Studies on the site and mode of TMPyP4 interactions with Bcl-2 promoter sequence G-Quadruplexes.
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.
Nagesh
Narayana
N
Centre for Cellular and Molecular Biology, Hyderabad, India. nagesh@ccmb.res.in
Buscaglia
Robert
R
Dettler
Jamie M
JM
Lewis
Edwin A
EA
D015075:2-Aminopurine; D000042:Absorption; D001483:Base Sequence; D002151:Calorimetry; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D005453:Fluorescence; D054856:G-Quadruplexes; D020125:Mutation, Missense; D017711:Nonlinear Dynamics; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics; D011166:Porphyrins / Q000737:chemistry*; D011401:Promoter Regions, Genetic; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry* / Q000235:genetics*; D012044:Regression Analysis; D013057:Spectrum Analysis; D013696:Temperature; D013816:Thermodynamics; D014466:Ultraviolet Rays
D009841:Oligonucleotides; D011166:Porphyrins; D019253:Proto-Oncogene Proteins c-bcl-2; C021096:tetra(4-N-methylpyridyl)porphine; D015075:2-Aminopurine