Abstract
The interactions of three related cationic porphyrins, TMPyP4, TMPyP3 and TMPyP2, with a WT 39-mer Bcl-2 promoter sequence G-quadruplex were studied using Circular Dichroism, ESI mass spectrometry, Isothermal Titration Calorimetry, and Fluorescence spectroscopy. The planar cationic porphyrin TMPyP4 (5, 10, 15, 20-meso-tetra (N-methyl-4-pyridyl) porphine) is shown to bind to a WT Bcl-2 G-quadruplex via two different binding modes, an end binding mode and a weaker mode attributed to intercalation. The related non-planar ligands, TMPyP3 and TMPyP2, are shown to bind to the Bcl-2 G-quadruplex by a single mode. ESI mass spectrometry experiments confirmed that the saturation stoichiometry is 4:1 for the TMPyP4 complex and 2:1 for the TMPyP2 and TMPyP3 complexes. ITC experiments determined that the equilibrium constant for formation of the (TMPyP4)1/DNA complex (K1 = 3.7 × 106) is approximately two orders of magnitude greater than the equilibrium constant for the formation of the (TMPyP2)1/DNA complex, (K1 = 7.0 × 104). Porphyrin fluorescence is consistent with intercalation in the case of the (TMPyP4)3/DNA and (TMPyP4)4/DNA complexes. The non-planar shape of the TMPyP2 and TMPyP3 molecules results in both a reduced affinity for the end binding interaction and the elimination of the intercalation binding mode.
Highlights
The promoter region of the B-cell lymphoma-2 (Bcl-2) gene is guanine and cytosine rich [1,2,3]
We looked at the interaction of three cationic porphyrins (TMPyP2, TMPyP3, and TMPyP4) with a WT 39-mer G-quadruplex forming sequence from the Bcl-2 promoter region
The CD spectra for the 39-mer Bcl-2 promoter sequence quadruplex is unchanged by the addition of saturating amounts of TMPyP2, TMPyP3, or TMPyP4 either in CD band intensity or in CD band wavelength
Summary
The promoter region of the B-cell lymphoma-2 (Bcl-2) gene is guanine and cytosine rich [1,2,3]. A 39 base pair region, located from 19 to 58 base pairs upstream of the Bcl-2 P1 promoter, plays a crucial role in the regulation of Bcl-2 transcription [5]. Dai et al used NMR and CD methods to demonstrate that the Bcl-2 39mer purine rich strand folds into multiple intramolecular Gquadruplex structures [6]. G-quadruplex structures are thought to be generally involved gene regulation, with G-rich sequences found upstream from as many as 40% of all human genes [7]. The exact nature of the interactions between cationic porphyrins and G-quadruplex DNA depends on the folding topology and base sequence of the G-quadruplex and on the molecular structure of the porphyrin [16]
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