Kontroliuojamo aktyvumo restrikcijos endonukleazių-tripleksą formuojančių oligonukleotidų konjugatai

Simple mutations within the coding region of critical human genes can lead to the formation of abnormal proteins, resulting in various diseases (e.g. cancer), in failure of an embryo to develop, or premature death. Genetic diseases can only be truly cured via restoration of defective gene function and one of the most promising strategies is based on homologous recombination. Naturally homologous recombination occurs with a low frequency (1 in 106 transfected cells), however it is known that DNA double-strand breaks enhance the efficiency of homologous recombination by several orders of magnitude (up to 10,000-fold). Therefore, gene therapy via homologous recombination requires new molecular tools that should be highly specific and rigorously controllable. In this work we have focused on the development of restriction enzyme-triple helix forming oligonucleotide (TFO) conjugates, where TFO provides specificity for the extended recognition site through the triple helix formation and addresses restriction enzyme to a particular target site where it introduces a double stranded break. We provide proof-of-concept demonstrations of two alternative strategies to control the DNA cleavage activity of restriction endonuclease-TFO conjugates, that allows adopt them in in vivo experiments. To this end we used restriction endonucleases MunI and Bse634I, which were structurally and biochemically characterized before in our laboratory. We successfully combined the restriction endonuclease photocaging and TFO-coupling to generate a photoswitchable MunI-TFO conjugate and provide the first demonstration that DNA cleavage activity of the caged MunI-TFO conjugate can be spatially and temporally regulated. We also generated Bse634I-TFO conjugate and demonstrated that two monomers assemble into the active dimer on the DNA providing a possible alternative for the catalytic module of the zinc finger nuclease. Moreover, in contrast to the FokI non-specific catalytic domain in zinc finger nucleases, Bse634I retains specificity for the cognate site and therefore is less prone to the off-site cleavage