F.M. Kirby Research Center for Functional Brain Imaging, Kennedy Krieger Institute, Baltimore, MD, USA.
The Russell H. Morgan Department of Radiology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Magn Reson Med. 2022 Aug;88(2):901-915. doi: 10.1002/mrm.29230. Epub 2022 Apr 5.
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.
饱和转移 MRI 先前已被用于通过水信号探测分子结合相互作用的信号增强。在此，我们详细描述了这种信号增强的基于接力核 Overhauser 效应（rNOE）的机制，开发了一种量化分子结合亲和力（即解离常数（Kd））的策略，并将该方法应用于检测几种带电小生物分子的静电结合。另一个目标是估计瞬时受体-底物结合的检测极限。
通过三步磁化转移模型对信号增强机制进行了定量描述，并进行了数值模拟以验证该理论。研究了精氨酸、胆碱和乙酰胆碱与阴离子树脂的结合平衡作为配体浓度、pH 值和盐含量的函数。通过拟合多个浓度数据确定平衡解离常数（Kd）。
数值模拟表明，信号增强足以检测亚毫摩尔（约 100 μM）浓度配体与低微摩尔水平分子靶标的分子结合。从精氨酸、胆碱和乙酰胆碱结合实验中测量的 rNOE 信号表明，几种磁化转移途径（内配体 rNOE 和分子间 rNOE）都可以贡献。由分子离子结合引起的 rNOE 受到 pH 值和盐浓度的影响。在所研究的三种阳离子中，分子结合强度以 70-160 mM 的范围表示。
使用 MRI 检测小底物的瞬时结合的能力为检测体内微摩尔水平的受体-底物结合铺平了道路。
