Development of a Synthetic Biosensor for Chemical Exchange MRI UtilizingIn SilicoOptimized Peptides

Abstract

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 (MTRasym) 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.

The Proton Resonance Enhancement for CEST imaging and Shift Exchange (PRECISE) family of RF pulse shapes for Chemical Exchange Saturation Transfer MRI

“…In the present study, we have focused on developing versatile pulse shapes for CEST MRI using an applied a gradient ascent algorithm to optimize the shapes for a range of Δω and k ca that are observed in the agents employed in our center including the ones evaluated but also glucose 9 , superCESTide 37, 38 , creatine 39 and others. We focused on optimizing for a 100 msec saturation pulse to be inserted into a pulse train for producing CEST MRI contrast, so these pulses are not explicitly optimized for the steady-state CEST sequences with low flip angle pulse and buildup of saturation which are also widely used on clinical scanners.…”

Computational peptide discovery with a genetic programming approach

“…In the future, by combining peptides obtained through the evolutionary algorithm with proteins, it may be possible to achieve a stable structure without compromising the potential for enhanced detection through CEST contrast. A similar approach has been successfully employed in the generation of de novo biosensors for CEST MRI by coupling proteins with peptides exhibiting high CEST potential [28].…”

“…, LLK (28) or LKLL (17). However, there are also motifs that incorporate other AA, such as LQS (10) or SLK (16).…”

“…Conversely, among the 3 peptides in experiment with 100 cycles, the peptide QDGSKKSLKSCK (QDGSK brown line in Figure 8) generated MTR asym 58% greater than the gold standard peptide K12 at 3.6ppm. An interesting observation is that this peptide contains only 25% K residues, which is important for increasing the diversity of the AA composition of genetically encoded reporters [28]. QDGSKKSLKSCK is also unique compared to other peptides since it has a distinct peak at 3.6 ppm, resulting from the amide exchangeable protons, with little or no contribution from amine or guanidine exchangeable protons resonating between 1.8-2.0 ppm.…”

“…Peptide biomarkers are used in different disease diagnostics such as cancer [20,21], type II diabetes [22] or neurodegenerative disorders such as Alzheimer disease [23]. Peptides are also used in imaging diagnostics as positron emission tomography [24], single-photon emission computerized tomography [25] and chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) [26,27,28].…”