Triplex with a twist: Through metadynamics calculations, the thrombin binding aptamer (TBA) has been shown to adopt a stable G‐triplex structural motif, in addition to the usual G‐quadruplex (see scheme). An 11‐mer oligonucleotide was also shown to form a stable G‐triplex, whose structural and thermodynamic properties have been characterized.

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Aptamer-based drugs represent an attractive approach in pharmacological therapy. The most studied aptamer, thrombin binding aptamer (TBA), folds into a well-defined quadruplex structure and binds to its target with good specificity and affinity. Modified aptamers with improved biophysical properties could constitute a new class of therapeutic aptamers. In this study we show that the modified thrombin binding aptamer (mTBA), (3')GGT(5')-(5')TGGTGTGGTTGG(3'), which also folds into a quadruplex structure, is more stable than its unmodified counterpart and shows a higher thrombin affinity. The stability of the modified aptamer was investigated using differential scanning calorimetry, and the energetics of mTBA and TBA binding to thrombin was characterized by means of isothermal titration calorimetry (ITC). ITC data revealed that TBA/thrombin and mTBA/thrombin binding stoichiometry is 1:2 for both interactions. Structural models of the two complexes of thrombin with TBA and with mTBA were also obtained and subjected to molecular dynamics simulations in explicit water. Analysis of the models led to an improvement of the understanding of the aptamer-thrombin recognition at a molecular level.

The complex between distamycin A and the parallel DNA quadruplex [d(TGGGGT)]4 has been studied by 1H NMR spectroscopy and isothermal titration calorimetry (ITC). To unambiguously assert that distamycin A interacts with the grooves of the quadruplex [d(TGGGGT)]4, we have analyzed the NMR titration profile of a modified quadruplex, namely [d(TGGMeGGT)]4, and we have applied the recently developed differential frequency-saturation transfer difference (DF-STD) method, for assessing the ligand-DNA binding mode. The three-dimensional structure of the 4:1 distamycin A/[d(TGGGGT)]4 complex has been determined by an in-depth NMR study followed by dynamics and mechanics calculations. All results unequivocally indicate that distamycin molecules interact with [d(TGGGGT)]4 in a 4:1 binding mode, with two antiparallel distamycin dimers that bind simultaneously two opposite grooves of the quadruplex. The affinity between distamycin A and [d(TGGGGT)]4 enhances ( approximately 10-fold) when the ratio of distamycin A to the quadruplex is increased. In this paper we report the first three-dimensional structure of a groove-binder molecule complexed to a DNA quadruplex structure.

The nature of the binding mode and stoichiometry of the TMPyP4 cationic porphyrin to G-quadruplex structures continues to be controversial, with no consensus model to date, especially for intramolecular G-quadruplexes from human telomeric sequences. Those sequences possess intricate polymorphism in solution that appears to be reduced under molecular crowding conditions in which the parallel structure appears to be the most populated one. We have performed a systematic study, in dilute solution and under molecular crowding conditions, of the binding reactions between TMPyP4 and four G-quadruplexes formed by different truncations of human telomeric DNA, with 5'- or 3'-flanking bases, using isothermal titration calorimetry and circular dichroism. The results clearly indicate that all of these G-quadruplexes are able to bind up to four TMPyP4 molecules. CD studies show that interaction with TMPyP4 promotes the conversion of the hybrid structures to an antiparallel conformation in dilute solution, while under molecular crowding conditions the interaction does not promote any conformational change. ITC reveals in both cases that the binding process comprises two sequential events, a first in which one molecule of TMPyP4 interacts with the quadruplex structures and a second in which three other molecules bind to the structures. The selectivity of TMPyP4 for the quadruplex relative to duplex DNA was also investigated under molecular crowding conditions showing that TMPyP4 has enhanced selectivity for quadruplex DNA compared to the duplex structure. This finding reinforces the potential applications of TMPyP4.