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

Abstract

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.

Protein Optimization Evolving Tool (POET) based on Genetic Programming

“…Furthermore, POET was successfully deployed to evolve peptides with new function, i.e., reporting MRI contrast from peptides at a resonance frequency that could not be detected otherwise. POET generated CESTides could potentially be assembled into the next generation of MRI reporter gene 52 with improved sensitivity over previous generations of reporters.…”

“…Furthermore, POET was successfully deployed to evolve peptides with new function, i.e., reporting MRI contrast from peptides at a resonance frequency that could not be detected otherwise. POET generated CESTides could potentially be assembled into the next generation of MRI reporter gene 52 with improved sensitivity over previous generations of reporters. Since POET requires only a small set of input peptide sequences and their corresponding biological quantitively measured function to evolve models that predict better peptide function, it is anticipated that POET can be used for evolving of peptides in numerous applications.…”

Using genetic programming to predict and optimize protein function

“…The magnetic susceptibility or Chemical Exchange Saturation Transfer (CEST) contrast of peptides is our goal here to show a proof-of-concept of the efficacy of the method. CEST (van Zijl & Yadav, 2011) is a magnetic resonance imaging (MRI) contrast approach in which peptides with exchangeable protons or molecules are saturated and (Gilad et al, 2022;Airan et al, 2012;Gilad et al, 2007;Perlman et al, 2020). POET here aims to aid DE by predicting better functioning CEST contrast proteins and replacing the rigorous and costly task of screening and mutagenesis.…”

Using Genetic Programming to Predict and Optimize Protein Function

“…CEST [10] is a Magnetic Resonance Imaging (MRI) contrast approach in which peptides with exchangeable protons or molecules are saturated and detected indirectly through enhanced water signals after transfer. The main advantage of CEST based proteins is that they can be encoded into DNA and expressed in live cells and tissue, thus allowing tracking non-invasively with MRI [11,12,13,14]. POET here aims to aid DE by predicting better functioning CEST contrast proteins and replacing the rigorous and costly task of screening and mutagenesis.…”