A Swiss Army Knife of Methods

Deciphering the structure, dynamics and interactions of non-canonical DNA

Eric Largy
eric.largy@u-bordeaux.fr

ARNA, INSERM U1212, CNRS UMR 5320, Université de Bordeaux

UFR des Sciences Pharmaceutiques, Université de Bordeaux

May 12, 2026

Hello from Bordeaux

Conformational polymorphism
of DNA

DNA can adopt many secondary structures

Largy et al., Chem. Rev. 2022, 122, 8, 7720

G-quadruplexes are particularly peculiar

 

G-quadruplexes are particularly peculiar

 

 

G4s are DNA/RNA drug targets distinct from dsDNA

Largy, E., Mergny, J.-L. et Gabelica, V. Role of Alkali Metal Ions in G-Quadruplex Nucleic Acid Structure and Stability. 2016, p.203‑258.

G4s may also be drugs themselves

PDB: 4TS2: Warner et al., Nat. Struc. Mol. Biol., 2014, 21, 658

G4s are very polymorphic…

…and in equilibrium

 

Topology, stability and stoichiometry determination

Topologies are poor structural descriptors

Topologies are poor structural descriptors

Largy, E., Guédin, A., Kabbara, A., Mergny, J.-L. et Amrane, S. Nucleic Acids Res., 2025, 53, gkaf953

Stability measurements are often innacurate

Stability measurements are often innacurate

\[ A_T=(a^FT+b^F) \times \frac{1}{1+exp(-\frac{\Delta H^0 (1- \frac{T}{T_m})}{RT})} + (a^UT+b^U) \times \frac{exp(-\frac{\Delta H^0 (1- \frac{T}{T_m})}{RT})}{1+exp(-\frac{\Delta H^0 (1- \frac{T}{T_m})}{RT})} \]

\[ A_T=\textcolor{forestgreen}{(a^FT+b^F)} \times \frac{1}{1+exp(-\frac{\Delta H^0 (1- \frac{T}{T_m})}{RT})} + \textcolor{forestgreen}{(a^UT+b^U)} \times \frac{exp(-\frac{\Delta H^0 (1- \frac{T}{T_m})}{RT})}{1+exp(-\frac{\Delta H^0 (1- \frac{T}{T_m})}{RT})} \]

\[ A_T=(a^FT+b^F) \times \frac{1}{1+exp(-\frac{\Delta H^0 (1- \frac{T}{\textcolor{coral}{T_m}})}{RT})} + (a^UT+b^U) \times \frac{exp(-\frac{\Delta H^0 (1- \frac{T}{\textcolor{coral}{T_m}})}{RT})}{1+exp(-\frac{\Delta H^0 (1- \frac{T}{\textcolor{coral}{T_m}})}{RT})} \]

\[ A_T=(a^FT+b^F) \times \frac{1}{1+exp(-\frac{\textcolor{steelblue}{\Delta H^0} (1- \frac{T}{T_m})}{RT})} + (a^UT+b^U) \times \frac{exp(-\frac{\textcolor{steelblue}{\Delta H^0} (1- \frac{T}{T_m})}{RT})}{1+exp(-\frac{\textcolor{steelblue}{\Delta H^0} (1- \frac{T}{T_m})}{RT})} \]

meltR allows high-throughput and robust Tm determination

SEC allows robust stoichiometry determination

 

Largy, E. et Mergny, J.-L. Nucleic Acids Res., 2014, 42, e149.

SEC allows robust stoichiometry determination

 

 

Largy, E. et Mergny, J.-L. Nucleic Acids Res., 2014, 42, e149.

