Nanomolar small-molecule detection using a genetically encoded 129Xe NMR contrast agent

Abstract

Genetically encoded magnetic resonance imaging (MRI) contrast agents enable non-invasive detection of specific biomarkers in vivo. Here, we employed the hyper-CEST ¹²⁹Xe NMR technique to quantify maltose (32 nM to 1 mM) through its modulation of conformational change and xenon exchange in maltose binding protein (MBP). Remarkably, no hyper-CEST signal was observed for MBP in the absence of maltose, making MBP an ultrasensitive "smart" contrast agent. The resonance frequency of ¹²⁹Xe bound to MBP was greatly downfield-shifted (Δδ = 95 ppm) from the ¹²⁹Xe_(aq) peak, which facilitated detection in E. coli as well as multiplexing with TEM-1 β-lactamase. Finally, a Val to Ala mutation at the MBP-Xe binding site yielded 34% more contrast than WT, with ¹²⁹Xe resonance frequency shifted 59 ppm upfield from WT. We conclude that engineered MBPs constitute a new class of genetically encoded, analyte-sensitive molecular imaging agents detectable by ¹²⁹Xe NMR/MRI.

Scheme 1. Ultrasensitive detection of a small molecule (maltose)-protein (MBP) interaction via hyper-CEST NMR. HP ¹²⁹Xe (green) binds maltose-bound MBP, where the unique Xe resonance frequency is saturated by shaped RF pulses. Xe exchange leads to depolarization of solution-phase Xe pool, thereby generating MR contrast (yellow peak).

Fig. 1. Xe (red sphere) bound to MBP_open (PDB ID ; 1LLS), with the N-terminal domain colored blue, C-terminal domain colored green, and linking segments colored orange. (Inset) detailed view of the Xe-binding cavity.

Fig. 2. (a) Hyper-CEST z-spectra of 80 μM MBP with and without 1 mM maltose in pH 7.2 PBS at 300 K, with z-spectra of buffer only with and without maltose shown for reference. Pulse length, τ_pulse = 3.8029 ms; field strength, B_1,max = 77 μT (b) saturation contrast for 100 nM WT MBP and 100 nM MBP(I329Y)–GFP as a function of percent MBP in maltose-bound closed conformation. For WT MBP, [maltose] = 0, 0.1, 0.3, 0.5, 1, 3, 1000 μM. For MBP(I329Y)–GFP, [maltose] = 0, 32, 72, 140, 5000 nM. Pulse length, τ_pulse = 1.0496 ms; field strength, B_1,max = 279 μT. The number of pulses increased linearly from 0 to 15 000.

Fig. 3. Hyper-CEST z-spectra of 27 μM MBP and 80 μM Bla with and without 1 mM maltose in pH 7.2 PBS at 300 K. Blue and green lines show Lorentzian fits to the Xe–Bla and Xe_(aq) peaks, respectively. Pulse length, τ_pulse = 3.8029 ms; field strength, B_1,max = 77 μT.