Incorporating noncanonical amino acids (ncAAs) into proteins can grant novel properties by encoding amino acids with additional functional groups and elements, in turn, facilitating the creation of novel proteins for applications in chemical biology and protein therapeutics. Current methods for doing so can incorporate two noncanonical amino acids into proteins and seek to overwrite the current genetic code by repurposing stop or rare codons through the introduction of repurposed archaeal tRNAs and aminoacyl tRNA synthetases (aaRSs). Attempting to overwrite an existing code results in competition with the machinery adapted to the native code, in turn reducing translational fidelity. For example, repurposing tRNA/aaRS pairs from archaea has resulted in two commonly used orthogonal translation units (OTUs) that are not recognized well by the native bacterial translational machinery (including EF-Tu and ribosomes). Instead of overwriting existing codons, we seek to create new ones. We have demonstrated that an unnatural base pair (UBP) can be stably replicated in a semi- synthetic organism (SSO) for this purpose. Transcription and translation of the UBP has been successfully accomplished using the current OTUs available from archaea. With many more codons containing an unnatural base available, we are now limited by the number and efficacy of the available OTUs. Therefore, the central goal of this proposal is to develop OTUs for the translation of unnatural codons.
In Specific Aim 1, the stability and orthogonality of the UBP in all sequence contexts is evaluated in RNA, providing insight into promising codons for translation.
Specific Aim 2 utilizes an unnatural nucleotide in the anticodon of tRNAs as a recognition motifs to reprogram E. coli aaRSs for translation of the UBP. Finally, Specific Aim 3 will switch amino acid specificity of the aaRSs developed in Aim 2 from a canonical amino acid to an ncAA relevant for chemical biology and protein therapeutics. Successful completion of these aims will provide insight into RNA biology, produce OTUs that will seamlessly integrate into the SSO?s translational machinery, and allow the production of proteins with multiple ncAAs. The creation and use of these OTUs has the potential to revolutionize the field of protein therapeutics by providing a robust platform for the production of proteins with several ncAAs.

Public Health Relevance

The proposed work seeks to develop orthogonal translation units for the decoding of unnatural base pairs in a semi-synthetic organism. This will enable the production of proteins with multiple noncanonical amino acids, enabling the discovery, development, and production of biologics and novel protein therapeutics for the diagnosis and treatment of human disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
5F31GM128376-03
Application #
9894806
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Brown, Anissa F
Project Start
2018-04-01
Project End
2021-03-31
Budget Start
2020-04-01
Budget End
2021-03-31
Support Year
3
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037