Hertoghs Kirsten M L, Ellis Jonathan H, Catchpole Ian R
Department of Gene and Protein Therapeutics, Discovery Research, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK.
Nucleic Acids Res. 2003 Oct 15;31(20):5817-30. doi: 10.1093/nar/gkg801.
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.
用于将功能基团连接到质粒DNA上的现有试剂非常有限。大多数试剂以非序列特异性方式结合质粒DNA，化学计量不确定，会影响DNA电荷和传递特性，或者涉及消除基因表达的化学修饰。首次描述了含有锁核酸（LNA）的寡核苷酸（ODN）以序列特异性方式结合超螺旋双链质粒DNA的设计和能力。证明LNA ODN结合质粒DNA的主要机制是通过链置换。在诸如粒子介导的DNA递送（基因枪）等过程中，LNA ODN比类似的肽核酸（PNA）“夹子”更稳定地结合到质粒DNA上。结果表明，在阳离子脂质介导的转染进入哺乳动物细胞后，LNA ODN仍与质粒DNA相关联。LNA ODN可以以序列特异性方式与DNA结合，因此结合不会干扰质粒构象或基因表达。通过“混合”硫代磷酸酯-LNA ODN将基于CpG的免疫佐剂连接到质粒上，可诱导巨噬细胞系RAW264.7产生肿瘤坏死因子-α。这一观察结果例证了一种重要的新型可控方法，可为基因递送和DNA疫苗接种的质粒添加功能。
