Physical Sciences︱Afaf El-Sagheer & Tom Brown Producing the first functioning synthetic DNA using

Click chemistry revolutionised very so often an observation, our ability to create custom experiment or discovery proteins, so could it do the same changes more than just scientific E DNA is made up of bases which match in understanding. Darwin, Copernicus, and for DNA? Professor Tom Brown pairs. These base pairs form the ‘rungs’ of at the and Newton’s breakthroughs, while scientific, the ladder-like structure.

Professor Afaf El-Sagheer at also had significance for philosophy, Morphart Creation/Shutterstock the University of Oxford and religion and metaphysics. Suez University are performing CHEMICAL LIGATION truly groundbreaking research Perhaps Friedrich Wöhler’s 1828 finding Since 2007, Afaf El-Sagheer and El-Sagheer and Brown’s triazole into the application of ‘click − that urea can be made from potassium Tom Brown have been pioneering an linkage is a unique example of a chemistry’ bioconjugation cyanate and ammonium chloride – should alternative approach to the chemical techniques to DNA. Their also be included in this list. Urea, in his synthesis of large DNA and RNA strands. biocompatible artificial DNA linkage technique allows the creation own words an ‘animal substance’, was the Their premise is that longer oligos of DNA and RNA constructs first to be created without any biological could be created from shorter oligos formed using chemical synthesis. larger than any other chemical starting materials, signalling the end for using chemical ligation rather than Friedrich Wohler created the first biological synthetic route and is more the doctrine of vitalism. enzymatic ligation. click reaction” by Sharpless, a pioneer T-triazole-T sequence was converted to flexible than the use of enzymes. substance from purely chemical materials. of click chemistry techniques. CuAAC a single ‘T’ in the PCR product. In other This is made possible by a In the decades since Wöhler, the list But this is no mean feat: it’s not possible creates a triazole linkage – a five- words, the linkage is not a very good novel chemical linkage which of biochemicals created using purely vitamin B12 and insulin; more recently for the chemical ‘links’ to be exact membered ring of atoms containing two mimic of the phosphodiester linkage is biocompatible with DNA- chemical means has grown longer, and larger molecules have become possible, replicas of the phosphoester linkage carbon and three nitrogen atoms. Initial found in natural DNA. In addition, manipulating cellular machinery the molecules themselves have grown including chemotherapies, antibiotics, and found in natural DNA. Instead, the experiments showed that it was possible producing oligos with the correct in E coli and human cells. larger and more complex. Notable even proteins. But despite our progress, chemical links in these DNA replicas need to combine various oligomers using a precursors on each end for the ligation early advancements include glucose, there is still no effective chemical synthesis to be ‘biocompatible’ – indistinguishable version of CuAAC which joined two ‘T’ step was difficult to accomplish. of probably the most famous biomolecule to natural enzymes from the real thing. nucleotides with a triazole linkage. of them all: Long DNA. Although one base was missing, the Finding a chemical reaction which These oligomers were then ‘amplified’ remarkable achievement was that The modern technique for constructing produces a biocompatible chemical using PCR – the biochemical process the PCR process, for the first time, DNA sequences involves using linkage is a major milestone in El-Sagheer used to replicate a DNA sequence. In successfully amplified triazole-link- biological enzymes to ligate (join and Brown’s work. The researchers PCR, the oligomer molecules containing containing oligomers. together) short synthetic sections outline in their 2009 research how they the triazole linkage need to be ‘read’ by Pablo Joanidopoulos/ShutterstockPablo of DNA, called ‘oligonucleotides’ use ‘click chemistry’, a set of chemical DNA polymerase, the key enzyme in the DEVELOPING BETTER LINKAGES or ‘oligos’. Multiple oligos can be reactions known to efficiently form replication process. If the triazole linkage With the promise of biocompatibility, El- assembled into genes and plasmids. bonds between biomolecules and is biocompatible, then the PCR process Sagheer and Brown began redesigning other molecules, a process known as should produce copies of the oligomer similar linkages, applying their technique Oligos are almost always produced bioconjugation. sequence. These resulting copies would to both DNA and RNA. using the solid-phase phosphoramidite not contain the triazole linkage. method. The error rate in this method CuAAC In 2010 work, they demonstrated that is reasonably low – enough to create El-Sagheer and Brown began working on LIMITATIONS OF CuAAC a redesigned triazole linkage could be oligos typically around 100 bases long. developing a variation of the ‘copper(I)- However, this version of the CuAAC used to synthesise ribozymes, which are However, at around the 150 bases mark, catalyzed azide-alkyne cycloaddition’ reaction had its limitations. While the a type of cellular machinery formed from errors start to become a problem. As a (CuAAC) to find a method which is DNA triazole linkage is read by DNA RNA. RNA is a similar molecule to DNA Some oligonucleotides are used as result, long segments of DNA require suitable for ligating oligomers. CuAAC polymerase in PCR, the process is slow, in many ways – it’s a polymer composed fluorescent biomarkers which can be useful in medical diagnosis. large numbers of oligos, requiring is the ‘classic’ click chemistry reaction, and results in the deletion of one of the of nucleic acids – but it does have some complex biochemical techniques. described as “the premier example of a two nucleotides around the triazole. The key differences which make long RNA

