Méthodes pour l’étude de structures, dynamiques et interactions d’acides nucléiques non canoniques
2025-12-19
Professional Journey
- M2 Intern - LSOSCO - 2008
- UMR 8123 CNRS-UCP-ESCOM
- PhD Student - U. Paris XI - 2008-2012
- UMR176 CNRS, Institut Curie
- Postdoc - UBC - 2012-2013
- Chemistry Dpt
- Postdoc - IECB - 2013-2015
- U869 CNRS/U. Bordeaux
- Research Scientist - Quality Assistance - 2015-2017
- R&D Dpt
- MCU - University of Bordeaux - 2017-present
- ARNA - INSERM U1212, CNRS UMR 5320, U. Bordeaux
Teaching & support duties
Topics
- Analytical Chemistry
- Regulatory aspects
- Biomolecules
- Scientific communication
- Data processing and visualization
Students
- Health Sciences
- Pharmacy (L2/L3)
- DEUST (Production, contrôle et qualité des produits de santé, L1/L2)
- TECSAN (Technologies pour la santé,L3)
- Master IPPS (Industries Pharmaceutiques et Produits de Santé, M1)
- Master DACQ (Développement Analytique et Contrôle Qualité, M2)
- CHU Bodeaux
- Préparateurs en pharmacie hospitalière
- Collège des écoles doctorales
- PhD students (all fields)
- Reference person @ ARNA for
- Research data; member of the Bordeaux Data Workshop
- Open Science
- Slides: slides.hdr.largy.fr (source code)
PhD at the Institut Curie/CNRS/University Paris XI
A quick introduction on G-quadruplexes
A quick introduction on G-quadruplexes
Moving targets
- Drug targets…
- Involved in biological processes at DNA/RNA levels
- Structurally different from duplexes
- …and drugs?
- Some aptamers are or contain a G4
- Very polymorphic
- Can fold into different conformers in equilibrium
- May not be fully folded
- Very dependent on cation concentration/nature
- Goal: selective binding to modulate biological functions
- Generally, \(\pi\)-stacking ligands on external tetrads
Career-defining PhD project goals
- Initially
- Synthesize metal complexes as G4 ligands
- Understand the influence of metal ions and aromatic ligand on binding selectivity.
- Synthesize ligands with an added value
- Topological selectivity
- Original binding mode
- Functionalization: fluorescence, biotinylation, halogenation,…
- Develop analytical tools to
- Characterize G4/ligand interactions
- Discover new G4 ligands
Fluorescent Interacalator Displacement Assay (FID)
FID is a straightforward method to assess binding selectivity
FID can give access to Kds
FID allows screening for G4 ligands
Metal complexes can selectively and covalently bind to G4s
Non-condensed ligands may allow selective groove binding
Non-condensed ligands may allow selective groove binding
Postdoc at the University of British Columbia
Towards sequence-selective binding with bifacial nucleosides
- Antisense strategy limited
- Requires strand displacement
- Not particularly energetically favorable
- Energetic gain by forming additional H-bonds
- Exploit H-bond donor/acceptor groups on both strands
- First generation JAT
- Donor/Acceptor network designed to target bp
- Suboptimal protonation state at physiological pH
- \(pK_{a} \approx 7.5\)
- Second generation NJAT
- Lowered pKa
- Improved binding
- Sequence-selective (Tm ~ 10°C > G/C)
- Adequate base-pairing geometry
- No significant distortion of duplex
Postdoc at IECB
SEC allows fast and robust stoichiometry determination
- Goal: method to assess the folding and stoichiometry of oligonucleotides
- Versatile
- DNA/RNA, secondary structures
- Buffers, Temperature
- Large concentration range
- Fast, simple, automatable
- Quantitative
- Versatile
- Excellent linearity
- Application on real-life RNA hairpins
- Here at 10°C (favors dimers)
- G4s display larger hydrodynamic radii distributions
- Robust stoichiometry assessment
- Real-life G4s can be messy
G4 conformations can be tuned with cations
- Cation-dependent G4 switches?
- Simple sequences!
- 222T: TGGGTTGGGTTGGGTTGGGT
- Terminal T to prevent end-stacking
- Antiparallel-to-parallel switch at low [KCl]
- Extensive characterization
- Fast (second-scale at 20°C)
- Large \(\Delta T_{m}\) between K+ and Na+
- \(E_{a}\) of interconversion by UV-vis!
- Conformer revealed at low KCl
- Antiparallel
- 2-tetrad
- Double switch!
Research Scientist at Quality Assistance
HDX/MS gives insights into protein structural dynamics
HDX/MS allows epitope mapping in regulated environments
MCU at the University of Bordeaux
DNA is amenable to HDX/MS
- H/D exchange ideal for structure probing
- Full sequence coverage
- Directly involved in folding
- Minimal primary/secondary structure change
- Rate of HDX should be structure dependent
- Native MS measurements
Coupling in-solution HDX to native MS
Exchange rates are sensitive to folding
The number of protected sites scales with the number of tetrads
The number of protected sites scales with the number of tetrads
The number of protected sites scales with the number of tetrads
Exchange protection scales with stability
Higher stability G4s exchange via local fluctuations
- Short-lived local fluctuations
- Local exchange: “EX2”
- Monomodal isotopic distributions
Lower stability G4s exchange via an unfolded conformer
- Longer-lived unfolded conformers
- Global exchange: “EX1”
- Multimodal isotopic distributions
- Each molecule visiting the exchange-competent conformation is revealed
Exchange-competent species resemble folding intermediates
Drift-tube IMS can separate analytes by shape
- Collisions between ions and gas
Drift-tube IMS can separate analytes by shape
- Collisions between ions and drift gas
- Compact structures travel faster
Same-mass conformer separation
Minor unfolding conformers can be revealed by HDX/IMS
HDX kinetics inform on binding modes
G4 absorbance spectra contain a wealth of data
Aptamer structures are seldom characterized
NMR provides (non-canonical !) starting models
NMR provides many constraints
- DNA (806)
- intra-residue
- inter-residue
- Dopamine (67)
- intramolecular
- intermolecular
A minimized ensemble that cannot be predicted (yet)
- CASP16
Critical Assessment of Techniques for Protein Structure Prediction- 107 models submitted
- 0 with RMSD < 10 Å