Shedding Light on the Interaction between TMPyP4 and Human Telomeric Quadruplexes

Abstract

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.

Spectroscopic study on the binding of porphyrins to (G4T4G4)4 parallel G-quadruplex

“…In our previous study on interactions of the d[AG 3 (T 2 AG 3 ) 3 ] sequence with TMPyP4 and TPrPyP4 (5,10,15,20-Tetrakis(N-propylpyridinium-4-yl)-21H,23H-porphyrin) in both dilute solution and crowding condition [23], the different binding mode was observed in the presence and absence of the crowding agent PEG 200, and there are different binding behaviors for TMPyP4 and TPrPyP4 molecules. Also a distinct binding behavior of TMPyP4 to the d[AG 3 (T 2 AG 3 ) 3 ] sequence was also reported recently in both dilute solution and crowding conditions [25]. It is therefore apparent that the binding mode of porphyrins to quadruplex molecules depends on the chemical features of the porphyrin, on the structure and composition of the target DNA, and on the solution conditions in the experiment.…”

“…Thus the stoichiometry of both porphyrins for the first and second binding site is 2. Interestingly, a low-affinity binding mode was also detected at stoichiometry greater than 1 for human telomeric AG 3 (T 2 AG 3 ) 3 or (TG 4 T)4 [13,18,25,26].…”

The Determination of Quadruplex Structures

“…Although there are very little comparative data available as yet, it does appear that quadruplex-ligand complexes in solution in molecular crowding conditions also follow this pattern. An example is the porphyrin TMPyP4 (Martino et al, 2009), whose telomeric quadruplex complex favors the more closely packed parallel topology, in accord with crystal-structure data (Parkinson, Ghosh & Neidle, 2007), albeit on a bimolecular TMPyP4-quadruplex complex. Thus it is tempting to conclude that crystal structures of human telomeric quadruplexes and their ligand complexes do reflect solution structures (and maybe even those in their biological environment), but that the solution condition needs also to be crowded or concentrated.…”

TMPyP4 Porphyrin Distorts RNA G-quadruplex Structures of the Disease-associated r(GGGGCC)n Repeat of the C9orf72 Gene and Blocks Interaction of RNA-binding Proteins

“…1, F-H). An induction of a 290 nm shoulder in a G-quadruplex detected by CD spectra in the presence of TMPyP4 has previously been reported as the appearance of antiparallel conformations in another G-rich sequence (42). Thus, in the presence of increasing concentrations of TMPyP4, the r(GGGGCC)n RNA undergoes a conformational change.…”