Double Incorporation of Non-Canonical Amino Acids in an Animal and its Application for Precise and Independent Optical Control of Two Target Genes

Proteins and the nucleic acids DNA and RNA are the most fundamental building blocks of life. Proteins are chains of amino acids made from 20 different building blocks, the canonical amino acids. The genetic code is the cipher that connects proteins to nucleic acids.

When proteins are produced, the sequence of nucleic acids is translated into a sequence of amino acids. There are 4 nucleotide bases of DNA and RNA organized in triplets. There are 64 triplets of which 61 are each assigned to one amino acid, while three triplets act as 'stop codons' and signal the end of the protein.

The genetic code is the link between triplet codons and amino acids. The 'decoding' of the nucleotide sequence happens using transfer RNA (tRNA). A tRNA molecule contains a triplet codon on one end and the corresponding amino acid at the other end. The amino acid is attached to tRNA by an enzyme called aminoacyl-tRNA-synthetase (aaRS). The tRNA triplet binds to an mRNA triplet and an amino acid is added to the growing protein chain.

Genetic code expansion is a technology whereby new tRNAs and aaRS are added to the cell, allowing it to make proteins using more than the 20 canonical amino acids. The additional amino acids, called non-canonical amino acids (ncAA), are chemically synthesized 'designer' amino acids with properties not found in nature. To specify where the ncAA should go within the amino acid sequence of a protein it is necessary to assign them to a nucleic acid triplet.

Since 61 triplets are already assigned, this is normally done by making use of one of the three stop codons.

We have previously established this technology in animals and we have used it to develop powerful in vivo tools that employ proteins modified by the introduction of ncAA. In this proposal we aim to take the next big step and develop a method allowing the use of multiple ncAA together, the first time this will be made possible in an animal.

For this we will develop i) additional aaRS/tRNA pairs for use in C. elegans, which can be used together with existing pairs without cross-reacting, and ii) we will establish new 'blank' nucleotide codons to determine where in the protein the ncAA will be incorporated. We will explore the use of triplet stop codons other than the widely used UAG (amber) stop, as well as nucleotide quadruplet codons.

We have recently shown that the use of quadruplet codons is possible in animals and can approach incorporation efficiencies close to that observed with traditional triplet codons.

We will then use the technology to upgrade a method we have previously developed for controlling gene expression with light. Using the new ability to employ two ncAA we will be able to switch on or off two genes independently of each other. This is an important breakthrough and will allow us and other researchers to, for example, control the activity of single nerve cells in C. elegans and tease apart the functioning of the C. elegans nervous system with a level of precision that is impossible using existing methods.

The technology we develop will also be immensely useful for researchers outside of C. elegans to study the basics of how life works in anything from bacteria to human cells.