Abstract
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.
Highlights
DNA has been extensively studied due to its outstanding function as carrier of genetic information
Choi et al reported that the fast dynamics of i-motif with a compact tetraplex is due to the intrinsic conformational changes at the fluorescent site, including the motion of the alkyl chain connecting the dye to DNA, whereas the slow intrachain contact formation observed from the open form is due to the DNA motion, corresponding to an early stage interaction in the folding process of the unstructured open form
Intramolecular folding dynamics of i-motif DNA were studied by combining time-resolved fluorescence, global fitting approach and multivariate methods, such as principal component analysis (PCA) and Multivariate curve resolution alternating least squares (MCR-ALS)
Summary
DNA has been extensively studied due to its outstanding function as carrier of genetic information. Repetitive DNA sequences have the potential to fold into non-B DNA or noncanonical structures such as hairpin, triplex, tetraplex or left-handed Z-form under certain experimental conditions. The study of these structures is of great interest because of their potential role in some diseases and aging phenomena [1]. The in vitro formation of tetraplex structures, such as the G-quadruplex and i-motif structures, in the DNA sequences corresponding to the end of the telomeres and the promoter regions of several oncogenes has been demonstrated [2,3,4]. The interest of DNA researchers in the i-motif structure decreased in past years due to the general thought that an acidic pH was necessary to stabilize the structure in vivo. The discovery of pH anomalies in some tumours and diseases such as amyotrophic lateral sclerosis [5,6], Huntington’s disease [7], epilepsy [8], brain tumour [9,10] and Parkinson’s disease [11] triggered again this interest, especially after a recent study that provides the first direct evidence of in vivo presence in human cells and control regulatory functions [12]
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