www.researchoutreach.org www.researchoutreach.org chains even more difficult to synthesise Repeating the test on E. coli cells than DNA. Crucially, RNA contains an deficient in DNA repair enzymes gave extra hydroxyl group at the sugar, which a similar survival rate, demonstrating Behind the Research can lead to unwanted side reactions that the success wasn’t down to the unless it is protected during solid linkage being ‘corrected’ by E. coli. The phase synthesis and deprotection. This link is truly biocompatible with DNA- hydroxyl group is also a ‘steric hindrance’, reading enzymes. Professor Afaf Professor meaning it physically gets in the way of molecules approaching the bond MEETING THE DEMANDS OF CRISPR The team tested their method using the El-Sagheer Tom Brown formation site. bacteria E. coli. El-Sagheer and Brown’s triazole linkage is a unique example of a biocompatible As a result, RNA chains longer than artificial DNA linkage formed using E: [email protected] T: +44 1865 275175 E: [email protected] T: +44 1865 275413 http://www.browngroupnucleicacidsresearch.org.uk/ 50 nucleotides are difficult to produce new triazole linkage: the DNA was chemical synthesis. Almost 200 years W: in a pure state, which doesn’t come accurately copied, with nucleotides on on from Wöhler’s synthesis of urea, the near to the length of biologically both sides of the triazole linkage included enzyme-free synthesis of DNA – or at functional RNA constructs like in the oligomer copies. least a functional mimic of it – is now Research Objectives ribozymes and riboswitches. possible, using click chemistry. Professors El-Sagheer and Brown investigate the chemical synthesis of long DNA and RNA strands. IN VIVO STUDIES Using the CuAAC reaction, El-Sagheer However, the true test of biocompatibility One area where the technique could and Brown were able to cross-link is within cells. In a crucial collaboration excel is in the emerging and promising separate strands of oligomers together with Ali Tavassoli and Pia Sanzone at technology of CRISPR gene editing. Detail to form two RNA constructs, both Southampton University, oligomers The technique relies on creating RNA almost 100 nucleotides in length. Both were ligated using the CuAAC to form a sequences called single guide RNAs Tom Brown Bio Funding the constructs – a hairpin ribozyme triazole-containing piece of DNA known (sgRNAs). When CRISPR-Cas9 leaves Department of Chemistry Tom studied for his chemistry degree and PhD at and Biological with cross-links, and a hammerhead to code for antibiotic resistance, which the laboratory and enters diagnostic University of Oxford Bradford University. He was appointed as a Lecturer Sciences Research Council ribozyme with the improved linkage at was inserted into the DNA of E. coli cells. and therapeutic use, sgRNAs will Chemistry Research Laboratory then Professor at Edinburgh University before (BB/J001694/2 and BB/ the active site – functioned like their be in high demand. But current use 12 Mansfield Road moving to Southampton University in 1995 then to R008655/1) natural equivalents. Overnight incubation on agar plates of enzymes to produce a library of Oxford OX1 3TA Oxford University in 2013. He is co-founder of three containing ampicillin revealed exactly sgRNAs is both time-consuming and UK Biotech companies (Oswel, Primer Design, ATDBio). Collaborators He has published over 400 papers and patents. • Ali Tavassoli After establishing the biocompatability what the team was hoping for: the plates difficult to scale. Afaf El-Sagheer • Pia Sanzone of the new linkage in RNA in 2010, with the triazole DNA added contained Department of Chemistry Afaf studied chemistry at Suez Canal University • Lapatrada Taemaitree this improved linkage was used to 96.5% of the colonies; in the negative These short sections of RNA are formed University of Oxford (Egypt) and did her PhD at Southampton University • Arun Shivalingam ligate three 100-base-long oligomers control it was only 1.6%. In other words, of two parts. Firstly, a section about 80 Chemistry Research Laboratory with Professor John Mellor then moved back to • Agnes Tyburn to construct a DNA molecule of 300 E. coli cells given artificial DNA were nucleotides long which binds to the Cas9 12 Mansfield Road Egypt to become a lecturer. She was promoted bases in length, and amplified using shown to be resistant to the antibiotic – protein, and is the same in all sgRNA Oxford OX1 3TA to Associate Professor in 2009 then Professor in PCR. Analysis of the resulting sequence and the triazole link is biocompatible with molecules. Secondly, a section about UK 2014 at Suez University. Afaf has been on a long confirmed the biocompatibility of the E. coli cellular machinery. 20 nucleotides long which is unique to and sabbatical as an academic visitor at Southampton the sequence of DNA near where the Chemistry Branch and Oxford universities since 2005. She has over edit should be made. This guides the Department of Science and Mathematics 150 publications. 200 years on from Wöhler’s synthesis Cas9 protein to the correct part of the Faculty of Petroleum and Mining of urea, the enzyme-free synthesis of DNA molecule. Engineering Afaf and Tom’s research interests are in Suez University oligonucleotide synthesis and its applications in long DNA – or at least a functional In their 2019 research published in Suez 43721 chemistry, biology and medicine (diagnostics and Communications, the group Egypt therapeutics). mimic of it – is now possible. detailed how the CuAAC reaction can be used to produce a library of sgRNAs in a single tube – by ligating the two parts of sgRNA oligomers using click References Personal Response chemistry. The advantage of using this What would it take for this procedure to become a

