Ligase-Free Synthetic Gene Assembly

CONTEXT
Synthetic biology is a pioneering avenue of research that traverses all of the disciplines of science to deliver new materials, pharmaceuticals, food and consumer products. An essential component to fabricating and applying biological systems in both research and commercial sectors is the ability to generate and manipulate DNA as a repository for genetic information. In order to make and modify proteins for biotechnological applications, the appropriate genetic code can be presented to an organism, however, chemists are still unable to synthesise sufficiently long DNA sequences to make entire genes and hence their useful protein products. In the past, biologists harnessed multiple enzymes, and a process called ligation, to fuse DNA segments into usable formats. Whilst this approach proved effective, there are still limitations in terms of time, efficiency and cost when applied to the large-scale synthesis of multiple genes. Hence there is now a clear opportunity to develop new, faster, accurate and cost-effective strategies for the complete synthesis of large DNA molecules using synthetic chemistry alone.

AIMS AND OBJECTIVES
Our project aims to employ readily available and inexpensive chemicals to connect small, overlapping strands of DNA in tandem to assemble synthetic genes. We will exploit our knowledge of how these chemicals behave and react in water to generate simple, efficient protocols for the gene assembly process. A range of chemical and biochemical techniques will be used to confirm that our ligation approach to strand joining has been successful. Ultimately we will prepare a synthetic gene and use it to make the protein that it encodes. The success of our research plan is based upon clear experimental evidence that demonstrates the validity of our strand ligation strategy. Our goal is to further enhance ligation efficiency by generating new and improved chemical building blocks to make the short DNA strands (known as oligonucleotides). These component oligonucleotides will be tested, the ligation conditions optimised, and then we will target our ultimate goal of a synthetic gene, to demonstrate the complete concept.

POTENTIAL APPLICATIONS AND BENEFITS
Our oligonucleotide ligation technology can be readily applied to DNA assembly to speed up and reduce the costs of gene and genome manufacture. Our approach will initially be targeted at improving gene synthesis, however, it could also benefit a wide range of nucleic acid-based approaches from molecular diagnostics to medical therapies and even in crop protection. Because our approach offers rapid chemistry, it will be ideal for monitoring large numbers of samples in parallel (high-throughput). It will also offer advantages to large-scale applications, such as the generation of larger quantities of ligated nucleic acids for gene therapies, and even for RNA-based crop protection agents.

These applications offer economic, environmental, societal and medical benefits across a wide range of users.

Synthetic biology envisages the construction of DNA molecules that code for genes, metabolic pathways and even complete genomes. However, the fabrication of such large assemblies is hampered by current approaches to mass production. We describe an entirely chemical approach to overcoming hurdles to rapid and efficient gene synthesis. Synthetic amine-terminated oligonucleotides will exploit Watson-Crick base pairing to bring their termini into close proximity for gene assembly. P(V) reagents that selectively react with amine groups will be deployed to ligate the oligos with a group mimicing the natural phosphate backbone. Second generation oligonucleotides will employ more nucleophilic hydrazine and methoxylamine groups that will improve ligation efficiency. The effectiveness of ligation using different P(V) reagents and conditions will be quantified by gel electrophoresis using fluorophore-labelled oligos. The 'read-through' properties of ligated oligonucleotides will be verified in vitro using T7 RNA polymerase and Taq DNA polymerase. Nucleotide sequencing will confirm accurate replication by these polymerases through the ligation sites. Synthetic gene assembly for in vivo applications will be established by placing both short and long inserts into a plasmid vector. The short insert clone will induce a frame-shift in the vector lacZ gene to allow a colour-based colony screening for insertion efficiency in parallel with existing enzymatic ligation methodologies. The longer gfp-based gene insert will be used to assess the validity of the approach towards the preparation of proteins. A fluorescence readout from colonies, alongside the colour-based screen, will reveal the efficacy of synthetic gene insertion, and nucleotide sequencing will be used to confirm the fidelity of insertion. Our approach has the potential to revolutionise gene construction and is readily adaptable to a myriad of other nucleic-acid based synthesis and screening technologies.

Who will Benefit from this research?

ACADEMICS
A range of academics both nationally and internationally will benefit from this study, including those engaged in: 1) basic cloning methods (geneticists and molecular biologists), 2) synthetic biology (molecular biologists and chemists) and nucleic acid tools (biologists, chemists and physicists). Further details on likely and potential applications are described under 'Academic Beneficiaries'.

PDRA
The PDRA will gain highly specialist training in nucleoside and oligonucleotide chemistry. These skills will support a major synthetic biology challenge, thus equipping the PDRA with cross-disiplinary expertise in bioconjugation, electrophoresis and cloning. This training will benefit the employability of the PDRA and the skills they can offer employers in both academic and commercial sectors.

INDUSTRY
A range of industrial users will benefit from the proposed working, and in the longer term, further applications that our ligation technology could underpin. The industrial beneficiaries could include: protein and enzyme production facilities, encompassing all those involved in overproduction of proteins through the creation of custom-made genes and rapid methods for directed mutagenesis. Our assembly-ligation method also allows the targeted insertion of 'random' nucleotide segments within pools of genes that could facilitate directed evolution and protein selection approaches towards the generation of new and improved biocatalysts.

SYNTHETIC BIOLOGISTS
This research offers significant short-term benefits to both academic and industrial users. Initially, training will be provided for a PDRA to engage with nucleic acids as foundations for synthetic biology. The technology will be IP protected by Durham University allowing scope for a UK industrial partner to exploit our approach. Applications of gene technologies include the preparation of pharmaceutical proteins (biologics) and enzyme catalysts (preparation of small molecules, technology applications and consumer cleaning products). These applications will offer benefits to downstream manufacturers, patients and day-to-day users of gene-derived products. The providers of oligonucleotides and the prerequisite building blocks will also benefit through the marketing of new, and greater numbers of, products.

How might they benefit from this research?

Academics will benefit through access to enhanced tools for cloning, synthetic biology and the construction of nucleic acids assemblies for basic and applied research. At Durham, opportunities will be created for the research team for future research funding and the university through IP exploitation.

The PDRA will benefit through training and improved employability prospects and earnings.

Industry will benefit through gaining a skilled employee. More significantly, industry will benefit through commercial exploitation of making oligonucleotide building blocks, the oligonucleotides themselves and the applications these systems offer across a range of science arenas.

Further details are explored in the Pathways to Impact document.