A Genetic Programming Approach to Engineering MRI Reporter Genes

Abstract

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.

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

“…Using the previously generated peptide library targeted for 3.6 ppm contrast from the Protein Optimization Engineering Tool (POET), 36 an amino acid sequence was generated by connecting the top-performing peptides end to end to create a 198 amino acid long protein (superCESTide). The amino acid sequence was then optimized for DNA expression in E. coli using the Azenta Life Sciences Codon Optimization tool on GENEWIZ (South Plainfield, NJ, USA).…”

“…The first scan is a water saturation shift referencing (WASSR) 41 scan used to generate a B 0 field map of the phantom and correct the CEST Z-spectra for any field inhomogeneity. 36 The second scan is a CEST Z-spectrum obtained from a modified rapid acquisition with relaxation enhancement (RARE) sequence, with a RARE factor of 25 and a T R of 12 000 ms; FOV = 40 mm  40 mm; matrix size = 48  48; resolution = 0.83 mm. Saturation pulses were applied as a block pulse for 4000 ms, and a saturation power of 3 μT covering saturation frequencies from À7 to 7 ppm offset from water in steps of 0.1 ppm.…”

“…To remedy this issue, we developed a machine learning algorithm that evolved peptides to optimize their CEST contrast while diversifying the amino acid residues in their sequences. 36,42 Having generated a wide range of 12 amino acid long peptides, questions about their expression in cells still existed. While these de novo peptides produced relatively high CEST contrast when suspended in solution, it was unknown if they would still create detectable contrast once expressed as a single genetic unit in a complex organism.…”

“…After studying a library of synthetic peptides, we identified a short list of peptides that produce a superior CEST contrast termed CESTides. 36 Here, we assembled them into a single gene that encodes a recombinant protein called superCESTide (Figure S1) with much more diverse amino acid composition than the past generation of CEST reporter genes.…”

Molecular Imaging with Aquaporin-Based Reporter Genes: Quantitative Considerations from Monte Carlo Diffusion Simulations

“…In synthetic biology, reporters based on fluorescent and bioluminescent proteins provide a natural approach for measuring and optimizing the performance of genetic systems . However, optical reporters are of limited use for tracking genetically engineered devices in living animals due to absorption and scattering of light in thick tissue. − Unlike optical methods, magnetic resonance imaging (MRI) can image deep tissues and generate volumetric scans with high spatial resolution. We recently developed an MRI-based reporter that enables imaging of genetic activity in deep tissues. , This reporter utilizes aquaporin-1 (Aqp1), a channel consisting of a ∼2.8 nm long and ∼0.3 nm wide pore that allows water molecules to diffuse freely albeit selectively across the plasma membrane. , In contrast to wild-type cells, which restrict water movement owing to the low permeability of the plasma membrane, cells engineered to express Aqp1 allow the free exchange of water (Figure a).…”