Background:
At AstraZeneca, our aim is to build a world class peptide discovery platform, combining phage and mRNA display technologies and amenable to the incorporation of a wide variety of non-canonical amino acids (ncAAs) in those display systems, from ncAAs capable of forming covalent bonds to enable new chemistries in peptides, such as cyclization, to ncAAs which can introduce novel physicochemical properties, such as altered hydrophobicity, charge, or backbone rigidity, to manipulate peptide solubility, cellular uptake, or overall structural dynamics.
Techniques like sense codon reprogramming and stop codon suppression are used to introduce ncAAs into the genetic code during in vitro translation. A critical step in the process is the creation of tRNAs that carry unnatural amino acids, allowing for their incorporation into proteins at specific, reprogrammed codon sites. To produce amino-acylated tRNAs for codon reprogramming, researchers utilize 2 main methods:
- Engineering of highly specific and efficient aaRS/tRNA pairs. This strategy requires careful optimization of the translation system to prevent competition from endogenous components and to minimize misincorporation and suffers from relatively low efficiency of ncAA incorporation. Furthermore, the engineering of highly specific and efficient aaRS/tRNA pairs remains a technical hurdle.
- Chemical acylation of tRNAs, in which purified tRNAs are reacted with activated ncAA derivatives, to generate misacylated tRNAs that can be directly used in cell-free translation systems. This technique bypasses the need for a dedicated synthetase and enables the incorporation of a wide array of non-canonical amino acids, including those that are synthetically challenging for enzymes to recognize.
We are seeking:
- Solutions to efficiently introduce ncAA allowing for the formation of thioether cyclised macrocycles in both phage and mRNA display systems.
- Solutions to efficiently introduce ncAA in an mRNA display system to bring in novel properties or stability to the peptide hist such as but not limited to: N-methylated amino acids, fluorosulphate-amino acid, Aib
The Challenge:
Provide solutions for the efficient incorporation of ncAAs in phage and/or mRNA display systems that allow for:
- Thioether macrocycle cyclisation
- Efficient incorporation of a range of amino-acids
The Solution:
Proposed solutions to this challenge should meet the following solution requirements to be considered for funding by AstraZeneca. The selected collaborator will need to provide AstraZeneca with reagents allowing for at least one of the following:
- Efficient incorporation of ncAAs allowing for thioether macrocycle cyclisation in a phage display system. This should go beyond the simple use of evolved tRNA synthetases and overcome some of the limitations of stop codon reprogramming
- Efficient incorporation of ncAAs allowing for thioether macrocycle cyclisation in an mRNA display/cell free protein expression system
- Incorporation of other types of ncAA such as but not limited to N-methylated amino acids, fluorosulphate-amino acid, Aib, in an mRNA display/cell free protein expression system
Solutions Requirements:
- Researchers must have the capability to carry out the work proposed (i.e. the research plan cannot be hypothetical in nature).
- Researchers should provide a statement of expertise and/or any facilities that will be used to execute the proposal. Upon selection, the researcher must be able to begin the project immediately.
- Researchers must have experimental proof-of-concept data, a description of the proposed methods for ncAA incorporation in suitable display systems, and evidence that demonstrates the feasibility of the approach.
- The proposal must contain at minimum proof-of-concept data showing introduction of a ncAA relevant for this proposal for peptide expression in a relevant system for their technology (in vitro transcription/translation or E.coli).
- The proposed solution must be amenable to technology transfer, offer licensing options or the possibility to buy final products for drug development purposes
- AstraZeneca will provide phage display/peptide expression constructs and experience where needed but expect respondents to have some phage display/cell free protein expression experience themselves.
