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Thibault Charnay

Specific recognition of double-stranded DNA: Development of luminescent probes

Published on 25 November 2022
Thesis presented November 25, 2022

DNA is the biomolecule that contains genetic information. Many diseases are linked to mutations in DNA that result in dysfunctional gene expression. Over the past twenty years, many efforts have been made in biochemistry to develop new tools to detect these mutations that could be interesting for biological or medical research.
This thesis project is part of this dynamic and aims to develop a luminescent probe for sequence-specific detection of double-stranded DNA, establishing a proof of concept for the recognition of a double-stranded DNA of 12 selected base pairs. It is based on the development of a system composed of two zinc finger proteins capable of binding to the target DNA, each recognising one half of the sequence. For the luminescence detection, these proteins are equipped with a pair of luminophores that allow resonant Förster-like energy transfer (FRET), which is only active when the proteins are bound to the DNA. First, the chemical synthesis of a luminophore-free protein pair was developed using a native chemical ligation assembly and the recognition of the chosen sequence was demonstrated by electrophoretic mobility shift assay (EMSA) experiments. Next, several bioconjugatable lanthanide complexes were synthesised and those with the best photophysical properties were selected to be grafted onto one of the two proteins, acting as the donor for this FRET system. The second protein was functionalized with an organic fluorophore acting as a FRET acceptor. Finally, we studied by luminescence and electrophoresis (EMSA) the interaction of these proteins with DNA of different sequences and obtained a first proof of concept of the sequence-specific detection by luminescence of a double-stranded DNA of 12 base pairs.

DNA, Zinc finger, Lanthanides, Native Chemical ligaiton, FRET, Detection

On-line thesis.