Synthetic Biology

Prof David P Hornby

Sheffield 222 4232
d.hornby@sheffield.ac.uk

Career History

Research Areas

1. Developing analytical methods for genomics and proteomics
2. Expanding the functional repertoire of natural macromolecular assemblies
3. Remodelling enzymes and toxins

1. Developing analytical methods for genomics and proteomics

As the Nobel Laureate Sydney Brenner said some years ago:
“Progress of science depends on new techniques, new discoveries and new ideas, probably in that order”

My group has been interested in the “automation” of Biochemical and Molecular Biology related methodology for many years. Working with a number of UK and US companies, we have initially developed methods for the isolation and analysis of DNA, which is incorporated into instrumentation for the detection of mutations. In addition, using reverse phase ion pair chromatography as a platform, we developed methods for a wide range of applications in the analysis of nucleic acid enzymology, in particular the analysis of modified nucleic acids. Building on this work we went on to adapt the technology for the quantitative analysis of gene expression, for the detection of differential methylation of genomic DNA and for the automation of molecular cloning (1-6). With the emergence of the functional role for RNA in molecular biology, we developed methods for the isolation and analysis of all forms of RNA together with methods for the interrogation of RNA structure.

In 2006, we described a novel capillary based method for macromolecular purification and analysis which remains under development in conjunction with Phynexus (www.phynexus.com), with whom my laboratory has enjoyed a long and productive collaboration. In this method, proteins, nucleic acids, large macromolecular assemblies and even cells and organelles, can be isolated rapidly in a biocompatible manner; by incorporating a modified TAP tag approach. We are currently attempting to apply this technology for the structural analysis of macromolecular assemblies in close collaboration with the X-ray crystallography and Cryo Electron Microscopy groups in the Krebs Institute at Sheffield.

In 2004, Professor Bernard Connoly’s group showed that a high fidelity, thermostable DNA polymerase could be induced to make introduce a relatively high frequency of mutations in PCR products (Biles & Connolly (Nucleic Acids (2004) 32:e176). We have recapitulated this property in the Pho DNA polymerase, and have designed in, an N-terminal single strand DNA binding polypeptide and a C-terminal Nickel binding domain taken from the human transcription and splicing factor p54. This Lo-Fi polymerase provides us with a robust reagent for experimental, “directed evolution” of a range of proteins. The application of this methodology has led us to uncover hitherto unknown determinants of base flipping in the family of cytosine specific DNA methyltransferases (see below). The combination of rapid affinity purification, biophysical interrogation, structural biology and directed evolution methods underpins our approach to Synthetic Biology (see below).

2. Expanding the functional repertoire of natural occurring polypeptides and macromolecular assemblies

It is one of the key aims of Synthetic Biology, to engineer novel polypeptides for Biotechnological, therapeutic and diagnostic purposes, drawing on our knowledge of naturally occurring proteins. However, predictive power is only robust when the knowledge base is comprehensive. The greatest successes to date in de novo molecular design are emerging from remodelled binding domains, where structural biology brings enormous insight (exemplified by the molecules of the human immune system). However, redesigning enzyme specificity remains a formidable challenge and this is an area that we are currently investigating using the cytosine-specific DNA methyltransferases (C5MTases) as a test bed.

The introduction of a methyl group at the C5 position of the cytosine ring, within the structural context of double-helical DNA results from a reaction catalysed by C5MTases. These enzymes were the first to expose the phenomenon of base flipping, when the Nobel Laureate (and Sheffield alumnus) Rich Roberts and his crystallography colleague, Xiaodong Cheng published the structure of HhaI DNA methyltransferases in complex with its target DNA duplex (Klimasauskas et al (1994) Cell 76 357-69). This discovery subsequently led to the realisation that base flipping underlies many nucleic acid transactions in replication, repair, recombination as well as modification. With an efficient purification technology together with a facile method for generating targeted random mutagenesis by PCR and semi-automated cloning procedures, we are now in a strong position to investigate the limitations of our knowledge of protein structure and function. In parallel we intend to use the principles of the design cycle used by engineers in order to introduce new functions into a range of proteins, protein assemblies and enzymes.

3. Remodelling enzymes and toxins

Current targets for Synthetic Biology in the laboratory include:

	DNA methyltransferases	Exploring base flipping determinants, specificity and the use of high affinity DNA binding mutants to interrogate response to genome damage
	Fc domains of antibodies	Exploring oligomerisation phenomena for aiding vaccine design
	Snake venom toxins	Investigating opportunities for therapeutic toxin design
	Bacteriophage	Investigating protein and nucleic acid assembly for vaccine design
	Yeast interactome	Developing methods for the isolation, interrogation and structural analysis of protein assemblies in model organisms

Selected Publications

Zhou, L., Cheng, X., Connolly, B.A., Dickman, M.J., Hurd, P.J. & Hornby, D.P. (2002) Zebularine: a novel DNA methylation inhibitor that forms a covalent complex with DNA Methyltransferases. J. Mol. Biol. 321 591-599

Hanna C, Gjerde D, Nguyen L, Dickman M, Brown P, Hornby D.P. (2006) Micro-scale open-tube capillary
separations of functional proteins. Anal. Biochem. 350, 128-37.

Dickman, M.J., Sedelnikova S.E., Rafferty, J.B. & Hornby, D.P. (2004) Rapid analysis of protein nucleic acid
complexes using MALDI TOF mass spectrometry and ion pair reverse phase liquid chromatography. J.
Biochem.Biophys.Methods 58 39-48

Xu W, Liu L, Brown NJ, Christian S, Hornby D. (2012) Quantum dot- conjugated anti-GRP78 scFv inhibits
cancer growth in mice. Molecules 17, 796-808.

A complete listing can be found by searching [Hornby, D] on PubMed