Yeinism/Shutterstock technique is that the Cas9-binding Taemaitree, L., Shivalingam, A., El-Sagheer, A.H. and Brown, commercially available product? oligomer could be produced at a T. (2019). Nat. Commun. 10, 1610. large scale, meaning only the 20-base El-Sagheer, A.H. and Brown, T. (2017). Chem. Commun. 53, The basic research has been carried out and patents oligomer needs to be tailored, and the 10700. have been filed. The next step is for an industrial partner to work on applications that could benefit from the two halves can be ligated. El-Sagheer, A.H. and Brown, T. (2012). Accounts of Chemical approach. This could be for example in CRISPR/Cas Research. 45 (8), 1258-1267. but there are many other potential areas where click- The resulting sgRNA allowed gene- El-Sagheer, A.H. et al. (2011). PNAS. 108 (28), 11338–11343. ligation could offer advantages, such as modified genes, editing in cells with no unexpected El-Sagheer, A.H. and Brown, T. (2010). PNAS. 107 (35), especially with epigenetic modification which can play a The team’s CuAAC reaction could help off-target effects. Perhaps CRISPR will be 15329–15334. vital role in gene therapy. produce key elements needed for CRISPR gene editing technology. where the triazole linkage leaves its mark El-Sagheer, A.H. and Brown, T. (2009). J. Am. Chem. Soc. on DNA chemistry. 131, 3958-3964.

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