A triazole mimic of a DNA phosphodiester linkage has been produced by templated chemical ligation of oligonucleotides functionalized with 5′-azide and 3′-alkyne. The individual azide and alkyne oligonucleotides were synthesized by standard phosphoramidite methods and assembled using a straightforward ligation procedure. This highly efficient chemical equivalent of enzymatic DNA ligation has been used to assemble a 300-mer from three 100-mer oligonucleotides, demonstrating the total chemical synthesis of very long oligonucleotides. The base sequences of the DNA strands containing this artificial linkage were copied during PCR with high fidelity and a gene containing the triazole linker was functional in Escherichia coli.replicated in bacteria | triazole DNA backbone | click chemistry | CuAAC reaction S olid-phase DNA synthesis (1, 2) is an advanced technology that has led to pioneering discoveries in biology and nanotechnology (3-8). Although automated solid-phase phosphoramidite synthesis is highly efficient, the accumulation of modifications (mutations) and failure sequences caused by side-reactions and imperfect coupling imposes a practical limit of around 150 bases on the length of oligonucleotides that can be made. Consequently very long synthetic oligonucleotides are not suitable for use in biological applications that require sequence fidelity, so combinations of shorter sequences are normally used in PCRmediated gene assembly (9, 10). This enzymatic method of DNA synthesis has the intrinsic limitation that site-specific chemical modifications can only be introduced in the primer regions of the resulting constructs. Certain unnatural analogues can be inserted throughout the PCR amplicon via modified dNTPs, but this process is essentially uncontrolled and does not allow combinations of different modifications to be incorporated at specific loci. Therefore, for biological studies, important epigenetic and mutagenic bases such as 5-methyl dC, 5-hydroxymethyl dC and 8-oxo dG are normally put into short oligonucleotides and subsequently inserted into larger DNA strands by enzymatic ligation. Templated enzymatic ligation of oligonucleotides can be used to produce large DNA fragments, but this is best carried out on a small scale. In addition, some modified bases are not tolerated by ligase enzymes. Enzymatic methods of gene synthesis are extremely important in biology, but a purely chemical method for the assembly of large DNA molecules would be an interesting and valuable addition to current tools, with the advantages of scalability, flexibility, and orthogonality.It has proved challenging to achieve clean and efficient chemical ligation of canonical DNA, although significant progress has been made using cyanogen bromide as a coupling agent (11,12). An interesting alternative approach is to design a chemical linkage that mimics the natural phosphodiester bond, and that can be formed in high yield in aqueous media from functional groups that are orthogonal to those naturally present in DNA. This goal has been pa...

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Combining enhanced π-stacking, H-bonding and electrostatic attraction in a single C-monomer greatly increases DNA duplex stability and massively destabilises mismatches.

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