Saturation transfer MRI has previously been used to probe molecular binding interactions with signal enhancement via the water signal (IMMOBILISE approach). Here, we detailed the relayed nuclear Overhauser effect (rNOE) based mechanisms of this signal enhancement by a four-pool magnetization transfer model, verified the model both using simulations and experimentally (using small charged molecules: arginine, choline, and acetyl-choline). The analytical model can be used to quantify molecular binding affinity, i.e., the dissociation constant (KD). The characterization of the transient binding of small natural substrates paves a pathway towards the detection of receptor-substrate binding in vivo using MRI.
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