Description of the PhD thesis project
Apurinic/apyrimidinic (AP, or abasic) sites
represent key intermediates in base excision DNA repair (BER), the repair
pathway responsible for the maintenance of genome integrity through removal of
oxidative and alkylated DNA damage. Quantification of the global level and
genomic mapping of AP sites are crucial for understanding the distribution of
different types of DNA damage (oxidized, alkylated or deaminated bases), both
endogenous and induced by anti-cancer drugs.
The available methods for the detection of AP
sites rely on the reaction of the open (aldehyde) form of AP sites with
aldehyde-reactive chemical probes possessing an oxyamine or a hydrazine group;
as a consequence, the reactivity of these probes with 5-formylcytosine and
5-formyluracil, naturally present in the genome, is a serious issue which
requires sophisticated protocols for avoiding cross-reactivity and can lead to
Along these lines, we have previously
developed a family of macrocyclic ligands, based on the bis-naphthalene
(“BisNP”) scaffold, that strongly and selectively bind to AP sites in DNA with
nanomolar affinity and, depending on the substitution pattern, induce DNA
strand cleavage at AP sites, or generate chemically stable covalent ligand–DNA
adducts through a mechanism different from other aldehyde-reactive probes
(Caron et al., Chem. Eur. J. 2019, 25, 1949).
In this project, we will exploit this scaffold
to develop novel chemical tools to evaluate the global level of AP sites in
genomic DNA and to map them in the genome.
Specifically, (i) non-covalent probes will be
designed to quantify the global level of AP sites through fluorescence
measurements, whereas (ii) covalent probes will be developed to capture and map
the distribution of AP sites in the genome through DNA sequencing. The utility
of these probes will be validated in cells treated with DNA-damaging drugs.
International, interdisciplinary &
intersectoral aspects of the project
This project spans across several
disciplines, namely organic chemistry (synthesis and characterization of the
probes), molecular biophysics and biochemistry (probe–DNA interaction studies)
and cellular biology (cell cultures).
The methods for quantification and genomic
mapping of AP sites developed within the framework of this project will be
protected through patent applications, if applicable.
Parts of the project, in particular the
development of protocols for isolation of AP-DNA from cells, or in-cell studies
of binding of probes to AP-DNA, will be performed in collaboration with labs
abroad (USA, Czech Republic).
1. Duskova K, Lejault P, Benchimol
É, Guillot R, Britton S, Granzhan A,
Monchaud D. DNA junction ligands trigger DNA damage and are synthetic lethal
with DNA repair inhibitors in cancer cells. J. Am. Chem. Soc. 2020, 142,
2. Caron C, Duong XNT, Guillot R,
Bombard S, Granzhan A. Interaction
of Functionalized Naphthalenophanes with Abasic Sites in DNA: DNA Cleavage, DNA
Cleavage Inhibition, and Formation of Ligand–DNA Adducts. Chem. Eur. J. 2019,
25, 1949–1962. https:// doi.org/10.1002/chem.201805555.
3. Krafcikova M, Dzatko S, Caron C, Granzhan A, Fiala R, Loja T,
Teulade-Fichou MP, Fessl T, Hänsel-Hertsch R, Mergny JL, Foldynova-Trantirkova
S, Trantirek L. Monitoring DNA–ligand interactions in living human cells using
NMR spectroscopy. J. Am. Chem. Soc. 2019, 141, 13281–13285.
4. Saha A, Bombard S, Granzhan A, Teulade-Fichou MP. Probing
of G-quadruplex structures via ligand-sensitized photochemical reactions in
BrU-substituted DNA. Sci. Rep. 2018, 8, 15814.
5. Kotera N, Poyer F, Granzhan A, Teulade-Fichou MP.
Efficient inhibition of human AP endonuclease 1 (APE1) via substrate masking by
abasic site-binding macrocyclic ligands. Chem. Commun. 2015, 51, 15948–15951.