Influence of pH and a porphyrin ligand on the stability of a G-quadruplex structure within a duplex segment near the promoter region of the SMARCA4 gene

In a previous work, the formation of G-quadruplex structures in a 44-nucleotide long sequence found near the promoter region of the SMARCA4 gene was reported. The central 25 nucleotides were able to fold into an antiparallel G-quadruplex structure, the stability of which was pH-dependent. In the present work, the effect of the presence of lateral nucleotides and the complementary cytosine-rich strand on the stability of this G-quadruplex has been characterized. Moreover, the role of the model ligand TMPyP4 has been studied. Spectroscopic and separation techniques, as well as multivariate data analysis methods, have been used with these purposes. The results have shown that stability of the G-quadruplex as a function of pH or temperature is greatly reduced in the presence of the lateral nucleotides. The influence of the complementary strand does not prevent the formation of the G-quadruplex. Moreover, attempts to modulate the equilibria by an external ligand led us to determine the influence of the TMPyP4 porphyrin on these complex equilibria. This study could eventually help to understand the regulation of SMARCA4 expression

Understanding the effect of the nature of the nucleobase in the loops on the stability of the i-motif structure

The nature and length of loops connecting cytosine tracts in i-motif structures may affect their stability. In this work, the influence of the nature of the nucleobases located in two of the loops of an intramolecular i-motif is studied using spectroscopy, separation techniques, and multivariate data analysis. Insertion of bases other than thymine induces an additional acid-base equilibrium with pKa~4.5. The presence of two guanine bases in the loops, placed opposite to each other, decreases the thermal stability of the structure. In contrast, thymine and cytosine bases in these positions stabilize the structur

Evaluation of the effect of polymorphism on G-quadruplex-ligand interaction by means of spectroscopic and chromatographic techniques

Guanine-rich sequences may fold into highly ordered structures known as G-quadruplexes. Apart from the monomeric G-quadruplex, these sequences may form multimeric structures that are not usually considered when studying interaction with ligands. This work studies the interaction of a ligand, crystal violet, with three guanine-rich DNA sequences with the capacity to form multimeric structures. These sequences correspond to short stretches found near the promoter regions of c-kit and SMARCA4 genes. Instrumental techniques (circular dichroism, molecular fluorescence, size-exclusion chromatography and electrospray ionization mass spectrometry) and multivariate data analysis were used for this purpose. The polymorphism of G-quadruplexes was characterized prior to the interaction studies. The ligand was shown to interact preferentially with the monomeric G-quadruplex; the binding stoichiometry was 1:1 and the binding constant was in the order of 105 M-1 for all three sequences. The results highlight the importance of DNA treatment prior to interaction studie

i-motif structures in long cytosine-rich sequences found upstream of the promoter region of the SMARCA4 gen

Cytosine-rich oligonucleotides are capable of forming complex structures known as i-motif with increasingly studied biological properties. The study of sequences prone to form i-motifs located near the promoter region of genes may be difficult because these sequences not only contain repeats of cytosine tracts of disparate length but also these may be separated by loops of varied nature and length. In this work, the formation of an intramolecular i-motif structures by a long sequence located upstream of the promoter region of the SMARCA4 gene has been demonstrated. Nuclear Magnetic Resonance, Circular Dichroism, Gel Electrophoresis, Size-Exclusion Chromatography, and multivariate analysis have been used. Not only the wild sequence (5'-TC3T2GCTATC3TGTC2TGC2TCGC3T2G2TCATGA2C4-3') has been studied but also several other truncated and mutated sequences. Despite the apparent complex sequence, the results showed that the wild sequence may form a relatively stable and homogeneous unimolecular i-motif structure, both in terms of pH or temperature. The model ligand TMPyP4 destabilizes the structure, whereas the presence of 20% (w/v) PEG200 stabilized it slightly. This finding opens the door to the study of the interaction of these kind of i-motif structures with stabilizing ligands or proteins

Study of light-induced formation of photodimers in the i-motif nucleic acid structure by rapid-scan FTIR difference spectroscopy and hybrid hard- and soft-modelling

The i-motif is a DNA structure formed by cytosine-rich sequences, very relevant from a biochemical point of view and potentially useful in Nanotechnology as pH-sensitive nanodevices or nanomotors. To provide a different view on the structural changes and dynamics of direct excitation processes involving i-motif structures, the use of rapid scan FTIR spectroscopy is proposed. Hybrid hard- and soft-modelling based on the Multivariate Curve Resolution by Alternating least squares (MCR-ALS) algorithm has been used for the resolution of rapid-scan FTIR spectra and the interpretation of the photochemically induced time-dependent conformational changes of i-motif structures. The hybrid hard- and soft-modelling version of MCR-ALS (HS-MCR), which allows the introduction of kinetic models to describe the process behavior, provides also rate constants associated with the transitions modeled. The results show that i-motif structures formed by short DNA sequences present higher structural changes upon UV irradiation than those formed by long sequences with additional structural stabilizing elements, such as hairpins

Study of conformational transitions of i-motif DNA using time-resolved fluorescence and multivariate analysis methods

