Use of locked nucleic acid oligonucleotides to add functionality to plasmid DNA.
The available reagents for the attachment of functional moieties to plasmid DNA are limiting. Most reagents bind plasmid DNA in a non-sequence- specific manner, with undefined stoichiometry, and affect DNA charge and delivery properties or involve chemical modifications that abolish gene expression. The design and ability of oligonucleotides (ODNs) containing locked nucleic acids (LNAs) to bind supercoiled, double-stranded plasmid DNA in a sequence-specific manner are described for the first time. The main mechanism for LNA ODNs binding plasmid DNA is demonstrated to be by strand displacement. 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 mammalian cells. LNA ODNs can bind to DNA in a sequence-specific manner so that binding does not interfere with plasmid conformation or gene expression. Attachment of CpG-based immune adjuvants to plasmid by 'hybrid' phosphorothioate-LNA ODNs induces tumour necrosis factor-alpha production in the macrophage cell line RAW264.7. This observation exemplifies an important new, controllable methodology for adding functionality to plasmids for gene delivery and DNA vaccination.
Hertoghs
Kirsten M L
KM
Department of Gene and Protein Therapeutics, Discovery Research, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK.
Ellis
Jonathan H
JH
Catchpole
Ian R
IR
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D045744:Cell Line, Tumor; D018899:CpG Islands / Q000235:genetics; D006224:Cricetinae; D004278:DNA, Superhelical / Q000737:chemistry / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry* / Q000235:genetics; D010957:Plasmids / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D004278:DNA, Superhelical; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D008156:Luciferases