Folding and binding landscapes of a parallel G-quadruplex
Circular dichroism and stopped-flow UV spectroscopies were used to investigate the thermodynamic stability and the folding pathway of d[TGAG3TG3TAG3TG3TA2] at 25 °C in solutions containing 25 mM KCl. Under these conditions the oligonucleotide folds into a thermally stable, all-parallel G-quadruplex topography containing three stacked quartets. K+-induced folding shows three resolved relaxation times, each showing distinctive spectral changes. Folding is complete within 200 s. These data indicate a folding pathway that involves at least two populated intermediates, one of which seems to be an antiparallel structure that rearranges to the final all-parallel conformation. Molecular dynamics reveals a topologically plausible folding pathway that involves conversion of an antiparallel intermediate to the final parallel form. The rate of unfolding was determined using complementary DNA assay to trap transiently unfolded states to form a stable duplex. As assessed by 1D-1H NMR and fluorescence spectroscopy, unfolding is extremely slow with only one observable rate-limiting relaxation time. The binding of the porphyrin N-Methyl Mesoporphyrin IX (NMM) to the folded G4 structure was studied by a variety of biophysical approaches. Binding is driven by a favorable enthalpy contribution. Stopped-flow kinetic studies revealed two kinetic steps, most consistent with a mechanism in which a bimolecular binding step is followed by a unimolecular rearrangement of the NMM-G4 complex. Supported by: NIH Grants GM077422 & CA35635, NIH/NCRR COBRE P20RR018733 and the James Graham Brown Foundation.