Development of a synthetic biosensor for chemical exchange MRI utilizing in silico optimized peptides.

Chemical exchange saturation transfer (CEST) MRI has been identified as a novel alternative to classical diagnostic imaging. Over the last several decades, many studies have been conducted to determine possible CEST agents, such as endogenously expressed compounds or proteins, that can be utilized to produce contrast with minimally invasive procedures and reduced or non-existent levels of toxicity. In recent years there has been an increased interest in the generation of genetically engineered CEST contrast agents, typically based on existing proteins with CEST contrast or modified to produce CEST contrast. We have developed an in silico method for the evolution of peptide sequences to optimize CEST contrast and showed that these peptides could be combined to create de novo biosensors for CEST MRI. A single protein, superCESTide, was designed to be 198 amino acids. SuperCESTide was expressed in E. coli and purified with size exclusion chromatography. The magnetic transfer ratio asymmetry generated by superCESTide was comparable to levels seen in previous CEST reporters, such as protamine sulfate (salmon protamine) and human protamine. These data show that novel peptides with sequences optimized in silico for CEST contrast that utilize a more comprehensive range of amino acids can still produce contrast when assembled into protein units expressed in complex living environments.

Development of a Synthetic Biosensor for Chemical Exchange MRI Utilizing In Silico Optimized Peptides.

Chemical Exchange Saturation Transfer (CEST) magnetic resonance imaging (MRI) has been identified as a novel alternative to classical diagnostic imaging. Over the last several decades, many studies have been conducted to determine possible CEST agents, such as endogenously expressed compounds or proteins, that can be utilized to produce contrast with minimally invasive procedures and reduced or non-existent levels of toxicity. In recent years there has been an increased interest in the generation of genetically engineered CEST contrast agents, typically based on existing proteins with CEST contrast or modified to produce CEST contrast. We have developed an in-silico method for the evolution of peptide sequences to optimize CEST contrast and showed that these peptides could be combined to create de novo biosensors for CEST MRI. A single protein, superCESTide 2.0, was designed to be 198 amino acids. SuperCESTide 2.0 was expressed in E. coli and purified with size-exclusion chromatography. The magnetic transfer ratio asymmetry (MTR asym ) generated by superCESTide 2.0 was comparable to levels seen in previous CEST reporters, such as protamine sulfate (salmon protamine, SP), Poly-L-Lysine (PLL), and human protamine (hPRM1). This data shows that novel peptides with sequences optimized in silico for CEST contrast that utilizes a more comprehensive range of amino acids can still produce contrast when assembled into protein units expressed in complex living environments.

A Genetic Programming Approach to Engineering MRI Reporter Genes.

Here we develop a mechanism of protein optimization using a computational approach known as "genetic programming". We developed an algorithm called Protein Optimization Engineering Tool (POET). Starting from a small library of literature values, the use of this tool allowed us to develop proteins that produce four times more MRI contrast than what was previously state-of-the-art. Interestingly, many of the peptides produced using POET were dramatically different with respect to their sequence and chemical environment than existing CEST producing peptides, and challenge prior understandings of how those peptides function. While existing algorithms for protein engineering rely on divergent evolution, POET relies on convergent evolution and consequently allows discovery of peptides with completely different sequences that perform the same function with as good or even better efficiency. Thus, this novel approach can be expanded beyond developing imaging agents and can be used widely in protein engineering.

Protein and peptide engineering for chemical exchange saturation transfer imaging in the age of synthetic biology.

At the beginning of the millennium, the first chemical exchange saturation transfer (CEST) contrast agents were bio-organic molecules. However, later, metal-based CEST agents (paraCEST agents) took center stage. This did not last too long as paraCEST agents showed limited translational potential. By contrast, the CEST field gradually became dominated by metal-free CEST agents. One branch of research stemming from the original work by van Zijl and colleagues is the development of CEST agents based on polypeptides. Indeed, in the last 2 decades, tremendous progress has been achieved in this field. This includes the design of novel peptides as biosensors, genetically encoded recombinant as well as synthetic reporters. This was a result of extensive characterization and elucidation of the theoretical requirements for rational designing and engineering of such agents. Here, we provide an extensive overview of the evolution of more precise protein-based CEST agents, review the rationalization of enzyme-substrate pairs as CEST contrast enhancers, discuss the theoretical considerations to improve peptide selectivity, specificity and enhance CEST contrast. Moreover, we discuss the strong influence of synthetic biology on the development of the next generation of protein-based CEST contrast agents.