Current research activity

The name of our research area (“Analytical Chemistry of Quadruplex DNA”) intends to synthesize in few words the activity that is currently carried out in our laboratory. We are interested in both, fundamentals and practical aspects of solution equilibria involving DNA sequences able to form quadruplex structures (G-quadruplex and i-motif).

G-quadruplex structures within guanine-rich regions of the genome, both in vitro as in vivo. The core of this structure is a tetrad composed of four guanine bases. G-quadruplexes may be formed by DNA sequences within just one, two or four nucleic acid strands. There is an increasing interest in the study of G-quadruplex because of their recent discovery in vivo and their possible role in biological processes like cancer. Also, as the folding of G-quadruplexes is mediated by cations, many applications in chemical analysis of these analytes have been already described.

G-quadruplex structure formed by three tetrads of four guanines each one (image from Wikipedia)

Although the guanine-rich strands are widely studied, the complementary cytosine-rich strands have received little attention. Cytosine-rich DNA strands may fold into the so-named i-motif structure. This structure consists of four cytosine tracts arranged in two parallel duplexes “zipped” together in antiparallel orientation. As G-quadruplexes, i-motif structures may be formed by DNA sequences within just one, two or four nucleic acid strands. The building block of the i-motif structure is the C·C+ base pair, where one of the cytosine bases is protonated. Because of this, the stability of i-motif is strongly pH-dependent. Recent interest has been shown in the study of i-motif structures due to their potential application in Nanotechnology and chemical analysis, as well as their possible roles in gene transcription. Cytosine-rich sequences have been observed in many genome regions, such as centromeric and telomeric regions of human chromosomes, as well as in the promoter regions of several genes. In April 2018, the in vivo presence of these structures was reported for the first time [Zeraati et al. Nature Chem. 2018; article in the Spanish journal "El Mundo"].

i-motif

Hypothetical i-motif structure present within a cytosine-rich sequence near the promoter region of SMARCA4 gene (Benabou et al. 2017). Cytosine bases are depicted as red spheres.

In our laboratory, we study the solution equilibria of guanine- and cytosine-rich sequences able to fold into G-quadruplex and i-motif structures. Hence, the conformational changes observed in these structures as a result of modifications of external variables (such as pH, temperature, ionic strength, cation concentration, ligand concentration, non-aqueous solvent…) are studied from the point of view of Analytical Chemistry (Alba et al. 2016).

For the study of solution equilibria involving G-quadruplex and i-motif structures we use different experimental approaches, as well as instrumental and data analysis techniques. Among the experimental approaches, classical acid-base or ligand titrations or meltings monitored spectroscopically are widely used. The instrumental techniques used include molecular absorption, molecular fluorescence and circular dichroism spectroscopies, separation techniques (Size Exclusion-HPLC and PAGE), and Surface Plasmon Resonance (SPR), among others. Finally, special focus is made on the use of multivariate data analysis methods for the analysis of spectroscopic data recorded along the experiments. It has been shown widely the advantages of using this approach over the classical, univariate approach (Benabou et al. 2019).

Example of the application of multivariate analysis to the study of the interaction of a ligand with DNA. (a) Experimental CD spectra. (b) Resolved distribution diagram of species and pure CD spectra for each one of the considered species (Navarro et al. 2020)

Also, we have studied the kinetics of pH-induced folding of different i-motif structures by means of ultrafast spectroscopic techniques hyphenated to multivariate analysis (Benabou et al. 2019). Finally, the interaction of ligands with both G-quadruplex and i-motif structures is also a matter of research (Mazzini et al. 2020).

Recently, we have been studying the solution equilibria involving the guanine- and cytosine-rich regions found near the promoter region of the SMARCA4 gene. The results obtained along the development of this project have been published in three research articles. Probably, the most relevant fact is the proposal of a G-quadruplex structure which folding is pH-mediated. The results pointed to an important role of two cytosine bases located at one of the loops with an apparent pKa value near 7.4. Interestingly, the protonation at these bases produced a dramatic decrease of the heterogeneous folding of the sequence at pH higher than 7.4, approximately.

Proposed structure of a G-quadruplex within a guarine-rich sequence near the promoter region of SMARCA4 gene (Benabou et al. 2019)

Our last article deals with the competition between intramolecular (G-quadruplex and i-motif structures) and the intermolecular Watson-Crick double helix within the guanine and cytosine-rich sequence near the promoter region of SMARCA4 gene. The influence of pH and a porphyrin ligand on these equilibria have been studied (Navarro et al. 2020).

In short-term, we are working on the synthesis of silver nanoclusters stabilized by cytosine-rich DNA sequences. These structures are highly fluorescent, which make them an interesting tool in chemical analysis.


	© Universitat de Barcelona	Edition: R.Gargallo Last update: 29.05.2020