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A Triazole Linkage That Mimics the DNA Phosphodiester Group in Living Systems

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A Triazole Linkage That Mimics the DNA Phosphodiester Group in Living Systems PERSPECTIVE A triazole linkage that mimics the DNA phosphodiester group in living systems Afaf H. El-Sagheer1,2 and Tom Brown1* 1 Department of Chemistry, University of Oxford, Chemistry Research Laboratory, 12 Mansfield Road, Oxford, OX1 3TA, UK 2 Chemistry Branch, Department of Science and Mathematics, Faculty of Petroleum and Mining Engineering, Suez Canal University, Suez 43721, Egypt Quarterly Reviews of Biophysics (2015), 48(4), pages 429–436 doi:10.1017/S0033583515000141 Abstract. We describe the development of a chemical process based on the CuAAC reaction (click chemistry) to ligate DNA strands and produce an unnatural triazole backbone linkage. The chemical reaction is templated bya complementary DNA splint which accelerates the reaction and provides the required specificity. The resultant 1,4-triazole linkage is read through by DNA and RNA polymerases and is biocompatible in bacterial and human cells. This work has implications for the synthesis of chemically modified genes and other large modified DNA and RNA constructs. Key words: triazole DNA, Biocompatibility, Click chemistry. Introduction The synthesis of large DNA constructs for applications in sequencing steps used commonly in the synthesis of large Synthetic Biology is routinely carried out by multiple cycles DNA constructs are essential to edit out these errors, adding of polymerase chain reaction (PCR) amplification of oligo- significantly to the cost of synthesis. This complex protocol nucleotide pools, mismatch repair, cloning, sequencing presents a significant bottleneck in the supply of long DNA and selection. Various approaches based on these principles strands, for which demand is escalating. Further improve- have been used to assemble entire bacterial and mitochon- ments in oligonucleotide synthesis chemistry could have a drial genomes and regions of eukaryotic chromosomes of major impact in this area. 1 megabase in length (Dymond et al. 2011; Gibson et al. A more demanding factor that should be considered into fu- 2010a, b). In efforts to minimize errors in the sequence of ture DNA synthesis strategies is the need for chemically the synthesized DNA, high-fidelity proofreading DNA poly- modified DNA. It has been established that epigenetic mod- merases are employed in the DNA amplification steps, and ifications to cytosine in eukaryotic DNA control levels of enzymatic mismatch repair is incorporated into the work- gene expression through mechanisms that are only partly flow (Kunkel, 2004). Using such protocols mutations are understood. The major epigenetic modifications are kept to a low level, in the best cases to around 1 error in 5-methylcytosine (mC) and 5-hydroxymethylcytosine 106. This is a remarkable achievement considering that (hmC) (Bachman et al. 2014; Branco et al. 2012), but solid-phase oligonucleotide synthesis (Caruthers, 1991), their chemically related ‘oxidation products’ 5-formylcyto- which provides the oligonucleotide-building blocks for sine (fC) and 5-carboxylcytosine (caC) are also present at gene synthesis, produces much higher error rates. These in- lower levels (Pfaffeneder et al. 2011). Our current under- clude nucleotide deletions due to inefficient coupling and standing is that these modified cytosine bases, along with nucleobase modifications arising from various side reac- A, G, C and T are the eight bases that constitute the tions. The time-consuming mismatch repair, cloning and human epigenome. Synthesizing DNA constructs (including entire genomes) containing modified cytosine nucleobases * Author for Correspondence: T. Brown, Department of Chemistry, should enable gene expression to be precisely tuned, leading University of Oxford, Chemistry Research Laboratory, 12 Mansfield Road, ffi Oxford OX1 3TA, UK. Tel.: +44 (0)0865 275413; Fax: +44 1865 285002; to more e cient engineered organisms and increased yields Email: [email protected] of gene products. This has implications for the production © Cambridge University Press 2015. