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
| Autor: | Hilary Mackay, Peter B. Uthe, John A Hartley, Toni Brown, Moses Lee, W. David Wilson, Justin B. Jones, Caroline O'Hare, Binh Nguyen, Kevin N. Blackmon |
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| Rok vydání: | 2007 |
| Předmět: |
Stereochemistry
Dimer Molecular Sequence Data DNA Footprinting DNA footprinting Nucleic Acid Denaturation Antiviral Agents Sensitivity and Specificity Biochemistry chemistry.chemical_compound Deoxyribonuclease I Organic chemistry Pyrroles Binding site Binding Sites Base Sequence Chemistry Circular Dichroism Distamycins DNase-I Footprinting Imidazoles Temperature Titrimetry Cooperative binding DNA Surface Plasmon Resonance Ligand (biochemistry) Kinetics Models Chemical Nucleic Acid Conformation Dimerization Linker |
| Zdroj: | Biochemistry. 46:11661-11670 |
| ISSN: | 1520-4995 0006-2960 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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