A universal DNA endonuclease

Xu, Shuang-Yong

Abstract

The goal of this proposal is to engineer universal restriction endonucleases with tailored cleavage specificities. The universal endonucleases will contain two functional components: 1) a specificity determinant and 2) a cleavage domain. The DNA binding specificity will be contributed by a modified singlestranded oligonucleotide (ss oligo) or a peptide nucleic acid oligomer (PNA). The cleavage domain will be derived from the cleavage domain of a type IIS restriction endonuclease (REase) or a non-specific nuclease. The first approach involves fusion of streptavidin and a nuclease cleavage domain. The chimeric nuclease, streptavidin-nuclease fusion protein, is linked to a biotinylated ss oligo through non-covalent binding. The cleavage activity of the chimeric nuclease will be examined on appropriate DNA substrates-. The second approach will explore the covalent attachment of an ss oligo or a PNA to the nuclease domain using inteinmediated protein ligation (IPL). The IPL reaction requires a cysteine which will be incorporated into the oligo or PNA during chemical synthesis. For in vivo applications of the universal nuclease, the oligonucleotide-Cys will be modified to contain locked nucleotides (LNA). Such LNA-oligos are resistant to endogeneous endonuclease and exonuclease attack. Accessory proteins such as RecA, helicase, and SSB will be evaluated to facilitate target DNA binding and cleavage. For isothermal applications of the universal nuclease at 50oC-70oC, the nuclease domain will be derived from thermophilic endonucleases.