The latest research on fundamental aspects of i-motif structures is reviewed with special attention to their hypothetical rolein vivo.

Nucleic acids have been among the first targets for antitumor drugs and antibiotics, and with the unveiling of new biological roles in regulation of gene expression, specific DNA and RNA structures have become very attractive targets, especially when the corresponding proteins are undruggable. Biophysical assays to test target structure and ligand binding stoichiometry, affinity, specificity and binding modes are part of the drug development pipeline. Mass spectrometry offers unique advantages as a biophysical method due to its ability to distinguish each stoichiometry present in a mixture. In addition, advanced mass spectrometry approaches (reactive probing, fragmentation techniques, ion mobility spectrometry, ion spectroscopy) provide more detailed information on the complexes. Here we review the fundamentals of mass spectrometry and all its particularities when studying non-covalent nucleic acid structures, and then review what has been learned thanks to mass spectrometry on nucleic acid structures, self-assemblies (e.g., duplexes or G-quadruplexes), and their complexes with ligands. 47

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The nature and the 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. The 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 structure.

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.

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 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'-TCTGCTATCTGTCTGCTCGCTGTCATGAC-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.

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 process behavior, provides also rate constants associated with the transitions modeled. The results show that UV irradiation does not produce degradation of the studied sequences but induces the formation of photodimers. The presence of these affect much more the stability of i-motif structures formed by short sequences than that of those formed by longer sequences containing additional structural stabilizing elements, such as hairpins.

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 10M for all three sequences. The results highlight the importance of DNA treatment prior to interaction studies.