Structural and Kinetic Profiling of Allosteric Modulation of Duplex DNA Induced by DNA-Binding Polyamide Analogues.

Khalid Aman,Glenn A Burley,Thomas Welte,John A Parkinson,Giacomo Padroni

doi:10.1002/chem.201805338

Khalid Aman, Glenn A Burley + Show 3 more

Open Access

https://doi.org/10.1002/chem.201805338

Copy DOI

Abstract

A combined structural and quantitative biophysical profile of the DNA binding affinity, kinetics and sequence‐selectivity of hairpin polyamide analogues is described. DNA duplexes containing either target polyamide binding sites or mismatch sequences are immobilized on a microelectrode surface. Quantitation of the DNA binding profile of polyamides containing N‐terminal 1‐alkylimidazole (Im) units exhibit picomolar binding affinities for their target sequences, whereas 5‐alkylthiazole (Nt) units are an order of magnitude lower (low nanomolar). Comparative NMR structural analyses of the polyamide series shows that the steric bulk distal to the DNA‐binding face of the hairpin iPr‐Nt polyamide plays an influential role in the allosteric modulation of the overall DNA duplex structure. This combined kinetic and structural study provides a foundation to develop next‐generation hairpin designs where the DNA‐binding profile of polyamides is reconciled with their physicochemical properties.

Highlights

DNA-binding polyamides (PAs) are cell-permeable transcriptional modulators which function by inhibiting RNA polymerase-mediated elongation and/or transcription factor binding to its target double-stranded DNA consensus sequence.[1]
We report a label-free biophysical assay to profile the affinity, sequence-selectivity and binding kinetics of PA·double-stranded DNA (dsDNA) interactions where the N-terminal heterocycle is systematically altered (PA1–4)
DNA duplexes (ODN1–3, Table 1) were immobilized on a gold surface and contained a fluorophore reporter positioned in close proximity to the proposed PA binding site

Summary

Introduction

DNA-binding polyamides (PAs) are cell-permeable transcriptional modulators which function by inhibiting RNA polymerase-mediated elongation and/or transcription factor binding to its target double-stranded DNA (dsDNA) consensus sequence.[1]. We have recently expanded the heterocyclic repertoire of current Py-Im hairpin PA designs to include N-terminal thia-. [+] These authors contributed to this work.

Results

Discussion

Conclusion