Development of a Series of Heteroaromatic Oligoamides to Target DNA Hairpin Structures

Details

Supervisors

Singleton, Michael L.

Faculty

Faculté des sciences

Degree label

Master [120] en sciences chimiques, à finalité approfondie

Abstract

enDynamic mutations arise from the expansion of naturally occurring polymorphic DNA repeat sequences, leading to fragile chromosomal sites and more than 40 neurological disorders, including Huntington’s disease and several forms of ataxia. A key feature underlying repeat expansion is the formation of an unrepaired hairpin slip-out. Large trinucleotide repeats (TNRs) generate highly dynamic hairpins able to evade recognition by the mismatch repair machinery, therefore promoting further genetic expansion. Consequently, aberrant proteins or toxic RNA species capable of sequestering essential cellular factors are generated, leading to progressive neurological and muscular symptoms. These mutations are called “dynamic” because the disease severity increases over time along with earlier age of onset across generations. In 2020, Nakamori et al. were able to induce repeats contraction in a CAG repeats DNA hairpin by designing a structure-specific ligand capable of stabilizing the dynamic structure. To do so, they targeted base pair mismatches inherent to the repetitive sequence. An alternative strategy would involve the design of sequence-specific ligands capable of simultaneously targeting the repeated sequence as well as characteristic geometric features of the hairpin. In this context, distamycin represents an attractive scaffold due to its well-established minor groove binding affinity and the sequence-recognition principles established by Dervan et al. for synthetic pyrrole-imidazole (Py-Im) oligoamides. This project therefore aims to evaluate distamycin derivatives as multivalent ligands targeting nucleic acid hairpin structures involved in repeat expansion disorders. A model hairpin (PDB ID: 2L5K) containing three targetable motifs (specific sequence, flipped unpaired bases, and base-pair mismatches) was selected. A combination of DFT calculations and molecular docking studies was implemented to design a series of ligand candidates. Structurally, these multivalent ligands consist of a minor groove-binding oligoamide trimer (anchor) linked to an unpaired-base targeting moiety. The minor groove anchor contained permutable heterocycles to investigate the influence of heterocycle identity on binding affinity. To target the unpaired thymine residue protruding from the hairpin loop, a modified 2,6-diaminopyridine (DAP) unit was introduced based on its ability to form complementary hydrogen-bonds or π-stacking interactions depending on linker length. A free amine was additionally incorporated to improve solubility and promote favorable DNA interactions. Among five initial designs, two ligands were successfully synthesized using solution-based synthesis (L1 and L2). Preliminary assessment of the ligands ability to stabilize DNA, performed by thermal shift assays (TSA), revealed significant differences in melting temperature for the oligoamides synthesized. The results obtained suggest the presence of a unique and saturable binding site on the model hairpin, with improved stabilization observed for the shorter linker variant (L1), consistent with docking predictions. Further investigation of the precise binding mode will nevertheless be needed to validate these observations. Overall, this work provides a potential methodological basis for the design of multivalent ligands targeting nucleic acid hairpin structures involved in repeat expansion disorders.