Final Report Summary - HNAEPISOME (Directed evolution of a synthetic episome based on hexitol nucleic acids (HNA))
A long-term goal of synthetic biology is the assembly of a cell from its individual components. A genetic element based on synthetic nucleic acids capable of stable propagation, a synthetic episome, is the minimal genetic element required for the systematic development of all cellular components of a synthetic organism based on artificial nucleic acids (XNAs). Such synthetic episomes require a processive XNA replicase (capable of synthesising XNA from an XNA template) as well as accessory proteins to maintain the XNA information stably – none of which are naturally available.
Directed evolution is a powerful strategy for the isolation of novel function and it is able to bypass knowledge gaps, such as the lack of detailed structure or incorrect mechanistic understanding of an enzyme. An approach to directed evolution is the selection of a desired activity from a large library of mutants. However, given the number of potential variants for even a small protein, it would not be possible to generate all possible variants – both in number and in technical challenge.
Here, we have solved the technical challenge of mutant generation developing a platform that can target any custom mutation to any custom site in a given gene, without constrains of sequence or position (Darwin assembly). For the development of an XNA replicase, a second problem emerges because not enough is known about DNA polymerases (our starting point) to accurately target sites that control substrate specificity. For that, we have developed new models of polymerase function as well as new structure-based approaches for protein engineering. We applied those methods to a hyperthermophilic DNA polymerase from T. kodakariensis generating a new XNA - phosphonomethyl, which, being modified in the phosphate moiety and capable of limited interaction with DNA, is the most orthogonal genetic material developed to date. Similarly, we isolated mesophilic HNA synthetases based on the Phi29 DNA polymerase, which outperform all previously engineered variants. In addition, we have developed novel strategies for directed evolution of proteins that are currently being tested and will be validated in subsequent projects.
We have improved in vitro selection platforms for DNA polymerases and ligases and we have also validated bacterial cell display as a viable ex vivo platform for the directed evolution of nucleic acid processing enzymes. In particular, the cell display platform we have developed is ideally suited for the selection of weak binders (as expected from DNA and XNA binding proteins) and has potential for the screening of protein-protein interactions. Those strategies are enabling technologies that can and will now be deployed against other targets.
Directed evolution is a powerful strategy for the isolation of novel function and it is able to bypass knowledge gaps, such as the lack of detailed structure or incorrect mechanistic understanding of an enzyme. An approach to directed evolution is the selection of a desired activity from a large library of mutants. However, given the number of potential variants for even a small protein, it would not be possible to generate all possible variants – both in number and in technical challenge.
Here, we have solved the technical challenge of mutant generation developing a platform that can target any custom mutation to any custom site in a given gene, without constrains of sequence or position (Darwin assembly). For the development of an XNA replicase, a second problem emerges because not enough is known about DNA polymerases (our starting point) to accurately target sites that control substrate specificity. For that, we have developed new models of polymerase function as well as new structure-based approaches for protein engineering. We applied those methods to a hyperthermophilic DNA polymerase from T. kodakariensis generating a new XNA - phosphonomethyl, which, being modified in the phosphate moiety and capable of limited interaction with DNA, is the most orthogonal genetic material developed to date. Similarly, we isolated mesophilic HNA synthetases based on the Phi29 DNA polymerase, which outperform all previously engineered variants. In addition, we have developed novel strategies for directed evolution of proteins that are currently being tested and will be validated in subsequent projects.
We have improved in vitro selection platforms for DNA polymerases and ligases and we have also validated bacterial cell display as a viable ex vivo platform for the directed evolution of nucleic acid processing enzymes. In particular, the cell display platform we have developed is ideally suited for the selection of weak binders (as expected from DNA and XNA binding proteins) and has potential for the screening of protein-protein interactions. Those strategies are enabling technologies that can and will now be deployed against other targets.