demonstrate large differences (up to 105-fold) in the association constants of these quadruplexes depending on primary sequence; the fastest samples exhibiting association rate equal or higher than the canonical TG4T quadruplex. In contrast, all sequences studied here unfold at a lower temperature than this quadruplex. Some sequences have thermodynamic stability comparable to the canonical TG4T tetramolecular quadruplex, but with faster association and dissociation. Sequence effects on the dissociation processes are discussed in light of structural data.

Effect of Base Sequence on G-Wire Formation in Solution

The formation and dimensions of G-wires by different short G-rich DNA sequences in solution were investigated by dynamic light scattering (DLS) and polyacrilamide gel electrophoresis (PAGE). To explore the basic principles of wire formation, we studied the effects of base sequence, method of preparation, temperature, and oligonucleotide concentration. Both DLS and PAGE show that thermal annealing induces much less macromolecular self-assembly than dialysis. The degree of assembly and consequently length of G-wires (5-6 nm) are well resolved by both methods for DNA sequences with intermediate length, while some discrepancies appear for the shortest and longest sequences. As expected, the longest DNA sequence gives the longest macromolecular aggregates with a length of about 11 nm as estimated by DLS. The quadruplex topologies show no concentration dependence in the investigated DNA concentration range (0.1 mM–0.4 mM) and no structural change upon heating.

Structural Probes in Quadruplex Nucleic Acid Structure Determination by NMR

Traditionally, isotope-labelled DNA and RNA have been fundamental to nucleic acid structural studies by NMR. Four-stranded nucleic acid architectures studies increasingly benefit from a plethora of nucleotide conjugates for resonance assignments, the identification of hydrogen bond alignments, and improving the population of preferred species within equilibria. In this paper, we review their use for these purposes. Most importantly we identify reasons for the failure of some modifications to result in quadruplex formation.

G-Quadruplex Nucleic Acids

G-quadruplexes are a family of four-stranded structuresstabilized by guanine quartets, in which four planar guaninesestablish a cyclic array of hydrogen bonds. They are ofspecial interest due to the increasing evidence for theirformation in vivo and their possible implication in biology,especially at telomeres and as contributors to gene regulation.Moreover, G-quadruplexes are also formed as a result ofself-assembling processes of guanosine derivatives, yieldingseveral interesting motifs such as G-ribbons and G-wireswhich have peculiar electrical conductivity properties thatare being explored as molecular wires.This special issue is initiated with two articles discussingthe self-assembling properties of guanosine derivatives whichdescribe the basic principles of G-quadruplex formation. Thefirst article by Neviani et al. analyzes the presence of severallevels of organization of guanosine derivatives carryingone or two lipophilic units as observed by light scatteringtechniques and transmission electron microscopy (TEM)experiments. The second article by Mariani et al. describesa study on quadruplex formation of 2 -deoxyguanosinemonophosphate by small-angle X-ray scattering techniques.

Rapid Stoichiometric Analysis of G-Quadruplexes in Solution

Guanine-rich sequences of nucleic acids may fold into secondarystructural folds called quadruplex architectures.These architectures are a rapidly growing theme of interestwith promising repercussions in our understanding of biologyand practical applications in medical fields, materialsscience, and biotechnology. Currently, there is thus interestin determining both topology and their atomic detail 3Dstructures. The establishment of solution conditions suitablefor structural studies of G-quadruplex architectures requiresthe determination of the level of oligomerization (stoichiometry)of DNA strands. Various analytical techniques arecurrently applied for the routine assessment of the stoichiometrythat generally include conditions not representativeof the environment in which the structural studies are performed.

Potential of Cellulose Functionalized with Carboxylic Acid as Biosorbent for the Removal of Cationic Dyes in Aqueous Solution

In the last decade, adsorption has been used to minimize the pollution caused by dyes, which represents a serious environmental problem. In this context, this work reports the preparation of phthalic anhydride-modified cellulose (PhCel), through the reaction of cellulose (Cel) with phthalic anhydride (Ph). The efficiency of the reaction was observed by elemental analysis, Fourier Transform Infrared (FTIR) spectroscopy, X-ray diffraction (XRD) and thermogravimetry/derivative thermogravimetry (TG/DTG). The adsorbent matrix (Cel and PhCel) was used in the removal of crystal violet (CV) and methylene blue (MB) dyes in aqueous medium. In the kinetic study, the experimental data obtained had the best fit to the pseudo-first-order model. In general, the isotherms obtained at different temperatures had a best fit to the model proposed by Langmuir, and the CV and MB adsorption process in adsorbent matrixes can be favored strictly by hydrogen bonds and/or electrostatic interactions for Cel and electrostatic interactions for PhCel.