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

Abstract

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 oneresidue 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 oneresidue stagger was observed with TIP-C7-amino. Selectivity of the leading thiazole was modulated by the head group, with Nterminal 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.

Indirect readout in drug-DNA recognition: role of sequence-dependent DNA conformation

“…Polyamides containing imidazole and pyrrole carboxamides are the synthetic counterpart with promising anticancer properties ( 34 ) of the natural netropsin and distamycin A, two antiviral drugs. The left panels of Figure 3 show the Z -score distribution of a DNA structure with sequence 5′-CCAGTACTGG-3′ bound by imidazole-pyrrole-hydroxypyrrole (Im/Py/Hp) polyamide ( top ), and the Z -score distribution of the corresponding free B-DNA crystal structure with minimum Z -scores ( bottom ).…”

Polyamide-Scorpion Cyclam Lexitropsins Selectively Bind AT-Rich DNA Independently of the Nature of the Coordinated Metal

“…It is thought that the formamide affects the way the molecule stacks as a dimer in the minor groove,[89] but poly-Py lexitropsins can bind as monomers [42]. The effect of removing the N -formyl group also varies with lexitropsin structure, and the effects are different for hairpin- and cross-linked lexitropsins [103]. As might be expected from these observations, the binding affinities observed for the novel lexitropsin conjugates in the present work imply that the removal of the terminal N -formamido is not prohibitive for binding.…”

Sequence‐Unrestricted, Watson–Crick Recognition of Double Helical B‐DNA by (R)‐MiniPEG‐γPNAs

“…[2] It was long recognized that while Watson-Crick (W-C) base-pairing provides a more direct and specific means for establishing sequence-specific interactions with nucleic acid biopolymers, such as DNA and RNA, it would be difficult to do so with intact double helical DNA because of the preexisting base pairs. [4] This effort has so far led to the development of several major classes of antigene molecules, with the likes of triplexforming oligonucleotides, [5][6][7] minor-groove binding polyamides, [8][9][10][11] and major-groove binding zinc-finger peptides. [12][13][14][15][16] While they can be designed to bind sequence specifically to dsDNA, there are still remaining issues with sequence selection, specificity and/or target length that have not yet been completely resolved, [8,13,[17][18][19] although some progress has been made in recent years.…”