Use of locked nucleic acid oligonucleotides to add functionality to plasmid DNA

The available reagents for the attachment of func-tional moieties to plasmid DNA are limiting. Most reagents bind plasmid DNA in a non-sequence-speci®c manner, with unde®ned stoichiometry, and affect DNA charge and delivery properties or involve chemical modi®cations that abolish gene expres-sion. The design and ability of oligonucleotides (ODNs) containing locked nucleic acids (LNAs) to bind supercoiled, double-stranded plasmid DNA in a sequence-speci®c manner are described for the ®rst time. The main mechanism for LNA ODNs binding plasmid DNA is demonstrated to be by strand dis-placement. LNA ODNs are more stably bound to plasmid DNA than similar peptide nucleic acid (PNA) `clamps ' for procedures such as particle-mediated DNA delivery (gene gun). It is shown that LNA ODNs remain associated with plasmid DNA after cationic lipid-mediated transfection into mam-malian cells. LNA ODNs can bind to DNA in a sequence-speci®c manner so that binding does not interfere with plasmid conformation or gene expres-sion. Attachment of CpG-based immune adjuvants to plasmid by `hybrid ' phosphorothioate±LNA ODNs induces tumour necrosis factor-a production in the macrophage cell line RAW264.7. This observation exempli®es an important new, controllable method-ology for adding functionality to plasmids for gene delivery and DNA vaccination