All living organisms transcribe and translate the information encoded by DNA by well-understood processes known as transcription and translation respectively. The process converts the information stored in DNA into protein functions that are essential catalysts for life supporting reactions. In this process, every three nucleotides in a gene are read as a specific code for the serial incorporation of the natural building blocks protein synthesis. This study aims at refactoring the translational machinery to evolve novel transfer-RNA species that will recognize not a three-, but four-nucleotide code for the specific translational incorporation of unnatural amino acids. The objective is to create proteins that feature novel catalytic functions and chemical properties to aid biochemical and biomedical research. In addition, this research will shed light on fundamental questions about detailed mechanisms of translation. The project will attract, train, and transform today's young talents into tomorrow's leaders in sciences at the interface of chemistry and biology. The scientific insights and new technologies obtained under the proposed research program will be valuable to a broad biochemistry and synthetic biology research community.
While the triplet codon is the predominant form of the current genetic code, non-canonical reading of a quadruplet codon is a natural process and can be achieved under experimental conditions. Transfer RNA (tRNA) plays important roles in the quadruplet codon decoding process. The work will use a directed evolution approach to identify tRNA mutants that are able to efficiently decode quadruplet codons in a genomically recoded bacterium. A systematic study of quadruplet codon decoding mechanism will be conducted, which is likely contribute to the understanding on how ribosomes recognize the reading frame. This research is further strengthen by expanding the cellular genetic code with multiple and mutually orthogonal quadruplet codons. This would allow for the incorporation of t unnatural amino acids with different physical and chemical properties. Applications of this technology could enable unprecedented in vivo study of proteins in living cells, and could enable novel synthetic biology applications.
