Efficient RNA synthesis by in vitro transcription of a triazole-modified DNA templatew

Chemical ligation of long synthetic alkyne and azide functionalised oligonucleotides by the CuAAC reaction is a new strategy for overcoming the size limit of DNA and RNA synthesised by solid-phase phosphoramidite chemistry. Remarkably, the latest triazole linker formed by this method (Fig. 1a) is correctly readthrough by DNA polymerases and is functional in bacteria. In order to investigate the molecular basis of this discovery we recently determined the high-resolution NMR structure of a DNA duplex containing this modification and compared it to a fully natural phosphodiester backbone. Although some structural and dynamic perturbations were observed around the triazole (Fig. 1b) the duplex adopted the normal B-conformation. The triazole N2 andN3 nitrogen atoms are located close to the position of the phosphodiester oxygens of normal DNA and we have proposed that they can substitute as hydrogen-bond acceptors in interactions with polymerases. We have shown that the triazole linkage is a plausible phosphodiester surrogate, but for an artificial DNA backbone linkage to be truly biocompatible it must also be functional in other fundamental biological processes, particularly DNA-templated RNA synthesis. This would enable long, fully synthetic, click-ligated DNA constructs to be used directly for the synthesis of biologically active RNA and proteins, and would constitute a significant advance. However, our previous in vivo experiments gave no direct information on transcription. Therefore, to further explore the biological potential of click-ligated oligonucleotides, and provide fundamental information on the transcription of chemically modified DNA, we have investigated in vitro RNA synthesis from DNA that contains a single triazole linkage. The triazole linkage in Fig. 1 allows the DNA duplex to adopt a normal B-conformation in which the Watson–Crick bases are paired and stacked within the helix. However, there is some distortion in the backbone at the site of the triazole. This causes displacement of the deoxyribose sugar to accommodate the longer linkage so that base stacking is preserved. It also leads to a small increase in the distance between the bases on either side of the triazole. We were interested to determine if these perturbations, along with the presence of the rigid triazole ring and lack of negative charge at the backbone, would disrupt interactions with the RNA polymerase sufficiently to prevent transcription. Although we have already shown that replication is not inhibited by the triazole linkage, it was not clear whether this would also be true for transcription. Unlike replication, transcription does not require an oligonucleotide primer; it is initiated by a 50-nucleotide triphosphate and is a fundamentally different process. We chose to work with the RiboMAX large scale RNA production system (Promega) containing the commonly used T7 RNA polymerase (T7-RNAP) which transcribes DNA downstream of a specific promoter sequence. T7-RNAP is an important enzyme for DNA-templated RNA synthesis; it is commonly used in biotechnology for the synthesis of small RNAs and to direct the expression of cloned genes. It does not require auxiliary proteins, and is structurally distinct from