Biophysical Characterization of the Human Telomeric (TTAGGG)4 Repeat in a Potassium Solution
Biochemistry, 46(15), 4654- 4660, May 2007
Cosimo Antonacci Department of Chemistry and Biochemistry
University of Louisville and Richard D. Sheardy
Quadruplex structures arise from the arrangement of four coplanar guanine bases in a Hoogsteen base pairing motif to create a central pore coordinated with monovalent cations. The termini of eukaryotic chromosomes contain structures, known as telomeres, which are capable of forming quadruplex structures. Quadruplexes have been implicated in a variety of disease states including cancer. The human telomeric repeat containing four stretches of three guanines display different conformational states in the presence of Na+ and K+ and an unknown number of species involved in the quadruplex to single strand transition. Through employment of circular dichroism spectroscopy, differential scanning calorimetry and singular value decomposition, the number of species present in the dissociation process is assessed. The results indicate three species in equilibrium during the melting process. A model for the heat induced denaturation proposes that the hybrid parallel-antiparallel quadruplex undergoes a transition to a single strand through an unknown folding intermediate.
The small molecule stabilization of telomeric sequences has been realized as a possible approach to the treatment of cancer. H2TMPyP has been observed to bind human telomeric repeat sequences with a stoichiometry of two per quadruplex. However, the literature seems to deviate in terms of affinity and mode. Spectroscopic (107 M-1) and calorimetric (104 M-1) methods utilized to assess affinity seem to produce binding constants differing on the order of 10 3 M-1. Some feel the porphyrin intercalates between the tetrads, while others purport external stacking of terminal tetrads. This work indicates a high affinity binding mode (106-10 8 M-1) of H2TMPyP to (TTAGGG)4 in various potassium rich solutions. Consistent with Scatchard analysis, the two binding sites are characterized as identical and non-intreracting binding sites. CD titrations, visible titrations, visible melts and 31P NMR results are consistent with end-stacked binding sites. In addition, the heat induced interconversion of conformational forms in the presence of porphyrin, as noted by CD melting, suggests the presence of a parallel stranded quadruplex intermediate.