Project Summary 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.