High-throughput thermodynamic fingerprinting of protein–ligand interactions by DNA-directed focal molography
- 1R & D, lino Biotech AG, Zurich, Switzerland.
Abstract
Thermodynamic characterization of biomolecular interactions is essential for understanding the enthalpic and entropic driving forces of molecular recognition, but established label-free techniques are limited either by bulk refractive-index sensitivity or by the lengthy thermal equilibration required to suppress it. Here, we used focal molography to investigate the temperature-dependent binding of the protein kinase A regulatory subunit (PKA-R) to cyclic AMP (cAMP) derivatives and to derive apparent thermodynamic signatures from kinetic measurements. We first validated the diffractometric readout under conditions that challenge refractometric sensors: the coherent mass density channel strongly suppressed temperature-induced bulk refractive-index effects and resolved binding in 50% human serum despite measurable non-specific adsorption, reducing the need for lengthy equilibration and buffer matching. We then combined focal molography with DNA-directed immobilization (DDI), allowing five cAMP derivatives to be presented in parallel on the same multiplexed chip and followed across five temperatures. This format yielded distinct, internally consistent apparent thermodynamic fingerprints for each derivative, separating ligands with similar affinities by their enthalpic and entropic contributions. Together, these results establish focal molography with DDI as a multiplexed workflow for comparative thermodynamic fingerprinting of biomolecular interactions at higher throughput.
Related
- High-throughput thermodynamic screening of oxide-scale adhesion across the CoCrFeMnNiAl high-entropy alloys
- Generalized Dunkl Quantum Systems with Energy-Dependent Interactions: Exact Solvability and Thermodynamic Properties
- Higher-order interactions renormalize the thermodynamic signature of neural population codes
- Chaos-Modulated Photothermal Fingerprinting of Pb(NO₃)₂-Induced Perturbations in Wastewater Using Phase-Space and Fractional Thermal-Memory Signatures
- Unimodality and Radial Monotonicity of the Magnetic Resonance Fingerprinting T1/T2 Matching Objective
BibTeX
@article{oehninger2026highthroughput,
title = {{High-throughput thermodynamic fingerprinting of protein–ligand interactions by DNA-directed focal molography}},
author = {John Oehninger and Simona Notova and Andreas Frutiger},
journal = {PLOS One},
year = {2026},
month = oct,
doi = {10.1371/journal.pone.0354008},
url = {https://doi.org/10.1371/journal.pone.0354008},
note = {CC BY},
}
RIS
TY - JOUR
TI - High-throughput thermodynamic fingerprinting of protein–ligand interactions by DNA-directed focal molography
AU - John Oehninger
AU - Simona Notova
AU - Andreas Frutiger
PY - 2026
DA - 2026/10/22
JO - PLOS One
DO - 10.1371/journal.pone.0354008
UR - https://doi.org/10.1371/journal.pone.0354008
AB - Thermodynamic characterization of biomolecular interactions is essential for understanding the enthalpic and entropic driving forces of molecular recognition, but established label-free techniques are limited either by bulk refractive-index sensitivity or by the lengthy thermal equilibration required to suppress it. Here, we used focal molography to investigate the temperature-dependent binding of the protein kinase A regulatory subunit (PKA-R) to cyclic AMP (cAMP) derivatives and to derive apparent thermodynamic signatures from kinetic measurements. We first validated the diffractometric readout under conditions that challenge refractometric sensors: the coherent mass density channel strongly suppressed temperature-induced bulk refractive-index effects and resolved binding in 50% human serum despite measurable non-specific adsorption, reducing the need for lengthy equilibration and buffer matching. We then combined focal molography with DNA-directed immobilization (DDI), allowing five cAMP derivatives to be presented in parallel on the same multiplexed chip and followed across five temperatures. This format yielded distinct, internally consistent apparent thermodynamic fingerprints for each derivative, separating ligands with similar affinities by their enthalpic and entropic contributions. Together, these results establish focal molography with DDI as a multiplexed workflow for comparative thermodynamic fingerprinting of biomolecular interactions at higher throughput.
SN - 1932-6203
ER -