Advancing directed protein evolution technology

In nature, proteins take millions and trillions of years to evolve¹, but scientists do not have this time when designing drugs for life-threatening diseases. Therefore, researchers use directed protein evolution technologies, to evolve and design proteins with specific functions under well-defined conditions and a practical time frame.

At PEGS Europe, I spoke to Victoria Goldenshtein, a PhD candidate in Biomedical Engineering at Duke University, US, who presented an engaging poster on her lab’s novel in vitro library display platform for directed protein evolution termed GRIP Display, which entails Gluing RNA to Its Proteins.

Motivations behind developing new technology

“We have developed GRIP Display with a very specific application in mind: optimisation of the binding kinetics of a large protein with already great nano-molar affinity to its binding partner²,” Goldenshtein explained.

She also highlighted that existing directed protein evolution technologies have several limitations, creating a need to develop a new platform, explaining that “traditional methods fall short in their ability to display large protein variants in vast numbers while maintaining a stable link between the displayed variant and its encoding genetic material, needed for protein identification.”

For example, in vivo platforms (such as Phage Display) require bacterial transformation, imposing a bottleneck of ~10⁹ variants, non-covalent genotype-to-phenotype in vitro platforms have been found to fall far short of the needed linkage stability, while covalent in vitro technologies require a multi-step process of chemical modification to achieve a stable link between RNA and protein, resulting in a low-yield procedure^3,4.

A novel directed protein evolution platform

To address the limitations, Goldenshtein and the team developed GRIP Display, “a novel one-pot in vitro display method that can rapidly generate and screen vast protein libraries (up to 10^14 variants), including large proteins, against any target of interest.” She explained that with GRIP Display the team was able to successfully modify their proprietary drug delivery technology named DART (Drugs Acutely Restricted by Tethering), which rapidly localises drugs to the surface of defined neurons in the brain.² 

How does GRIP Display work?

“GRIP Display utilises a high-affinity interaction between a small peptide motif and a short RNA hairpin sequence borrowed from a virus. This interaction has previously been used in various applications such as biochemical assays and live cell imaging, but not in a library display context,” explained Goldenshtein when asked about the technology’s functionality. The conjugated mRNA serves as a unique identifier for each variant, enabling fast identification of the best binders for further characterisation and development.

“However, displaying biologics in a library poses its own set of challenges, such as ensuring linkage fidelity where each mRNA molecule is bound to its corresponding protein, and linkage stability that lasts for hours under a wide range of conditions.”

At PEGS Europe, Victoria Goldenshtein presented an engaging poster on her lab’s novel in vitro library display platform for directed protein evolution termed GRIP Display.

To overcome these challenges, the team focused their efforts on technology optimisation, resulting in a patent-pending three-dimensional mRNA design and improved avidity of the peptide-RNA tandems. This allows each individual mRNA to be glued to its own protein without crosstalk or swapping with other RNA/protein pairs.

Next steps

In addition to publishing this research, Goldenshtein hopes to validate this technology in clinically relevant fields such as cancer therapeutics development using nanobodies, short antibody fragments and small peptides. Looking to the future, Goldenshtein would like to focus on the overall goal of targeting the undruggable G protein-coupled receptors (GPCRs).

References

1. Agozzino L, Dill KA. Protein evolution speed depends on its stability and abundance and on chaperone concentrations. Proceedings of the National Academy of Sciences. 2018;115(37):9092–7.
2. Shields BC, Kahuno E, Kim C, et al. Deconstructing behavioral neuropharmacology with cellular specificity. Science. 2017;356(6333).
3. Bhide M, Guillemin N, Comor L, et al. Library-based display technologies: where do we stand? Mol Biosyst. 2016;12(8):2342-2358. doi:10.1039/c6mb00219f
4. Jijakli K, Khraiwesh B, Fu W, et al. The in vitro selection world. Methods. 2016;106:3-13. doi:10.1016/j.ymeth.2016.06.003