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http:// Downloaded from https://www.cambridge.org/corecreativecommons.org/licenses/by/3.0/),. IP address: which 170.106.35.229 permits unrestricted, on 01 Oct re-use,2021 at distribution,00:22:58, subject and to reproduction the Cambridge in anyCore medium, terms of use, provided available the at original work is https://www.cambridge.org/core/termsproperly cited. https://doi.org/10.1017/S0033583515000141 of economically important biomolecules, such as proteins and linkage that mimics the natural phosphodiester bond and small molecule drugs. We should also not overlook the possi- which can be formed in high yield in aqueous media from func- bility that other hitherto undiscovered DNA modifications tional groups that are orthogonal to those present in DNA. might exist, and could exert an additional level of control This greatly broadens the chemical space that can be explored. over the genome. In addition, the synthesis of DNA contain- The templated reaction between 3′-phosphorothioate and ing chemically modified bases or sugars has many potential 5′-tosylate (or iodide) modified oligonucleotides has been stud- applications in biology and nanotechnology. This makes the ied (Herrlein et al. 1995; Xu & Kool, 1998), but this chemistry synthesis of chemically modified DNA even more important. does not satisfy all of the key requirements for robust chemical DNA synthesis which are: (i) the use of functional groups that The PCR-based strategies described above are not viable for are stable in aqueous media; (ii) the ability to synthesize bifunc- the synthesis of epigenomes. This is because they do not tional oligonucleotides with reactive groups at each end for use permit the site-specific incorporation of cytosine derivatives. in multiple ligation reactions; (iii) a trigger to initiate the lig- All four modified cytosine bases form stable base pairs with ation reaction when participating oligonucleotides have been guanine, so there is no obvious way to control their relative carefully hybridized to complementary splints under thermo- incorporation during PCR amplification. For example, mix- dynamic control (to arrange the DNA strands in the desired tures of dCTP and dmCTP will produce PCR products with order by templated pre-assembly); and (iv) the creation of a random mixtures of C and mC opposite the G bases in the stable backbone linkage. template. To address this problem, a combination of PCR amplification followed by controlled insertion of modified An efficient chemical ligation method would increase the oligonucleotides can be used for locus-specific insertion of maximum achievable size of chemically synthesized oligonu- modified cytosines, but it would be extremely tedious and cleotides, and could in principle be optimized for the total inefficient to make large DNA constructs by this strategy. chemical synthesis of large DNA constructs. It could be of Templated ligation using ligase enzymes to assemble DNA value in nanotechnology, but its potential in biology will entirely from synthetic oligonucleotides is another option. only be fully realized if DNA strands containing the artificial This requires the availability of highly pure oligonucleotides, linkages can act as templates in replication and transcription. precise control of stoichiometry between the constituent However, after billions of years of evolution the phosphodie- oligonucleotides and highly efficient and robust ligase ster linkage has become ubiquitous in the DNA and RNA of enzymes. It is an area that is rife for development. living systems, and there is no reason a priori to expect DNA polymerases to tolerate an artificial backbone. Moreover, any Given that every nucleotide in a DNA construct synthesized change to the structure of DNA caused by the presence of a by ligation of oligonucleotides would originate from chemi- modified backbone is likely to alter the biophysical properties cal synthesis, and the only remaining enzymatic steps would of the duplex, including its thermodynamic stability and the involve joining 5′-phosphate to 3′-hydroxyl groups of adjac- shape of the helix. Despite these reservations there is reason ent DNA strands, a simple question arises. Can the entire to believe that alterations to the chemical structure of the DNA synthesis process, including DNA strand ligation, be DNA backbone can be tolerated in biology; some DNA poly- carried out chemically without the need for enzymes? This merases have the capacity to read through damaged DNA, would be challenging because although it is possible to and engineered enzymes can perform this feat with remark- synthesize oligonucleotides up to 200 nucleotides in length able efficiency (d’Abbadie et al. 2007). This provided us for ligation, the aforementioned limitations of solid-phase with further impetus for the design of a biocompatible synthesis make it impossible to make them in a pure state. DNA backbone linkage. This is in part due to the physical properties of the solid supports on which the synthesis is conducted, but it is also a consequence of the limitations of the chemistry, namely imperfect coupling and side-reactions that create Design and evolution of a biocompatible mutagenic modifications to the nucleobases. For long oligo- fi nucleotides such modifications cannot be removed by puri- modi ed DNA linkage − fication, and error rates as high as 10 2 have been reported In order to produce a biocompatible chemical linkage we (Hecker & Rill, 1998). Circumventing the problem by as-
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