title: Promoter scanning of the human COX-2 gene with 8-ring polyamides: unexpected weakening of polyamide-DNA binding and selectivity by replacing an internal N-Me-pyrrole with β-alanine.
abstract: Rules for polyamide-DNA recognition have proved invaluable for the design of sequence-selective DNA binding agents in cell-free systems. However, these rules are not fully transferrable to predicting activity in cells, tissues or animals, and additional refinements to our understanding of DNA recognition would help biomedical studies. Similar complexities are encountered when using internal β-alanines as polyamide building blocks in place of N-methylpyrrole; β-alanines were introduced in polyamide designs to maintain good hydrogen bonding registry with the target DNA, especially for long polyamides or those with several GC bp (P.B. Dervan, A.R. Urbach, Essays Contemp. Chem. (2001) 327-339). Thus, to clarify important subtleties of molecular recognition, we studied the effects of replacing a single pyrrole with β-alanine in 8-ring polyamides designed against the Ets-1 transcription factor. Replacement of a single internal N-methylpyrrole with β-alanine to generate a β/Im pairing in two 8-ring polyamides causes a decrease in DNA binding affinity by two orders of magnitude and decreases DNA binding selectivity, contrary to expectations based on the literature. Measurements were made by fluorescence spectroscopy, quantitative DNA footprinting and surface plasmon resonance, with these vastly different techniques showing excellent agreement. Furthermore, results were validated for a range of DNA substrates from small hairpins to long dsDNA sequences. Docking studies helped show that β-alanine does not make efficient hydrophobic contacts with the rest of the polyamide or nearby DNA, in contrast to pyrrole. These results help refine design principles and expectations for polyamide-DNA recognition.
authors.affiliation: Department of Chemistry & Biochemistry, Center for Nanoscience, University of Missouri-St. Louis, One University Blvd., St. Louis, MO 63121, USA. bashkinj@umsl.edu
authors.forename: James K
authors.initials: JK
authors.lastname: Bashkin
authors.forename: Karl
authors.initials: K
authors.lastname: Aston
authors.forename: Joseph P
authors.initials: JP
authors.lastname: Ramos
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Koeller
authors.forename: Rupesh
authors.initials: R
authors.lastname: Nanjunda
authors.forename: Gaofei
authors.initials: G
authors.lastname: He
authors.forename: Cynthia M
authors.initials: CM
authors.lastname: Dupureur
authors.forename: W
authors.initials: W
authors.lastname: David Wilson
mesh_terms: D001483:Base Sequence; D051546:Cyclooxygenase 2 / Q000737:chemistry* / Q000235:genetics; D004247:DNA / Q000737:chemistry*; D018983:DNA Footprinting; D052162:Human papillomavirus 16 / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D055029:Inverted Repeat Sequences / Q000235:genetics; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D009757:Nylons / Q000138:chemical synthesis / Q000737:chemistry*; D011401:Promoter Regions, Genetic; D050784:Proto-Oncogene Protein c-ets-1 / Q000737:chemistry* / Q000235:genetics; D011758:Pyrroles / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance; D015091:beta-Alanine / Q000737:chemistry*
chemical_list: C494041:ETS1 protein, human; D009757:Nylons; D050784:Proto-Oncogene Protein c-ets-1; D011758:Pyrroles; D015091:beta-Alanine; D004247:DNA; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human