Highly specific DNA-interacting enzymes with programmable specificity
Different enzyme families are used in vitro and in vivo for the manipulation of DNA. Restriction endonucleases, exonucleases, DNAseI, DNA repair enzymes and DNA methyltransferases, amongst others, belong to these enzyme families. However, the targeted and exclusive addressing of specific individual genes using these enzymes has not been possible, because the recognition site of the enzyme occurs severalfold in the target DNA.
The invention presented here produces, on the contrary, enzyme conjugates, which specifically interact with target DNA: It also provides methods for manufacturing these enzyme conjugates. Using a linker, enzymes are covalently bonded to enzyme conjugates with a specificity anchor. Here the specificity anchor refers to, for example, oligonucleotide derivatives or peptide nucleic acids that, adjacent to the recognition site for the enzyme residue, are characterised by a triplex forming site (TFS) on the target DNA, whereby the specificity anchor and the linker determine in which position on the target DNA and in which distance to the enzyme residue the triplex formation between the TFS of the target DNA and the triplex forming specificity anchor of the conjugate takes place. As a result, the specificity of a enzyme conjugate according to the current invention can be ?programmed? so that ideally only one single recognition site exists on the target DNA for the enzyme conjugate. Even if the recognition site for the conjugate appears more than once, the number of positions in which the target DNA binds with the conjugate is significantly smaller than the number of recognition sites for the unconjugated enzyme.
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