Optimization of CEST MRI Reporter Protein Design Using Cation‐Pi Networks

Nucleic acid-based therapeutics, such as oncolytic virotherapy or gene therapy, would benefit greatly from a reporter gene that induces endogenous production of a protein biomarker to noninvasively track the delivery, persistence, and spread with imaging. Several chemical exchange saturation transfer (CEST) reporter proteins detectable by magnetic resonance imaging (MRI) have been demonstrated to have high sensitivity. However, to date none can provide strong CEST contrast at a distinct resonance from that of endogenous proteins, limiting their specificity. We investigated proteins and peptides containing tyrosine (Tyr), tryptophan (Trp), and lysine (Lys) residues that demonstrate CEST contrast shifted far downfield (4-10 ppm) from water. Although Tyr, Trp, and Lys exchangeable protons are typically not detectable under physiological conditions, those in our tested molecules are, having exchange rates of 400-2500 s^-1. The large chemical shift dispersion and rapid exchange rates are attributed to unique hydrogen bonding and cation-π network interactions. These discoveries set the stage for designing a stable reporter protein with high detection specificity and sensitivity that can facilitate the in vivo monitoring of viral and gene therapies using MRI.