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

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.