Drug Target Review’s Ria Kakad recently spoke with Victoria Goldenstein about her lab’s novel IVF library display platform for directed evolution of proteins called GRIP display – sticking RNA to its proteins.
In nature, proteins evolve over millions and trillions of years1, but scientists don’t have that time when designing drugs for life-threatening diseases. Therefore, researchers use directed protein evolution technologies to develop and design proteins with specific functions under well-defined conditions and a practical time frame.
At PEGS Europe, I spoke with Victoria Goldenstein, a PhD student in biomedical engineering at Duke University, USA, who presented a fascinating poster on her lab’s novel IVF a library display platform for directed protein evolution called GRIP Display, which involves sticking RNA to its proteins.
Motives behind the development of new technologies
“We developed the GRIP Display with a very specific application in mind: optimizing the binding kinetics of a large protein with already high nanomolar affinity for its binding partner2”, Goldenstein explained.
She also emphasized that existing technologies for directed protein evolution have several limitations, creating a need to develop a new platform, explaining that “traditional methods fail to display large protein variants in huge numbers while maintaining a stable relationship between the displayed variant and its coding genetic material needed to identify proteins.
For example, Live platforms (such as Phage Display) require bacterial transformation, imposing a barrier of ∼109 variants, non-covalent genotype to phenotype IVF The platforms were found to be far from the required bond stability while covalent IVF technologies require a multi-step chemical modification process to achieve a stable RNA-protein association, resulting in a low-yield procedure3,4.
A novel directed platform for protein evolution
To address the limitations, Goldenshtein and team developed the GRIP Display, “a new one-pot option.” IVF display method that can rapidly generate and screen huge protein libraries (up to 10^14 variants), including large proteins, against any target of interest. She explained that with the GRIP Display, the team was able to successfully modify their proprietary drug delivery technology, called DART (Drugs Acutely Restricted by Tethering), which rapidly localizes drugs to the surface of certain neurons in the brain.2
How does GRIP Display work?
“GRIP Display uses 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 the context of a library display,” Goldenstein explained when asked about the functionality of the technology. The conjugated mRNA serves as a unique identifier for each variant, allowing rapid identification of the best binders for further characterization and development.
“However, displaying biological products in a library poses its own set of challenges, such as ensuring binding fidelity, where each mRNA molecule is bound to its corresponding protein, and binding stability that lasts for hours under a wide range of conditions .”
At PEGS Europe, Victoria Goldenstein presented a fascinating poster on her lab’s novel IVF a library display platform for directed protein evolution called GRIP Display.
To overcome these challenges, the team focused their efforts on technological optimization, which led to a patent-pending three-dimensional design of the mRNA and improved avidity of the peptide-RNA tandems. This allows each individual mRNA to be attached to its own protein without crossing or exchanging with other RNA/protein pairs.
next steps
In addition to publishing this research, Goldenshtein hopes to validate this technology in clinically relevant areas, such as developing cancer therapeutics using nanobodies, short antibody fragments and small peptides. Looking to the future, Goldenshtein would like to focus on the general goal of targeting non-druggable G protein-coupled receptors (GPCRs).
Victoria Goldenstein is an analytical chemist by education. Her undergraduate research involved investigating the affinity of zinc finger proteins for heavy metals. While pursuing her master’s degree, she joined the Mathiowitz lab at Brown University, where she learned polymer nanoencapsulation techniques and studied factors influencing the interaction of nanoparticles with gastrointestinal mucin to develop new oral drug delivery systems. At Duke University, she is a postdoctoral fellow in the Tadross lab, focusing on improving drug delivery systems based on high-affinity biologics.
References
- Agozzino L, Dill KA. The rate of protein evolution depends on its stability and abundance and on chaperone concentrations. Proceedings of the National Academy of Sciences. 2018; 115 (37): 9092–7.
- Shields BC, Kahuno E, Kim C, et al. Deconstructing behavioral neuropharmacology with cell specificity. Science. 2017;356(6333).
- Bhide M, Guillemin N, Comor L, et al. Library-based display technologies: where are we? Mol Biosyst. 2016; 12 (8): 2342-2358. doi:10.1039/c6mb00219f
- Jijakli K, Khraiwesh B, Fu W, et al. The IVF selection world. Methods. 2016; 106: 3-13. doi:10.1016/j.ymeth.2016.06.003