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. Recent progress in DNA polymerase engineering has successfully isolated variants with expanded substrate spectra capable of efficiently synthesising hexitol nucleic acids (HNA) from DNA templates, and capable of synthesising DNA from HNA templates. Together, they demonstrate that HNA can serve as a genetic material. However, the unavoidable DNA intermediate in HNA replication and their limited processivity greatly limit the potential of these polymerases for the development of an HNA episome.
We are developing novel selection methodologies that will enable us to systematically evolve all components required to establish a viable HNA episome supporting maintenance and replication in vitro. Those include a processive HNA-directed HNA polymerase as well as other cis- (DNA sequences) and trans- (proteins including polymerase) factors.
Rewriting the genetic code through aminoacyl tRNA synthetase engineering
Two classes of molecules are essential to all life on Earth: nucleic acids and proteins. Nucleic acids, such as DNA, are our genetic material and serve as a repository for all information required by each cell to function. That information includes the instructions for the synthesis of all proteins, which are the cellular workhorses and catalyse essential chemical reactions as well as orchestrating how the cells sense and respond to the environment. The genetic code describes how specific DNA sequences are translated into specific protein sequences – such that given a DNA sequence, it is possible to exactly deduce the sequence of the protein generated. The genetic code emerged so early in evolution that, bar minor exceptions, it is universal – a given DNA sequence will generate the same protein in most organisms.
Although conversion of genetic information into a specific protein sequence is a complex multi-step process, a single family of enzymes, aminoacyl-tRNA synthetases (aaRSs), lie at the heart of it – they enforce the genetic code by linking specific nucleic acid sequences to specific protein building blocks (amino acids). We are developing new selection methodologies to enable the directed evolution of synthetases with reassigned amino acid specificity in vitro – providing the first step towards rewriting the natural genetic code.