Recently, the presence of i-motif structures at C-rich sequences in human cells and their regulatory functions have been demonstrated. Despite numerous steady-state studies on i-motif at neutral and slightly acidic pH, the number and nature of conformation of this biological structure are still controversial. In this work, the fluorescence lifetime of labelled molecular beacon i-motif-forming DNA sequences at different pH values is studied. The influence of the nature of bases at the lateral loops and the presence of a Watson-Crick-stabilized hairpin are studied by means of time-correlated single-photon counting technique. This allows characterizing the existence of several conformers for which the fluorophore has lifetimes ranging from picosecond to nanosecond. The information on the existence of different i-motif structures at different pH values has been obtained by the combination of classical global decay fitting of fluorescence traces, which provides lifetimes associated with the events defined by the decay of each sequence and multivariate analysis, such as principal component analysis or multivariate curve resolution based on alternating least squares. Multivariate analysis, which is seldom used for this kind of data, was crucial to explore similarities and differences of behaviour amongst the different DNA sequences and to model the presence and identity of the conformations involved in the pH range of interest. The results point that, for i-motif, the intrachain contact formation and its dissociation show lifetimes ten times faster than for the open form of DNA sequences. They also highlight that the presence of more than one i-motif species for certain DNA sequences according to the length of the sequence and the composition of the bases in the lateral loop

Application of analytical and chemometric methodologies to study complex bioanalytical processes involving DNA i-motif structures

[eng] 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.[cat] La estructura de l’ADN coneguda com “i-motif” es forma en seqüències riques en bases citosina (C). L’esquelet de l’i-motif està format per parells de bases C·C+ intercalats i estabilitzats per ponts d’hidrogen. S'ha demostrat la formació in vitro d'aquesta estructura en seqüències d'ADN corresponents a les regions promotores de diversos oncògens, com el c-kit, el c-myc o el bcl-2. Recentment, s'ha demostrat la primera evidència de la seva presència in vivo. La present Tesi Doctoral tracta de l'aplicació de metodologies analítiques i quimiomètriques per estudiar processos bioanalítics complexos que en els que intervenen aquestes estructures. Les seqüències estudiades corresponen a les regions promotores dels gens nmyc i SMARCA4. D'una banda, s'ha estudiat l'estabilitat de les estructures formades per aquestes seqüències segons variacions de pH, temperatura, força iònica o presència de lligands en condicions d'estat estacionari. D'altra banda, s'ha avaluat el potencial d'espectroscòpia ultraràpida per a l'estudi de processos cinètics ràpids provocats per la llum. Al llarg de la Tesi, els mètodes de resolució multivariant, ja sigui basats en models flexibles, rígids o híbrids, s'han utilitzat àmpliament per modelitzar els processos d'interès. Els estudis d'estat estacionari han demostrat que l'estabilitat davant el pH o les variacions de temperatura de les tres diferents seqüències riques en citosina esmentades anteriorment depèn molt del nombre de parells de bases de C·C+, però també de la contribució d'altres factors, com ara la composició de la base i la longitud dels bucles. A partir d'estudis ultraràpids, el procés fotoquímic induït per la irradiació de llum UV i l'IR d'escaneig ràpid s'ha relacionat amb la formació de fotoproductes dimèrics en seqüències plegades i desplegades. L'estudi dels processos seguits mitjançant fluorescència resolta en el temps ha demostrat l’existència de més d’una espècie associada amb l’estructura i-motif en el cas de les seqüencies curtes. Finalment, l'aplicació de mètodes de resolució multivariant, basats tant en models rígids o flexibles, han permès extreure informació química valuosa dels processos evolutius de l'ADN. A més, s'ha demostrat que l'adaptació i l'aplicació del modelatge híbrid ha permet calcular les constants cinètiques i detectar transicions dependents de la temperatura

Variable-temperature size exclusion chromatography for the study of the structural changes in G-quadruplex

The conformational equilibria of a guanine-rich sequence found at the promoter region of the human c-kit oncogene are studied by means of circular dichroism spectroscopy (CD) and variable-temperature Size Exclusion Chromatography (SEC). It is shown that the wild sequence ckit21 exists as a mixture of monomeric and multimeric G-quadruplex. Appropriate mutation of several bases in the wild sequence produces the shift from parallel to antiparallel G-quadruplex, as well as the disappearance of multimeric species. The shift from the antiparallel to the parallel conformation induced by temperature is reflected in both CD and SEC profiles

Understanding the effect of the nature of the nucleobase in the loops on the stability of the i-motif structure

The nature and length of loops connecting cytosine tracts in i-motif structures may affect their stability. In this work, the influence of the nature of the nucleobases located in two of the loops of an intramolecular i-motif is studied using spectroscopy, separation techniques, and multivariate data analysis. Insertion of bases other than thymine induces an additional acid-base equilibrium with pKa~4.5. The presence of two guanine bases in the loops, placed opposite to each other, decreases the thermal stability of the structure. In contrast, thymine and cytosine bases in these positions stabilize the structur