Going further with native MS

Native MS probes non-covalent interactions

 

 

Adapting to a potassium-deficient buffer

ericlarg4.github.io/G4_database

Ghosh, Largy, Gabelica, Nucleic Acids Res., 2021, 49, 2333

G4 conformations can be tuned with cations

222T: TGGGTTGGGTTGGGTTGGGT

Largy, E., Marchand, A., Amrane, S., Gabelica, V. et Mergny, J.-L. J. Am. Chem. Soc., 2016, 138, 2780

The interconversion occurs at low [KCl]

Largy, E., Marchand, A., Amrane, S., Gabelica, V. et Mergny, J.-L. J. Am. Chem. Soc., 2016, 138, 2780

The interconversion is fast

Largy, E., Marchand, A., Amrane, S., Gabelica, V. et Mergny, J.-L. J. Am. Chem. Soc., 2016, 138, 2780

The interconversion arises from large ΔTm

Largy, E., Marchand, A., Amrane, S., Gabelica, V. et Mergny, J.-L. J. Am. Chem. Soc., 2016, 138, 2780

The interconversion can be monitored by UV

Largy, E., Marchand, A., Amrane, S., Gabelica, V. et Mergny, J.-L. J. Am. Chem. Soc., 2016, 138, 2780

The interconversion can be monitored by nMS

Largy, E., Marchand, A., Amrane, S., Gabelica, V. et Mergny, J.-L. J. Am. Chem. Soc., 2016, 138, 2780

The combination of methods allows thermodynamic
and kinetic characterization of complex system

Largy, E., Marchand, A., Amrane, S., Gabelica, V. et Mergny, J.-L. J. Am. Chem. Soc., 2016, 138, 2780

Combining methods to decipher ligand interactions

G4 can be targeted by small molecules

Most G4 ligands are \(\pi\)-stacking on external tetrads

Largy et al., Chem. Rev. 2022, 122, 8, 7720

Sometimes luck does better than reason

König, A., Laffilé, V., Thore, S., Mackereth, C., Yatsunyk, L., Ferrand, Y., Largy, E., Gabelica, V., Nucleic Acids Res., 2025, gkaf1365

Binding preference to parallel topology

36 oligonucleotides × 4 foldamers = 144 spectra

König, A., Laffilé, V., Thore, S., Mackereth, C., Yatsunyk, L., Ferrand, Y., Largy, E., Gabelica, V., Nucleic Acids Res., 2025, gkaf1365

Selectivity confirmed by high-throughput nMS K titrations

6 oligonucleotides × 7 foldamers × 7 foldamer concentrations = 294 spectra

König, A., Laffilé, V., Thore, S., Mackereth, C., Yatsunyk, L., Ferrand, Y., Largy, E., Gabelica, V., Nucleic Acids Res., 2025, gkaf1365

Not completely crystal clear binding

 

QQPQ complexed to 222T adopting a parallel topology. PDB: 8QN2.

König, A., Laffilé, V., Thore, S., Mackereth, C., Yatsunyk, L., Ferrand, Y., Largy, E., Gabelica, V., Nucleic Acids Res., 2025, gkaf1365

NMR confirms the stacking on both ends

QQPQ complexed to 2LK7 (and not 222T) and T → U variants (missing H7, see boxes).

König, A., Laffilé, V., Thore, S., Mackereth, C., Yatsunyk, L., Ferrand, Y., Largy, E., Gabelica, V., Nucleic Acids Res., 2025, gkaf1365

Final models supported by MD simulations

König, A., Laffilé, V., Thore, S., Mackereth, C., Yatsunyk, L., Ferrand, Y., Largy, E., Gabelica, V., Nucleic Acids Res., 2025, gkaf1365

Foldamers allow precisal structural control
of artifical protein assemblies

Sigl, J. et al. Nat. Chem., accepted

SEC and nMS are methods of choice
to determine stoichiometries

Sigl, J. et al. Nat. Chem., accepted

Foldamers also bind proteins in unexpected ways

Solution structure of Nanofitin C10 in complex with a 12unit aromatic oligoamide foldamer | PDB: 9T0O | Sigl, J. et al. Nat. Chem., accepted
737 non-ambiguous distance + 114 dihedral angle restraints

Native HDX/MS
from proteins to nucleic acids

HDX/MS gives insights
into protein structural dynamics

Adapted from Wang, Y. © 2024 Rapid Novor, Inc.

