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

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.

Biological role for existence of overlapping structures in RNA is possible yet remains very less explored. G-rich tracts of RNA form G-quadruplexes while GC-rich sequences prefer stem-loop structures. Equilibrium between alternate structures within RNA may occur and influence its functionality. We tested 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 stemloop leads to deregulated mature miRNA levels, providing readout of existence of 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 Mg 2+ ions suggesting an ion-dependent structural shift. Further in cellulo studies with and without TmPyP 4 (RNA G-quadruplex destabilizer) demonstrated that miRNA biogenesis is modulated by G-quadruplex-stem-loop equilibrium in a subset of premiRNAs. Our combined analysis thus provides evidence for formation of non-canonical Gquadruplexes in competition with canonical stem-loop structure inside the cell and its effect on miRNA maturation in a comprehensive manner.

The 5'-end of the P1 promoter of the B-cell lymphoma-2 (bcl-2) gene contains a highly guaninecytosine-rich region, which has a role in the regulation of bcl-2 transcription. Whereas the guanine-rich region has been the focus of recent studies, little attention has been paid to the cytosine-rich strand. Here we examine the structural transitions of the cytosine-rich sequence by means of acid-base, mole-ratio and melting experiments monitored by molecular absorption, circular dichroism, and NMR spectroscopies. Two intramolecular i-motif structures have been detected in the pH range 2-7, with maximal formation at pH 4 and 6, respectively. At pH 7.6 the majority species has been associated with a hairpin involving Watson-Crick base pairs. Upon addition of the quadruplex-interacting ligand TmPyP4, bcl-2c structures at pH 6.1 and 7.6 yield identical interaction species with stoichiometries 1:2 (DNA:ligand) and logarithms of formation constant 12.4+/-0.2 and 11.7+/-0.1, respectively. The initial i-motif structure at pH 6.1 is lost upon interaction with TmPyP4.

A successful application is reported of the multivariate curve resolution alternating least-squares method (MCR-ALS) for the analysis of nucleic acid melting and salt-induced transitions. Under conditions where several structures co-exist in a conformational equilibrium, MCR-ALS analysis of the UV and circular dichroism (CD) spectra at different temperatures, ionic strength and oligonucleotide concentration allows for the resolution of concentration profiles and pure spectra of the different species. The methodology is illustrated by the case of the cyclic oligonucleotide d. The melting transition of this molecule at different oligonucleotide concentrations was studied at 0, 2 and 10 mM MgCl2 by UV and CD spectroscopy. In addition, salt titration experiments were carried out at 21.0 and 54.0 degrees C. The MCR-ALS analysis indicates that three different conformations of this molecule co-exist in solution. In agreement with previous NMR studies, these conformations were assigned to a monomeric dumbbell-like structure, a dimeric four-stranded conformation and a disordered (random coil) structure. The MCR-ALS methodology allows for a detailed analysis of how this equilibrium is affected by temperature, salt and oligonucleotide concentration.

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The influence of pH, temperature and the cationic porphyrin TMPyP4 on the conformational equilibria of the cytosine-rich strand of the human telomeric sequence 5'-(C(3)TA(2))(4)-3', as well as those of the sequence 5'-(C(3)TT(2))(4)-3', was studied. The presence of adenine bases at the loops causes the formation of two different intramolecular i-motif structures with a pH-transition midpoint around 4.6, which stability is lower than the i-motif formed by the sequence 5'-(C(3)TT(2))(4)-3'. The stoichiometries of the complexes formed by these i-motif structures with TMPyP4 are also influenced by the presence of adenine at the loops.