Application of analytical and chemometric methodologies to study complex bioanalytical processes involving dna i-motif structures
- Autores: Sanae Benabou Zdaou
- Directores de la Tesis: Raimundo Gargallo Gómez (dir. tes.), Anna Maria De Juan Capdevila (dir. tes.)
- Lectura: En la Universitat de Barcelona ( España ) en 2018
- Idioma: español
- Tribunal Calificador de la Tesis: José Luis Cortina Pallás (presid.), Nuria Escaja Sanchez (secret.), Federico Marini (voc.)
- Programa de doctorado: Programa de Doctorado en Química Analítica y Medio Ambiente por la Universidad de Barcelona
- Materias:
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- Resumen
The i-motif is a DNA structure formed by cytosine-rich sequences that consists of parallel-stranded duplexes held together by intercalated base pairs. The in vitro formation of this structure in DNA sequences corresponding to the promoter regions of several oncogenes, such as c-kit, c-myc or bcl-2, has been demonstrated. Recently, the first direct evidence for its in vivo presence in human cells and control regulatory functions has been proven. This structure is not only interesting from a biophysical and biomedical point of view, but also for their potential application in Analytical Chemistry or Nanotechnology.
The present Doctoral Thesis deals with the application of analytical and chemometric methodologies to study complex bioanalytical processes involving DNA i-motif structures. The sequences studied correspond to those found at cytosine-rich regions found near the promoter regions of the nmyc and SMARCA4 genes. On the one hand, the stability of the i-motif structures formed by these sequences according to variations of pH, temperature, ionic strength, or presence of ligands in steady-state conditions has been studied. On the other hand, the potential of ultrafast spectroscopies for the study of fast kinetic processes triggered by light has been evaluated. Through the Thesis, curve resolution methods, either based on soft-, hard- or hybrid-modelling have been used extensively to model the biochemical processes of interest.
The steady-state studies have demonstrated that the stability against pH or temperature variations of the three different kinds of cytosine-rich sequences mentioned above is strongly dependent on the number of the C·C+ base pairs, but also on the contribution of other factors, such as the base composition and length of the loops and the presence of additional stabilising structures (hairpins) in the DNA sequence. The studies performed at ultrafast time scales have revealed that the photochemical process induced by UV-lamp irradiation and monitored by rapid-scan FTIR involves the formation of dimeric photoproducts in folded and unfolded sequences. The study of processes monitored by time-resolved fluorescence in the scale of picoseconds has shown that i-motif relaxation is detected by the presence of fast lifetimes in the pH range between 4 and 6, associated with intrinsic conformational changes at the fluorescent site. In this last study, one or two different i-motif structures have been detected in the nmyc and in the shortest DNA sequences studied, respectively.
Finally, the application of multivariate resolution methods, based either on hard- or soft-modelling, has allowed the recovery of valuable chemical information from evolutionary processes of DNA. Besides, the adaptation and application of hybrid hard- and soft-modelling has been shown to be a useful approach to detect intermediate temperature-dependent conformational transitions and to avoid the effect of baseline drifts in the estimation of the melting temperature, as well to retrieve rate constants from the kinetic information present in rapid-scan FTIR difference spectra.
