The i-motif structure is formed in cytosine-rich sequences, its building block being the cytosine·cytosine + base pair. This structure is particularly stable at pH values below physiological (∼7.4) and, because of that, it has not attracted as much biological interest as other non-canonical structures such as the G-quadruplex. Nowadays, the proposal of potential roles in vivo, as well as nanotechnological applications, has produced an increasing interest in its study. In this context, the present work provides an overall picture of the i-motif structure. Those aspects related to formation and stability, such as chemical modifications or the interaction with ligands, are discussed. Special attention has been made to the i-motif structures that could have a hypothetical role in vivo, such as those present near the promoter region of several oncogenes.

The present paper reviews the recent advances in and applications of experimental techniques used to study interactions between G-quadruplex structures and ligands that are potentially of pharmaceutical interest. Several instrumental techniques are used to study such interactions. The application of spectroscopic techniques such as molecular absorption, circular dichroism, molecular fluorescence, mass spectrometry and nuclear magnetic resonance are reviewed and we discuss the type of information (qualitative or quantitative) that can be obtained from the use of each technique. Additionally, the application of complementary techniques such as surface plasmon resonance, isothermal titration calorimetry and different methods based on biochemistry is considered. For each technique, the main applications are presented and they are classified according to the family of the ligand and the type of G-quadruplex forming sequence (human telomeric or promoter region of oncogenes) considered.

The biological role of the existence of overlapping structures in RNA is possible yet remains very unexplored. G-Rich tracts of RNA form G-quadruplexes, while GC-rich sequences prefer stem–loop structures. The equilibrium between alternate structures within RNA may occur and influence its functionality. We tested the equilibrium between G-quadruplex and stem–loop structure in RNA and its effect on biological processes using pre-miRNA as a model system. Dicer enzyme recognizes canonical stem–loop structures in pre-miRNA to produce mature miRNAs. Deviation from stem–loop leads to deregulated mature miRNA levels, providing readout of the existence of an alternate structure per se G-quadruplex-mediated structural interference in miRNA maturation. In vitro analysis using beacon and Dicer cleavage assays indicated that mature miRNA levels depend on relative amounts of K⁺ and Mg²⁺ ions, suggesting an ion-dependent structural shift. Further in cellulo studies with and without TmPyP₄ (RNA G-quadruplex destabilizer) demonstrated that miRNA biogenesis is modulated by G-quadruplex to stem–loop equilibrium in a subset of pre-miRNAs. Our combined analysis thus provides evidence of the formation of noncanonical G-quadruplexes in competition with canonical stem–loop structure inside the cell and its effect on miRNA maturation in a comprehensive manner.

Under the appropriate experimental conditions of pH and temperature, cytosine-rich segments in DNA or RNA sequences may produce a characteristic folded structure known as an i-motif. Besides its potential role in vivo, which is still under investigation, this structure has attracted increasing interest in other fields due to its sharp, fast and reversible pH-driven conformational changes. This “on/off” switch at molecular level is being used in nanotechnology and analytical chemistry to develop nanomachines and sensors, respectively. This paper presents a review of the latest applications of this structure in the field of chemical analysis.