Je tiens à exprimer mes sincères remerciements à :Monsieur le Professeur Éric Deutsch, de m'avoir fait l'honneur de présider cette thèse. Monsieur le Professeur Ahmed Idbaih et Monsieur le Professeur Keith Ligon, d'avoir pris le temps de diriger et encadrer cette thèse. Monsieur le Docteur Franck Bourdeaut, Madame la Professeure Magali Svrcek, et Monsieur le Professeur Alex Duval, d'avoir pris le temps de juger ce travail. Monsieur le Docteur Franck Bielle, et Monsieur le Professeur Marc Sanson, pour leur participation à ces travaux. Une partie importante de ces travaux a été réalisée au Dana-Farber Cancer Institute et je tiens à remercier ici très sincèrement mes collègues de Boston pour leur amitié et leurs efforts déterminants dans l'obtention de ces résultats, en particulier Keith Ligon pour son accueil au sein de son laboratoire, ses conseils et ses encouragements.
[Ru(Me4phen)2dppz]2+ serves as a luminescent “light switch” for single base mismatches in DNA. The preferential luminescence enhancement observed with mismatches results from two factors: (i) the complex possesses a 26-fold higher binding affinity towards the mismatch compared to well-matched base pairs, and (ii) the excited state emission lifetime of the ruthenium bound to the DNA mismatch is 160 ns versus 35 ns when bound to a matched site. Results indicate that the complex binds to the mismatch through a metalloinsertion binding mode. Cu(phen)22+ quenching experiments show that the complex binds to the mismatch from the minor groove, characteristic of metalloinsertion. Additionally, the luminescence intensity of the complex with DNA containing single base mismatches correlates with the thermodynamic destabilization of the mismatch, also consistent with binding through metalloinsertion. This complex represents a potentially new early cancer diagnostic for detecting deficiencies in mismatch repair.
We report a bifunctional fluorescent probe that combines a rhodium metalloinsertor with a cyanine dye as the fluorescent reporter. The conjugate shows weak luminescence when free in solution or with well matched DNA but exhibits a significant luminescence increase in the presence of a 27-mer DNA duplex containing a central CC mismatch. DNA photocleavage experiments demonstrate that, upon photoactivation, the conjugate cleaves the DNA backbone specifically near the mismatch site on a 27-mer fragment, consistent with mismatch targeting. Fluorescence titrations with the 27-mer duplex containing the CC mismatch reveal a DNA binding affinity of 3.1 × 106 M−1, similar to that of other rhodium metalloinsertors. Fluorescence titrations using genomic DNA extracted from various cell lines demonstrate a clear discrimination in fluorescence between those cell lines that are proficient or deficient in mismatch repair. This differential luminescence reflects the sensitive detection of the mismatch repair-deficient phenotype.
DNA base pair mismatches occur naturally in cells as a result of incorporation errors and damage. Most cells are able to identify and correct these mistakes before replication, allowing for high genome fidelity between cellular generations. In some forms of cancer, however, proteins involved in the machinery of mismatch repair (MMR) undergo mutation, making those cells unable to correct mismatches and leading to an increase in mutations. Since higher mismatch frequency serves as an early indicator of cancer progression, for many researchers mismatches have provided a novel target for the design of organic and inorganic small-molecule therapeutics. In particular, transition metal complexes have shown great promise in this context owing to their valuable spectroscopic and photophysical properties and flexibility with respect to modification of their coordination spheres. Thus far, experimental designs have ranged from targeting the thermodynamic destabilization of mismatched sites to the hydrogen-bonding pattern of specific mismatched base pairs. Here, we review the diversity, practical application, and evolution of mismatch-targeting small molecules, with an emphasis on rhodium metalloinsertors and luminescent ruthenium compounds. Importantly, we highlight the discovery of metalloinsertion, a noncovalent DNA binding mode that is specific towards destabilized sites, such as mismatches, within the DNA duplex.
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