Label-Free Measurement of Ligand Interactions Using SABRE Hyperpolarization at Low Magnetic Fields

The protein-ligand binding affinity is evaluated using low-cost, low-field nuclear magnetic resonance (NMR) spectroscopy at 0.85 mT. Strong signals are achieved through the hyperpolarization of ¹H nuclei by parahydrogen-based signal amplification by reversible exchange (SABRE). The interaction is monitored by tracking the ubiquitous hydrogen signal. Despite the hyperpolarization, a key challenge of the label-free detection at low field is the distinction of signals in the absence of chemical shift. ¹H signals of ligands are measured after deuterating the coligand and solvent and numerically accounting for orthohydrogen signal, which is produced during the hyperpolarization process. Spin-spin (R₂) relaxation rates quantify the protein-ligand interaction. Since R₂ in a milli-Tesla field does not include an exchange contribution, the calculation of the ligand dissociation constant K_D is simplified compared to traditional high-field NMR. The technique for monitoring ¹H signals is generalizable to detect any ligand that is competitively binding to the protein. It expands the application of low-field NMR for high-throughput screening and for studies of biochemical processes that involve ligand interactions.