The binding mode and stoichiometry of the cationic porphyrin TMPyP4 to G-quadruplex structure are still controversial to date, mainly due to the intricate polymorphism of G-rich sequences in the different conditions of solution. Here in the presence of the molecular crowding agent PEG, the binding interaction of TMPyP4 and another porphyrin derivative TPrPyP4 with four-stranded parallel (G4T4G4)4 G-quadruplex was studied systematically using circular dichroism, visible absorption titration, and steady-state and timeresolved fluorescence spectroscopies. The results show that each (G4T4G4)4 molecule is able to bind four TMPyP4 or TPrPyP4 molecules. Two types of independent and nonequivalent binding sites with the higher and lower binding affinity are confirmed, and the stronger and weaker binding constants are 2.74×108 and 8.21×105 M−1 for (G4T4G4)4-TMPyP4, 2.05×108 and 1.05×106 M−1 for (G4T4G4)4-TPrPyP4, respectively. The two porphyrin molecules stack on the two ends of G-quadruplex with the higher binding affinity, another two porphyrins bind weakly to the two external grooves.

Understanding the nature of the interaction between small molecules and G-quadruplex DNA is crucial for the development of novel anticancer drugs. In this paper, we present the first data on time-resolved fluorescence anisotropy study on the interaction between a water-soluble cationic porphyrin H2TMPyP4 and four distinct G-quadruplex DNAs, that is, AG3(T2AG3)3, thrombin-binding aptamer (TBA), (G4T4G4)2, and (TG4T)4. The anisotropy decay curves show the monoexponential for free H2TMPyP4 and the biexponential upon binding to the excess amount of G-quadruplex DNAs. The biexponential anisotropy decay can be well interpreted using a wobbling-in-the-cone model. The orientational diffusion of the bound H2TMPyP4 is initially restricted to a limited cone angle within the G-quadruplex DNAs, and then an overall orientational relaxation of the G-quadruplex DNA-H2TMPyP4 complexes occurs in a longer time scale. It was found that the dynamics of the restricted internal rotation of bound H2TMPyP4 strongly depends on the ending structures of the G-quadruplex DNAs. According to the order parameter (Q) calculated from the wobbling-in-the-cone model, we deduce that the degree of restriction around the bound H2TMPyP4 follows the order of TBA>(TG4T)4>AG3(T2AG3)3>(G4T4G4)2. Especially, based on the maximum order parameter (Q) of bound H2TMPyP4 within TBA, a new sandwich-type binding mode for TBA-H2TMPyP4 complex was proposed in which both terminal G-quartet and T•T base pair stack on the porphyrin ring through π-π interaction. This study thus provides a new insight into the interaction between G-quadruplex DNAs and H2TMPyP4.

The structural interconversion between the G-quadruplex and duplex in vivo is an important subject. In the present study, we used human telomeric DNA duplex composed of GGG(TTAGGG)3/CCC(TAACCC)3 as a model system to investigate its properties under near physiological conditions by spectroscopic methods. Circular dichroism and fluorescence spectra demonstrated that G-quadruplex structure can be formed from duplex at near physiological pH (pH 7.4), salt concentration (150 mM Kþ), and temperature (37℃) in the presence of molecular crowding agent PEG (400 g/l), whereas the G-quadruplex structure cannot be formed at 25 C in buffer containing 150 mM Kþ in the presence of PEG. It is found that the formation rate of G-quadruplex structure depends on the temperature and the concentrations of both PEG and Kþ. This work suggests that human telomeric G-quadruplex structure may be potentially formed from Watson–Crick duplex in vivo.

Interactions of 5,10,15,20-Tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphyrin (TMPyP4) and 5,10,15,20-Tetrakis(N-propylpyridinium-4-yl)-21H, 23H-porphyrin (TPrPyP4) with the parallel four-stranded (TG4T)4 G-quadruplex DNA in 100 mM K+-containing buffer were studied using circular dichroism (CD) spectroscopy, visible absorption titration, and steady and time-resolved fluorescence spectroscopies. The results show that the binding stoichiometric ratios of both TMPyP4 and TPrPyP4 to (TG4T)4 are 3:1. Two types of independent and nonequivalent binding sites with the higher and lower binding affinities are confirmed, and the stronger and weaker binding constants are 9.44×107 and 6.94×105 M−1 for (TG4T)4-TMPyP4 complex, 7.86×107 and 6.35×105M−1 for (TG4T)4-TPrPyP4 complex, respectively. For both TMPyP4-(TG4T)4 and TPrPyP4–(TG4T)4 complexes, one porphyrin molecule stacks on the one end of G-quadruplex with the higher binding affinity, another two porphyrins bind weakly to the two external grooves. The size of cation side arms around porphyrin core almost fails to affect the binding mode, stoichiometry and affinity of porphyrin to (TG4T)4 G-quadruplex in 100 mM K+-containing buffer.

Structures of G-quadruplex DNAs can be typically stabilized by monovalent cations such as K+, Na+. Some divalent and trivalent cations, such as Sr2+, Pb2+, Tb3+ and Eu3+, can also induce the formation of G-quadruplex DNA. Here we show that Zn2+ can induce the human telomeric sequence AG3(T2AG3)3 to fold the G-quadruplex structure by UV absorbance difference spectra and circular dichroism (CD) spectroscopy. At micromolar concentrations, the Zn2+-induced changes in the UV absorbance difference spectra and CD spectra are the characteristics of antiparallel G-quadruplexes although the long wavelength CD maximum is around 285 nm rather than the typical value of 295 nm. The binding stoichometry of Zn2+ per one AG3(T2AG3)3 molecule is four. To our knowledge, the structural transition of human telomeric sequence induced by Zn2+ was observed for the first time.