DNA sequence recognition in the minor groove by crosslinked polyamides: The effect of N-terminal head group and linker length on binding affinity and specificity.
Development of sequence-reading polyamides or "lexitropsins" with comparable DNA-binding affinities to cellular proteins raises the possibility of artificially regulated gene expression. Covalent linkage of polyamide ligands, with either a hairpin motif or crosslinking methylene bridge, has greatly improved binding affinity by ensuring their side-by-side register. Whereas hairpin polyamides have been investigated extensively, the optimized structure of crosslinked polyamides remains to be determined. This study examines a series of thiazole-imidazole-pyrrole (TIP) monomers and crosslinked dimers to evaluate the effects on selectivity and binding affinity of different N-terminal head groups attached to the leading thiazole ring and differing methylene linker lengths. Quantitative footprinting of a DNA sequence, containing potential match and mismatch sites for both maximum overlap and one-residue stagger binding modes, allowed measurement of binding constants at each putative site. Within an N-terminal amino TIP series, C7 and C8-linked compounds bound most strongly to these sites, whereas maximum binding affinity was observed for a C6 linker with a formyl head group. A C5 linker gave weak binding with either head group. A hydrogen or acetyl head group abrogated binding. Binding was confirmed by gel shift analyses. The highest specificity for the maximum overlap site over the one-residue stagger was observed with TIP-C7-amino. Selectivity of the leading thiazole was modulated by the head group, with N-terminal formyl TIP exhibiting up to 3-fold specificity for AGT over TGT, suggesting that N-formyl-thiazole may provide sequence discrimination of adenine over thymine. Moreover, the leading head group and methylene linker length significantly influences the binding characteristics of crosslinked polyamides.
O'Hare
C Caroline
CC
Cancer Research Campaign Drug-DNA Interactions Research Group, Department of Oncology, Royal Free and University College Medical School, UCL, 91 Riding House Street, London, W1W 7BS, United Kingdom.
Mack
Darcy
D
Tandon
Manju
M
Sharma
Sanjay K
SK
Lown
J William
JW
Kopka
Mary L
ML
Dickerson
Richard E
RE
Hartley
John A
JA
D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D003850:Deoxyribonuclease I / Q000737:chemistry / Q000378:metabolism; D006838:Hydrocarbons; D006860:Hydrogen Bonding; D007700:Kinetics; D008697:Methane / Q000031:analogs & derivatives* / Q000737:chemistry; D008956:Models, Chemical; D015233:Models, Statistical; D009690:Nucleic Acid Conformation; D009757:Nylons / Q000737:chemistry; D011485:Protein Binding; D012091:Repetitive Sequences, Nucleic Acid
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D006838:Hydrocarbons; D009757:Nylons; C030011:carbene; D004247:DNA; D003850:Deoxyribonuclease I; D008697:Methane