Introduction

Electrostatic interactions play a fundamental role in mediating molecular interactions and are essential in many biochemical processes.¹ It was previously shown that transient molecular binding can be imaged using a saturation transfer (ST) approach for enhancing sensitivity,² the so-called IMMOBILISE (for “IMaging of MOlecular BInding using Ligand Immobilization and Saturation Exchange”) method. More recently, it was shown that this approach can be used to image the binding of small charged molecules via electrostatic interactions to immobile ionic receptors.³
Here, the mechanism of water signal enhancement through molecular binding was quantitatively described and verified using simulations and experimentally using select small charged molecules (arginine, choline, and acetylcholine). The derived model was used to quantify binding affinities for these molecules based on the IMMOBILISE signal.

Materials and Methods

Theory
In the “IMMOBILISE” approach², the aliphatic protons of free ligands are first selectively labelled using a radio-frequency (RF) pulse, and the signal labelling is transferred to water via several possible binding-mediated pathways (Figure 1). Within free fast-tumbling ligands, intramolecular NOE transfer is very weak. However, upon binding to immobile receptors, the cross-relaxation becomes very efficient (spin diffusion) thus allowing the labelled ligands to couple to water. The following four-pool model was proposed to describe the “IMMOBILISE” process,
H_aL ^k'_on↔^k_off H_aLRH_e ^σ_ae↔^σ_ea H_aLRH_e ^k_ew↔^k_we H_w
where k_on and k_off are ligand binding on/off rates. At equilibrium, k_on[R][L]=k_off[LR], where [R], [L] and [LR] are the concentrations of free ligands, free receptor, and bound receptor, respectively. Total receptor concentration [R_T] = [LR] + [R]. and σ_ae are σ_ea the effective cross relaxation rates from receptor-bound-ligand aliphatic protons (H_aLR) to either exchangeable protons (LRH_e) or bound water in the macromolecule and back. k_ew and k_we are effective saturation transfer rates from LRH_e to free water (H_w) and back, corresponding to either the proton or water molecular exchange rates.
The evolution of the z-magnetization for each pool can be described by a set of modified Bloch equations.³ The analytical solution of the IMMOBILISE signal was obtained:
$$$IMMOBILISE={rNOE}_{max}\bullet\frac{\left[L\right]}{\left[L\right]+K_D} $$$ (1)
where rNOE_max is a constant that determines the maximum detected signal, K_D ($$$K_D=\frac{k_{off}}{k_{on}}$$$), the dissociation constant.

Sample preparation
Various concentrations (10, 20, 50, 100, and 200 mM) of arginine (Arg), choline (Cho), and acetyl-choline (ACh) were dissolved in phosphate-buffered saline (PBS), pH of 7.2 and mixed with ion-exchange media (Macro-Prep CM Support, with functional groups -COO^-). The mixture was pH-adjusted and transferred to the imaging tube for ST measurements.

CEST MRI
Experiments were performed on a Bruker Avance III 17.6 T vertical bore MR scanner at 20°C, using a 4 s CW saturation pulse followed by RARE MRI readout. Z-spectra were acquired within ±6 ppm relative to water in steps of 0.1 ppm, and normalized by the signal intensity without saturation. The B₁ was varied from 0.3 to 2.0 μT. Z-spectral components were analyzed using multi-Lorentzian fitting as described previously.⁴

Results

Using the four-pool Bloch equation modeling, numerical simulations were performed (Figure 2). CEST Z-spectra were shown for binding equilibria with varied free ligand concentration ([L]), binding affinity (K_D), and exchange rate (k_ew), respectively. The simulated IMMOBILISE signal increases with [L] and reaches a plateau when receptors get saturated; is highest with a K_D in the mM range; and depends on k_ew. IMMOBILISE signal is detectable for binding events with slow binding rates ( k'_on, within about 10¹ to 10⁶ s^-1), medium affinity (K_D, within about 10^-7 to 1 M), and above sub-millimolar level of receptor sites ([R_T], larger than about 10^-4 M). The analytical solution for IMMOBILISE signal (Eq. 1) is also verified by numerical simulations.
The results of IMMOBILISE experiments showing the interaction between Arg, Cho, and ACh and negatively charged ion-exchange resin (-COO^- functional group) are in Figure 3. The Z-spectra show the detection of Arg aliphatic protons at -0.9, -1.6, and -2.0 ppm, Cho aliphatic protons at -1.6 ppm, and ACh aliphatic protons at -1.6 and -2.6 ppm. ACh has no exchangeable protons, proving one pathway to be intermolecular rNOEs.
The IMMOBILISE signals were measured as a function of free ligand concentration and the curves were fit (Figure 4) using Eq. 1 to estimate K_D. The results (Table 1) show that the K_D values fitted from three individual peaks of Arg were the same within error (~130 mM). ACh has a higher binding affinity (smaller K_D, ~70 mM for the aliphatic proton at -1.6 ppm) with the resin (-COO^-) compared to Cho (~160 mM).

Discussion

The quantification of electrostatic molecular binding using IMMOBILISE MRI was demonstrated using simulations and small charged molecules. Simulation results show that molecules with medium affinity (within about 10^-7 to 1 M) can provide detectable IMMOBILISE signals, which is comparable to the K_D of Arg, Cho, and Ach for the resin binding studied here. Experimentally, we showed that the molecular binding of sub-mM to mM level concentration of receptor sites to molecular targets can be detected by the signal enhancement mechanism, enabling the possibility of in vivo studies of molecular binding using IMMOBILISE MRI.

Acknowledgements

No acknowledgement found.