The development of catalytic DNA (DNAzymes) for the treatment of infectious disease.
PHE ePoster Library. Pine A. Apr 10, 2019; 257504
Angela Pine
Angela Pine
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Abstract
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Abstract DNAzymes are single stranded DNA molecules that bind to target mRNA via Watson-Crick base pairing. The '10-23' DNAzyme contains a catalytic core of conserved 15 nucleotide sequence enabling the cleavage of mRNA between any unpaired purine and a paired pyrimidine, in the presence of a divalent cation. The cleavage of mRNA decreases protein expression and thus can serve as a viable treatment option for many diseases whereby the causative agent can be targeted by gene downregulation.DNAzymes do not exist in nature and are usually identified using in vitro selection via multiplex assays and examination of kinetic data. This is a time consuming, costly and inefficient process often only leading to a small number of suitable molecules, that may not even be the best. We are developing a computational tool to predict the most efficient DNAzymes against any transcript. In order to do this we compared published DNAzymes against HPV16 with those designed using our program. In vitroassays on short FAM labelled RNA were performed alongside total RNA and full length in vitrotranscribed HPV16 RNA. Lead DNAzymes were also co-transfected into a HPV16 positive cell line, CaSki, alongside published counterparts and their efficiencies compared. We were able to show our program-designed DNAzymes had a cleavage efficiency of up to 94%.As proof of concept we have also developed DNAzymes against an oncogene. Here we have seen in vitroknockdown of the target mRNA of up to 99% and we are currently investigating DNA nanoflower, utilising both aptamer and DNAzyme sequences, as method to deliver these molecules in vivo. Funding Funding for this project has come from a University of Essex scholarship and the Rosetree's Foundation.
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