We will review the principles for designing an optimal bioorganic probes that are based on the specific targets. In addition we will discuss how to design a genetically encoded probe for a specific scientific question.
Genetically encoded CEST probes
TARGET AUDIENCE : Researchers and clinicians who are interested in molecular and cellular MRI, particularly in the non-invasive monitoring of gene expression, cell therapy and transplantation, as well as pre-clinical drug screening, with advanced MRI-based techniques. OBJECTIVES: (a) To understand the basics of bioorganic CEST probes. (b) To learn the basics of gene expression in living systems. (c) To review and discuss the different options to genetically encode CEST based probes. PURPOSE: In the past decade we have experienced a substantial technological growth in DNA synthesis capabilities that resulted in significant reduction in costs and increase in length of DNA accompanied with accelerated rate of production(1). This allowed replacing the traditional molecular biology techniques such as cloning from foreign organism, mutagenesis, directed evolution etc. Such DNA synthesis technologies can clearly integrate into the field of molecular imaging. One prominent application is the design of new genetically encoded reporters, probes and biosensors. Genetically encoded entities have the advantage that they can assist in visualize biological processes in living organisms. METHODS: Here we will discuss the feasibility of these technologies of reporters and biosensors based on chemical exchange saturation transfer magnetic resonance imaging (CEST-MRI)(2). While CEST MRI is a good model system, synthetic genes can be use for designing imaging tools for variety of imaging modalities. Especially because many of the probes for CEST MRI are proteins that can be encoded by genes and the requirements for making them good imaging probes are well defined. The methodology is usually composed of the following steps: (a) Identifying a biological target. (b) Identifying a potential probe for the target (protein, substrate antigen etc.). (c) Determining the CEST properties of the probe in vitro (with and without the target). (d) [Optional] modify to optimize the exchange rate to improve the CEST contrast. (e) Transferring the gene to the target tissue, e.g., transfecting/transducing cells in culture; viral or non-viral gene delivery to a live animal or generating transgenic mouse. (f) Imaging the subject with the appropriate pulse sequences. (g) Image processing and data analysis. RESULTS AND DISCUSSION: Initial studies demonstrated that a synthetic gene could be used as a reporter gene for MRI. A know set of properties were used to design, clone and express a lysine rich protein(3) and demonstrate it’s applicability to monitor promoter specific expression(4) and oncolytic virotherapy(5). Another application was to transform the human protamine-1 into a CEST based reporter gene. In this case, to validate the reporter in bacterial system the gene was optimized and reversed engineered for expression in prokaryotes. Again a synthetic gene was produced that utilized the bacterial codons in an optimal manner(6). A synthetic gene that encode for eight monomers of the phosphorylation site of the protein kinase A (PKA) was synthesized and was used as a biosensor for the enzyme activity(7). Finally, supercharging the green fluorescent protein transformed it into a reporter gene suitable for MRI(8). Alternatively, several substrates for different enzyme have been proposed as CEST probes that are able to detect the activity of these enzymes. Among them are probes for Cytosine Deaminase(9), the Herpes Simplex Virus type 1 Thymidine Kinase(10) as well as β-Galactosidase and β-Glucuronidase(11). CONCLUSION: By understanding the features of the imaging target, it is practical today to design an optimal reporter gene based on the study goal, or even, in many cases, to design a genetically encoded probe for a specific scientific question. These examples and others demonstrate that synthetic biology can be used for pushing the envelope and increasing the arsenal of molecular imaging tools(12).