HDX/MS allows epitope mapping
in regulated environments

 

Belarif, L. et al. Nature Communications, 2018, 9, 4483

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

Largy and Gabelica, Anal. Chem., 2020, 92 (6), 4402–10

Coupling in-solution HDX to native MS

Largy and Gabelica, Anal. Chem., 2020, 92 (6), 4402–10

Largy and Ranz, Anal. Chem., 2023, 95, 9615–22

Exchange rates are sensitive to folding

 

 

Largy and Gabelica, Anal. Chem., 2020, 92 (6), 4402–10

The number of protected sites scales
with the number of tetrads

Largy, Ranz, Gabelica, J. Am. Chem. Soc. 2023, 145, 49, 26843

The number of protected sites scales
with the number of tetrads

Largy, Ranz, Gabelica, J. Am. Chem. Soc. 2023, 145, 49, 26843

The number of protected sites scales
with the number of tetrads

Largy, Ranz, Gabelica, J. Am. Chem. Soc. 2023, 145, 49, 26843

Exchange protection scales with stability

Largy, Ranz, Gabelica, J. Am. Chem. Soc. 2023, 145, 49, 26843

Higher stability G4s exchange via local fluctuations

Largy, Ranz, Gabelica, J. Am. Chem. Soc. 2023, 145, 49, 26843

Lower stability G4s exchange via unfolded conformers

Largy, Ranz, Gabelica, J. Am. Chem. Soc. 2023, 145, 49, 26843

Exchange-competent species
resemble folding intermediates

Largy, Ranz, Gabelica, J. Am. Chem. Soc. 2023, 145, 49, 26843

Drift-tube IMS can separate analytes by shape

Drift-tube IMS can separate analytes by shape

Minor conformers revealed by HDX/IMS

 

 

HDX kinetics inform on binding modes

Beating structure prediction models?

Feeding structure prediction models

Aptamer structures are seldom characterized

Nakatsuka N., et al., Science, 2018, 362, 319-324

NMR provides (non-canonical !)
starting models

Hoi Pui Chao, E., Largy, E., Kaiyum, Y. A., Nguyen, M.-D., Vialet, B., Dauphin-Ducharme, P., Johnson, P. E., Mackereth, C.D., Nat. Commun., in revision

NMR provides many constraints

  • DNA (806)
    • intra-residue
    • inter-residue
  • Dopamine (67)
    • intramolecular
    • intermolecular

Hoi Pui Chao, E., Largy, E., Kaiyum, Y. A., Nguyen, M.-D., Vialet, B., Dauphin-Ducharme, P., Johnson, P. E., Mackereth, C.D., Nat. Commun., in revision

MD simulations informs on dynamics

Hoi Pui Chao, E., Largy, E., Kaiyum, Y. A., Nguyen, M.-D., Vialet, B., Dauphin-Ducharme, P., Johnson, P. E., Mackereth, C.D., Nat. Commun., in revision

A minimized ensemble that cannot be predicted

  • CASP16
    Critical Assessment of Techniques for Protein Structure Prediction
    • 107 models submitted
    • 0 with RMSD < 10 Å

Kretsch R.C., et al., Proteins, 2025, prot.70043

Hoi Pui Chao, E., Largy, E., Kaiyum, Y. A., Nguyen, M.-D., Vialet, B., Dauphin-Ducharme, P., Johnson, P. E., Mackereth, C.D., Nat. Commun., in revision

The structural landscape
of DNA is large

A robust method toolbox is required to explore it

Acknowledgements

Université de Bordeaux

  • Cameron Mackereth
    • Samir Amrane
    • Stéphane Thore
  • Yann Ferrand
    • Vincent Laffilé

Université de Bordeaux → Institut Polytechnique de Paris

  • Jean-Louis Mergny
    • Aurore Guedin-Beaurepaire
    • Marie Toulisse

Université de Bordeaux → Université de Genève

  • Valérie Gabelica
    • Frédéric Rosu
    • Clarisse Fourel
    • Anirban Ghosh
    • Romane Guisiano
    • Alexander König
    • Adrien Marchand
    • Matthieu Ranz
    • Liliya Yatsunyk

York University

  • Philip Johnson
    • Emily Chao

Université de Sherbrooke

  • Philippe Dauphin Ducharme

LMU München

  • Yvan Huc
    • Jonas Sigl

Quality Assistance &
Ose Immunotherapeutics

Funding sources

Thank you for your attention!