Award details

Synthetic Biology facility at the MRC Laboratory of Molecular Biology

Reference	BB/M000842/1
Principal Investigator / Supervisor	Dr Hugh Pelham
Co-Investigators / Co-Supervisors	Professor Jason Chin, Dr Philipp Holliger
Institution	University of Cambridge
Department	LMB Protein and Nucleic Acids
Funding type	Research
Value (£)	1,970,000
Status	Completed
Type	Research Grant
Start date	28/07/2014
End date	31/03/2016
Duration	20 months

Abstract

The objective of this work is to develop, test and expand new technologies based on the encoded synthesis of proteins containing non-natural amino acids and nucleic acids with novel backbone chemistry and/or bases (XNAs). Such approaches enable the creation of proteins with novel functional properties, such as reactive residues that can be cross-linked, or fluorescently labelled; photo-activated groups; defined post-translational modification; or photo-caged enzymatic activity, either by expression in E. coli or in situ in cells and organisms. They also allow the selection and evolution of novel nucleic acid aptamers with resistance to biological and chemical degradation. Ultimately, encoded synthesis of completely novel defined polymers, by engineered ribosomes in cells or by polymerases in vitro should be possible. The common approach involves the evolution of the new activities required to for polymerases to copy DNA to XNA and vice versa, or for aminoacyl-tRNA synthetases to recognise novel amino acids and tRNAs, and for the creation of bacterial strains containing orthogonal ribosomes that recognise only specifically engineered mRNAs, and have altered properties such as the reading of multiple quadruplet codons. Generally, this involves the combinatorial synthesis of many variant DNA sequences, and selection of genes with the right properties in vitro or in bacteria. Chemical synthesis of novel amino acid derivatives and probes is a key requirement. Assaying the incorporation of novel amino acids, and additional events such as protein-protein crosslinking, depends heavily on mass spectrometry. Many other experiments, such as specific protein labelling, photo-control of signalling pathways and the fate and properties of selected aptamers will be based on advanced optical microscopy.

Summary

Living cells have genes made of DNA. DNA is a molecule comprising a string of bases of four different types, and the sequence of these bases in a gene directs the synthesis of a particular protein, which consists of a string of amino acids of twenty different types. Three bases encode one amino acid, this code being read by a cellular machine, the ribosome, which makes the protein. Some triplets do not encode amino acids, but act as stop signals. It is possible to engineer cells such that one of these stop triplets instead encodes a synthetic amino acid, which can have novel chemical properties. Genes can be chemically synthesised with any sequence, so by introducing the appropriate triplet proteins can be specified that have novel properties, such as an enzyme that can be turned on by light. Synthetic biology also allows the creation of new kinds of ribosome in bacteria, engineered to translate only particular synthetic genes, which can read four bases at a time instead of three, thus significantly expanding the range of new amino acids that can be encoded. This technology not only makes possible a wide range of experiments to study the functions of cells and organisms, but also allows the production of novel proteins for therapeutic or other use, such as antibodies with drugs attached at specific sites. Ultimately, such engineering could produce entirely new encoded polymers with many potential uses. DNA too can be altered in novel ways. By engineering the enzymes that copy DNA, it is possible to produce, in the test tube, "XNA" molecules that retain the double helical structure of DNA but are made from chemically different precursors, and hence have a different chemical structure that is resistant to natural and chemical degradation, and can have other novel properties. By making billions of variants of a short sequence of bases, one can select molecules that have a desirable property, such as inhibiting an enzyme or catalysing a reaction, copy theminto DNA, and identify the particular sequence. These molecules can be chemically synthesised, and used as potential drugs or for many other purposes. The development of these technologies depends on the synthesis of novel DNA sequences and genes, and many sophisticated forms of analysis including mass spectrometry and advanced microscopy to determine the properties of the novel proteins and XNA molecules that result, and their effects on cells and organisms. This award will provide the advanced equipment required to automate gene synthesis, engineer new functions, and test the many different applications of the new technology.

Impact Summary

Who will benefit from this research? Research scientists in wide areas of structural, cell and molecular biology, both in the UK and internationally. Staff and shareholders of pharmaceutical and biotech companies. Patients who ultimately are treated with products derived from this technology. How will they benefit? Researchers will have access to powerful new tools for discovery, in the form of reagents, strains and protocols. By expanding the natural genetic code to allow the programming of new chemistries into proteins, either expressed and purified or in situ in cells and organisms, many different experiments will be enabled, including the ability to control important cellular processes with light, to understand the biochemical roles of natural protein modifications, to identify protein-protein interactions, to visualise the distribution of proteins at high resolution by fluorescent labelling, to make specific, controllable and irreversible inhibitors. These tools in turn will lead to new discoveries in basic biology. Companies will be able to license the technologies and use them to develop innovative products. These may include, for example, defined antibody-drug conjugates for treating cancer. Aptamers formed of non-natural nucleic acids will be resistant to degradation (or can be designed to be degraded to toxic products), and potentially offer an alternative to some antibody drugs, with the potential for novel functions such as cell penetration, targeted chemotherapy, or sequence-specific (i.e. gene-specific) effects. Patients will benefit from novel treatments based on these technologies. Ultimately, as with other technologies such as monoclonal antibodies, there may be uses, such as in pregnancy testing, which extend to the general public. By training young scientists to combine chemistry with biology the facility will produce highly skilled interdisciplinary experts, of potential benefit to both future academic research groups and companies.

Committee	Not funded via Committee
Research Topics	Microbiology, Synthetic Biology, Technology and Methods Development
Research Priority	X – Research Priority information not available
Research Initiative	DNA Synthesis (DNASyn) [2014]
Funding Scheme	X – not Funded via a specific Funding Scheme

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