Detection of electrostatic molecular binding using the water proton signal

Saturation transfer MRI has previously been used to probe molecular binding interactions with signal enhancement via the water signal. Here, we detail the relayed nuclear overhauser effect (rNOE) based mechanisms of this signal enhancement, develop a strategy of quantifying molecular binding affinity, i.e., the dissociation constant ( $$ ), and apply the method to detect electrostatic binding of several charged small biomolecules. Another goal was to estimate the detection limit for transient receptor-substrate binding.

The signal enhancement mechanism was quantitatively described by a three-step magnetization transfer model, and numerical simulations were performed to verify this theory. The binding equilibria of arginine, choline, and acetyl-choline to anionic resin were studied as a function of ligand concentration, pH, and salt content. Equilibrium dissociation constants ( $$ ) were determined by fitting the multiple concentration data.

The numerical simulations indicate that the signal enhancement is sufficient to detect the molecular binding of sub-millimolar (∼100 μM) concentration ligands to low micromolar levels of molecular targets. The measured rNOE signals from arginine, choline, and acetyl-choline binding experiments show that several magnetization transfer pathways (intra-ligand rNOEs and intermolecular rNOEs) can contribute. The rNOEs that arise from molecular ionic binding were influenced by pH and salt concentration. The molecular binding strengths in terms of $$ ranged from 70-160 mM for the three cations studied.

The capability to use MRI to detect the transient binding of small substrates paves a pathway towards the detection of micromolar level receptor-substrate binding in vivo.