Construção de um sistema de veiculação e expressão de DNAzimas para Escherichia coli: um protótipo para a fagoterapia

Abstract

Antisense oligonucleotides have great potential for use as therapeutic agents. The DNAzymes 10-23 are antisense molecules with improved chemical stability and catalytic efficiency. This work developed a system for DNAzymes expression regulated by two promoters built within a phagemid, which was called pDESCP. The expression of MoMuLV-RT enzyme occurs in a constitutively way through the pA1 promoter. The second cassette, regulated by the lac promoter, expresses an RNA transcript that will give origin at DNAzyme. MoMuLV-RT will act on the RNA substrate, and then DNAzymes are produced by reverse transcription. The DNAzyme ftsZ, used to validate this system, was efficiently expressed and able to change the morphology of Escherichia coli, which went from bacillary to filamentous form. When properly packaged by M13 phage, pDESCP was used to transfect different strains of E. coli F+. In vitro kinetic assays involving DNAzymes with target on transcripts of alanine racemase genes of E. coli, alr e dadX, showed no conclusive results. This made it impossible for these were tested on the phagemid. The forwarding of pDESCP for E. coli F+ and the possibility of inserting new DNAzymes in its structure make this phagemid an important prototype for phage therapy. Provided that lethal DNAzymes are developed against E. coli, these can be inserted into pDESCP and turn it into a powerful tool able to prevent the transmission of pathogenic genes from bacteria that transfer by conjugation