Studies on the Site and Mode of TMPyP4 Interactions with Bcl-2 Promoter Sequence G-Quadruplexes

Abstract

TMPyP4 (Mesotetra(N-methyl-4-pyridyl)porphine) is known to have a high affinity for G-quadruplex DNA. However, there is still some controversy over the exact site(s) and mode(s) of TMPyP4 binding to G-quadruplex DNA. We examined TMPyP4 interactions with seven G-quadruplex forming oligonucleotides. The parent oligonucleotide is a 27-mer with a wild-type (WT) G-rich sequence of the Bcl-2 P1 promoter mid-region (5'-d(CGG GCG CGG GAG GAA GGG GGC GGG AGC-3')). This sequence folds into at least three unique loop isomer quadruplexes. The two mutant oligonucleotides used in this study are shorter (23-mer) sequences in which nonquadruplex core bases were eliminated and two different (-G-G-) --> (-T-T-) substitutions were made to restrict the folding complexity. The four additional mutant oligonucleotides were labeled by substituting a 2-aminopurine (2-AP) base for an A or G in either the first three-base lateral loop or the second five- or seven-base lateral loop (depending on the G-->T mutation positions). Spectroscopic and microcalorimetric studies indicate that four molecules of TMPyP4 can be bound to a single G-quadruplex. Binding of the first two moles of TMPyP4 appears to occur by an end or exterior mode (K approximately 1 x 10(7) M(-1)), whereas binding of the third and fourth moles of TMPyP4 appears to occur by a weaker, intercalative binding mode (K approximately 1 x 10(5) M(-1)). As the mid-loop size decreases from seven to five bases, end binding occurs with significantly increased affinity. 2-AP-labeled Bcl-2 promoter region quadruplexes show increased fluorescence of the 2-AP base on addition of TMPyP4. The change in fluorescence for 2-AP bases in the second half of the TMPyP4 titration lends support to our previous speculation regarding the intercalative nature of the weaker binding mode.

Interaction of Pyrrolobenzodiazepine (PBD) Ligands with Parallel Intermolecular G-Quadruplex Complex Using Spectroscopy and ESI-MS

“…Among the porphyrins, TMPyP4 was known to have better interaction with G-quadruplex DNA [19], [20]. From circular dichroism experiments, it was evident that the interaction of the PBD ligands with G-quadruplex DNA was in the order; PBD2>PBD1.…”

Phenyl 1,2,3-Triazole-Thymidine Ligands Stabilize G-Quadruplex DNA, Inhibit DNA Synthesis and Potentially Reduce Tumor Cell Proliferation over 3′-Azido Deoxythymidine

“…The mode-1 interaction occurs with higher binding constant (K d ≈10 −7 M) and with more favourable entropy change. The low affinity interaction (mode-2) is due to either π-π stacking interaction of nucleoside triazole ligands with guanines of quadruplex DNA or due to binding of ligands to adenosine bases in the loop (end loop binding with binding constant in the range of K d ≈10 −5 M), which occurs with more favourable enthalpy change [57].…”

Bcl-2 Promoter Sequence G-Quadruplex Interactions with Three Planar and Non-Planar Cationic Porphyrins: TMPyP4, TMPyP3, and TMPyP2

“…The nonlinear regression fitting was done using CHASM data analysis program developed in our laboratory [24]. The ITC fitting procedures used here have been described previously [17,19,25–27]. …”

“…The Lewis group reported that the binding stoichiometry of the cationic porphyrin (5, 10, 15, 20-meso-tetra (N-methyl- 4-pyridyl) porphyrin), TMPyP4, to the c-MYC and Bcl-2 promoter region G-quadruplexes is 4:1 at saturation [17–19]. Their microcalorimetric and spectroscopic results were consistent with two TMPyP4 binding modes for both the c-MYC and Bcl-2 promoter quadruplexes; external or end binding and intercalation [17–19].…”

“…The Lewis group reported that the binding stoichiometry of the cationic porphyrin (5, 10, 15, 20-meso-tetra (N-methyl- 4-pyridyl) porphyrin), TMPyP4, to the c-MYC and Bcl-2 promoter region G-quadruplexes is 4:1 at saturation [17–19]. Their microcalorimetric and spectroscopic results were consistent with two TMPyP4 binding modes for both the c-MYC and Bcl-2 promoter quadruplexes; external or end binding and intercalation [17–19]. Wei et al has similarly suggested that TMPyP4 binds to G-quadruplex DNA by a combination of binding modes that include external (end) stacking within the loop region and intercalation between G-tetrads with complex binding ratios of both 2:1 and 4:1 between TMPyP4 and G-quadruplex DNA [20].…”