Custom tRNA Synthesis with Unnatural Amino Acids

This feature of the biochemical flexibility of the cellular translation provides an easy route to the synthesis of drug-like modified peptides and novel biopolymers. A novel translation system has recently been developed that allows for the synthesis of peptides that are composed primarily of unnatural amino acids (UAAs) by utilizing ribosomes. With the rapid development and expansion of protein engineering techniques, a series of new possibilities have been created for studying or constructing the reactivity, stability and spectroscopic properties of engineered proteins using UAAs. Currently, UAA incorporation has been applied in numerous fundamental research applications. The introduction of UAAs into target homologues can selectively activate, repress or reversibly regulate target homologues. Through enzymatic reactions, UAA can be loaded onto tRNA, which enables many structurally and chemically different unnatural amino acids to bind site-specifically, facilitating many applications including protein imaging, protein engineering and protein structure studies, and functional regulation.

Fig 1. Modified translation system obtained with the UAA incorporation. (Zhao H, 2021)

Application of tRNA Incorporated with UAAs

In recent years, a series of approaches have been developed to modulate protein function through genetically incorporated UAAs. Incorporated UAAs are able to modulate protein function through translational, optical or chemical control. The incorporation of UAAs in tRNA can endow polypeptide sequences with novel structural and functional features, which enables ribosomes to synthesize highly modified drug-like peptides.

Fig 2. Single-site and multi-site incorporation of UAAs. (Zhao H, 2021)

Enzymatic Aminoacylation of tRNA with UAAs

The chemical aminoacylation of tRNAs allows amino acid analogs to enter the translation pathway. In this process, aminoacyl-tRNA synthetase (AARS) plays a crucial role in the enzymatic aminoacylation of tRNA. A large number of amino acid analogs of interest can therefore be incorporated into peptides with the employment of mutant AARS. We offer site-specific incorporation services to deliver UAA in response to nonsense or four-base (frameshift) codons by utilizing tRNA/aminoacyl-tRNA synthetase (aaRS) pairs. Orthogonal tRNAs can be identified to transfer specific UAAs by orthogonally inhibiting the tRNA/aaRS pair. Our method allows for rapid qualitative analysis of aminoacyl-tRNA synthesis using multiple unnatural amino acid analogs in a single reaction mixture.

Standard workflow:

• Synthesis of UAA with appropriate bioorthogonal handles.
• Rational design for site-specific incorporation of UAA into tRNA.
• Purification and characterization of modified proteins.

Our Capabilities

Protein Engineering Techniques

We provide synthetic strategies and services for unnatural amino acids to generate modified proteins with altered function and methods for their incorporation. BOC Sciences' experts provide the latest protein engineering techniques for incorporating UAA with bioorthogonal handles such as:

• Azides for click reactions or Staudinger ligation
• Alkynes for click reactions
• Ketones for amine reactions
• Aryl halides for palladium-catalyzed cross-couplings
• Alkenes for metathesis

Identification of Available UAAs

BOC Sciences provides an aminoacyl-tRNA synthetase (AARS)-based assay with the application of matrix-assisted laser desorption/ionization - mass spectrometry (MALDI-MS) to rapidly identify UAAs that can beenzymatically charged onto tRNA. Finally, our experts screen and identify commercially available unnatural amino acids as well as unnatural backbone and side-chain analogs that can be enzymatically charged onto tRNA.