The enzymatic synthesis and bioconjugation of 5'-AzaRNAs

• Main Author: Williamson, David
• Published: Durham University 2007
• Subjects: 547.7
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.442554

As research into the interactions and reactivity of RNA species often requires the bioconjugation of reporter or substrate compounds to the oligonucleotide with high regiospecificity, we envisage uses for RNA molecules modified to incorporate reactive aza groups at the 5'-terminus. By incorporating groups with novel reactivity at specific locations within the RNA molecule, 'handles' are provided to facilitate the specific bioconjugation of the RNA to a wide range of compounds. We achieved the modification through the use guanosine derivatives, modified to feature 5’-aza groups, as initiators in the T7 RNA polymerase mediated synthesis of RNA. The figure above shows the structures of the three initiator species used in our research. The synthesis of GANP from Amino-G was achieved via the development of a regiospecific amino-phosphorylation reaction, which takes place in aqueous solution and yields the potentially hydrolytically unstable GANP in high yield (90 %).Through the use of Amino-G and GANP we were able to improve on literature methods for the synthesis of 5'-AminoRNA, raising the observed level of incorporation from 20 % to a maximum of 88 %(^45). The use of Azido-G enabled us to achieve the unprecedented direct incorporation of an azide group into the 5'-terminus of RNA species (37 %). We successfully demonstrated that the azide group could be reduced to give 5'-AminoRNA or used directly in a copper mediated bioconjugation reaction.