title: Sequence recognition in the minor groove of DNA by covalently linked formamido imidazole-pyrrole-imidazole polyamides: effect of H-pin linkage and linker length on selectivity and affinity.
abstract: The polyamide N-formamido imidazole-pyrrole-imidazole (f-ImPyIm) binds with an exceptionally high affinity for its cognate site 5'-ACGCGT-3' as a stacked, staggered, and noncovalent cooperative dimer. Investigations are presented into its sequence specificity and binding affinity when linked covalently as an H-pin "dimer". Five f-ImPyIm cross-linked analogues with six to nine methylene linkers and an eight-linked ethylene glycol linker were examined to investigate the effect of linkage and linker length on DNA binding. Thermal denaturation studies on short DNA hairpins showed preferential binding by both f-ImPyIm (DeltaTm = 7.8 degrees C) and its cross-linked derivatives (DeltaTm > 30 degrees C) at 5'-ACGCGT-3', indicating sequence specificity was retained on linkage. DNase I footprinting confirmed strict cognate site selectivity and demonstrated that affinity increased with linker length (f-ImPyIm-9 = f-ImPyIm-8 = f-ImPyIm-EG-8 > f-ImPyIm-7 > f-ImPyIm-6). The eight- and nine-linked derivatives bound at 100-fold lower concentrations at the cognate site relative to f-ImPyIm-6, and with 10-fold higher affinity than unlinked f-ImPyIm. Use of an ethylene glycol linkage in f-ImPyIm-EG-8 to improve solubility slightly increased the cognate site affinity relative to those of f-ImPyIm-8 and f-ImPyIm-9, although some selectivity was lost at high ligand concentration. CD demonstrated that cognate site binding by eight and nine-linked compounds occurred in the minor groove. SPR analysis gave a binding affinity (K) for f-ImPyIm-EG-8 at the cognate site of 2 x 10(10) M-1, representing a 100-fold increase relative to that of f-ImPyIm. This study demonstrates that the high-affinity cooperative binding of f-ImPyIm can be enhanced significantly by suitable covalent linkage, while maintaining its strict cognate site selectivity.
authors.affiliation: Cancer Research UK Drug-DNA Interactions Research Group, Department of Oncology, University College London, 91 Riding House Street, London W1W 7BS, United Kingdom.
authors.forename: C Caroline
authors.initials: CC
authors.lastname: O'Hare
authors.forename: Peter
authors.initials: P
authors.lastname: Uthe
authors.forename: Hilary
authors.initials: H
authors.lastname: Mackay
authors.forename: Kevin
authors.initials: K
authors.lastname: Blackmon
authors.forename: Justin
authors.initials: J
authors.lastname: Jones
authors.forename: Toni
authors.initials: T
authors.lastname: Brown
authors.forename: Binh
authors.initials: B
authors.lastname: Nguyen
authors.forename: W David
authors.initials: WD
authors.lastname: Wilson
authors.forename: Moses
authors.initials: M
authors.lastname: Lee
authors.forename: John A
authors.initials: JA
authors.lastname: Hartley
mesh_terms: D000998:Antiviral Agents / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry*; D018983:DNA Footprinting; D003850:Deoxyribonuclease I / Q000494:pharmacology; D019281:Dimerization; D004214:Distamycins / Q000737:chemistry; D007093:Imidazoles / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D011758:Pyrroles / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D012680:Sensitivity and Specificity; D020349:Surface Plasmon Resonance; D013696:Temperature; D017186:Titrimetry
chemical_list: D000998:Antiviral Agents; D004214:Distamycins; D007093:Imidazoles; D011758:Pyrroles; C030000:stallimycin; D004247:DNA; D003850:Deoxyribonuclease I