21 Scientific Article The Israel Chemist and Chemical Engineer Issue 9 · January 2023 · Tevet 5783 reporter probe pair. More specifically, we capitalized on the vast existing knowledge of a genetically engineered reporter system used in nuclear imaging and composed of a deoxyribonucleoside kinase (dNK) as the reporter gene and synthetic imageable deoxyribonucleoside (dN) as the reporter probe [35, 36]. Our MRI reporter system, which we termed GeneREFORM (stands for GENet ical ly Engineered REporters FOR multicolor-MRI), is composed of orthogonal reporter probe/ reporter gene pairs of dN/dNK. The dN/dNK system relies on the fact that imageable dN is trapped upon its phosphorylation solely in cells engineered to express the heterologous dNK transgene. For GeneREFORM, the synthetic dNs used as the putative reporter probes were the CEST-MRI detectable pyrrolo-deoxycytidine (pdC), a deoxycytidine analog and 5-methyl-dihydrothymidine (5-MDHT), a thymidine analog. Importantly, pdC and 5-MDHT generate CEST contrast from their -NH protons that resonate at Dw = 6 ppm (-NH of pdC)  and Dw = 5 ppm (-NH of 5-MDHT) [38, 39], allowing their use as artificial MRI colors in CEST MRI (Figure 7a,b). With the aid of a structure- and evolution-based protein design method, called PROSS , followed by active-sitespecific mutagenesis of promiscuous dNKs, namely Dm-dNK and HSV1-TK, two highly active and fully orthogonal dNKs were developed and obtained. The Dm-dNK_7C (PDB Entry - 6YBH) variant was found to be highly active with regard to its ability to phosphorylate pdC with negligible capability Genetically engineered reporters for multicolor MRI of gene expression The revolution of multicolor imaging of multiplex biological systems would not be complete without the development of fluorescent proteins as reporter genes. In this regard, the evolution of the green fluorescent protein (GFP) from its first isolation from the Aequorea victoria jellyfish, through the cloning of its gene and its expression as an imageable reporter in cells, to the extension of the fluorescent protein family to a color palette beyond green, has changed science and resulted in the founders being awarded the Nobel prize in chemistry. Realizing the challenges of fluorescent imaging, genetically engineered MRI reporters have been developed as potential alternatives for non-invasive spatial maps of reporter-gene expression . Nevertheless, although extensively developed and demonstrated in multitude animal models, MRI-based reporter-gene signals lack the orthogonal, color-like display capabilities of luminescent reporters. To accomplish multicolor MRI mapping of transgene expression, we aimed to design a set of orthogonal reporter genes that produce multicolor MRI signals that can be spectrally resolved and spatially mapped . To this end, we used a genetically engineered reporter system composed of a reporter gene/ a b c d Figure 6. ParaGEST. (a) The dynamic exchange process between a paramagnetic-CD and a fluorinated-guest that allows paraGEST MRI. (b) z-spectra of solutions containing Ln-CD hosts and a 19F-guest. Data were obtained with the fluorinated guest para-trifluoromethylbenzylamine. Modified from Reference 30, which is licensed under a Creative Commons Attribution 4.0 International License http://creativecommons.org/licenses/by/4.0/. Figure 7. GeneREFORM design. (a) MTRasym plots (i.e., CEST effects representation) of 5-MDHT and pdC emphasizing the negligible level of overlap of CEST peaks. (b) CEST-MRI maps of 5-MDHT, pdC, and PBS solutions obtained at ∆w = 5 and 6 ppm. (c) Crystal structure of Dm-dNK_7C, presenting its mutations, and inset showing the magnification of the active site with pdC. (d) HSV1-TK_7B structure, presenting its mutations (computational model) and magnification of the active-site vicinity with docked 5-MDHT (right). Modified with permission from Reference 34.