Studies on the site and mode of TMPyP4 interactions with Bcl-2 promoter sequence G-quadruplexes

Narayana Nagesh, Robert Buscaglia, Jamie M. Dettler, Edwin A. Lewis

Research output: Contribution to journalArticlepeer-review

51 Scopus citations


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 sevenbase 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 ≈ 1 × 107 M-1), whereas binding of the third and fourth moles of TMPyP4 appears to occur by a weaker, intercalative binding mode (K ≈ 1 × 105 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.

Original languageEnglish (US)
Pages (from-to)2628-2633
Number of pages6
JournalBiophysical Journal
Issue number11
StatePublished - Jun 2 2010
Externally publishedYes

ASJC Scopus subject areas

  • Biophysics


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