Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.
Nucleic Acids Res. 2012 Mar;40(5):2152-67. doi: 10.1093/nar/gkr885. Epub 2011 Nov 8.
Anti-miRs are oligonucleotide inhibitors complementary to miRNAs that have been used extensively as tools to gain understanding of specific miRNA functions and as potential therapeutics. We showed previously that peptide nucleic acid (PNA) anti-miRs containing a few attached Lys residues were potent miRNA inhibitors. Using miR-122 as an example, we report here the PNA sequence and attached amino acid requirements for efficient miRNA targeting and show that anti-miR activity is enhanced substantially by the presence of a terminal-free thiol group, such as a Cys residue, primarily due to better cellular uptake. We show that anti-miR activity of a Cys-containing PNA is achieved by cell uptake through both clathrin-dependent and independent routes. With the aid of two PNA analogues having intrinsic fluorescence, thiazole orange (TO)-PNA and [bis-o-(aminoethoxy)phenyl]pyrrolocytosine (BoPhpC)-PNA, we explored the subcellular localization of PNA anti-miRs and our data suggest that anti-miR targeting of miR-122 may take place in or associated with endosomal compartments. Our findings are valuable for further design of PNAs and other oligonucleotides as potent anti-miR agents.
反义 miR 是与 miR 互补的寡核苷酸抑制剂，已被广泛用作研究特定 miR 功能的工具和潜在的治疗方法。我们之前曾表明，含有几个附着赖氨酸残基的肽核酸 (PNA) 反义 miR 是有效的 miR 抑制剂。我们以 miR-122 为例，报告了用于有效靶向 miR 的 PNA 序列和附着氨基酸要求，并表明末端游离巯基（如半胱氨酸残基）的存在可显著增强抗 miR 活性，主要是由于更好的细胞摄取。我们表明，含有半胱氨酸的 PNA 的抗 miR 活性是通过细胞摄取通过网格蛋白依赖和独立途径实现的。借助两种具有固有荧光的 PNA 类似物，噻唑橙 (TO)-PNA 和 [双-o-(氨基乙氧基)苯基]吡咯胞嘧啶 (BoPhpC)-PNA，我们探索了 PNA 抗 miR 的亚细胞定位，我们的数据表明，miR-122 的抗 miR 靶向可能发生在或与内体隔室相关。我们的发现对进一步设计作为有效抗 miR 剂的 PNA 和其他寡核苷酸具有重要价值。
