Dipartimento di Chimica Organica e Industriale, Università di Parma, Parco Area delle Scienze 17A, 43124 Parma, Italy.
Chembiochem. 2012 Jun 18;13(9):1327-37. doi: 10.1002/cbic.201100745. Epub 2012 May 25.
A series of 18-mer peptide nucleic acids (PNAs) targeted against micro-RNA miR-210 was synthesised and tested in a cellular system. Unmodified PNAs, R(8) -conjugated PNAs and modified PNAs containing eight arginine residues on the backbone, either as C2-modified (R) or C5-modified (S) monomers, all with the same sequence, were compared. Two different models were used for the modified PNAs: one with alternated chiral and achiral monomers and one with a stretch of chiral monomers at the N terminus. The melting temperatures of these derivatives were found to be extremely high and 5 M urea was used to assess differences between the different structures. FACS analysis and qRT-PCR on K562 chronic myelogenous leukaemic cells indicated that arginine-conjugated and backbone-modified PNAs display good cellular uptake, with best performances for the C2-modified series. Resistance to enzymatic degradation was found to be higher for the backbone-modified PNAs, thus enhancing the advantage of using these derivatives rather than conjugated PNAs in the cells in serum, and this effect is magnified in the presence of peptidases such as trypsin. Inhibition of miR-210 activity led to changes in the erythroid differentiation pathway, which were more evident in mithramycin-treated cells. Interestingly, the anti-miR activities differed with use of different PNAs, thus suggesting a role of the substituents not only in the cellular uptake, but also in the mechanism of miR recognition and inactivation. This is the first report relating to the use of backbone-modified PNAs as anti-miR agents. The results clearly indicate that backbone-modified PNAs are good candidates for the development of very efficient drugs based on anti-miR activity, due to their enhanced bioavailabilities, and that overall anti-miR performance is a combination of cellular uptake and RNA binding.
针对 micro-RNA miR-210 的一系列 18 个碱基的肽核酸（PNA）被合成并在细胞系统中进行了测试。未修饰的 PNA、R(8)修饰的 PNA 和含有 8 个精氨酸残基的骨架修饰的 PNA，无论是 C2 修饰（R）还是 C5 修饰（S）单体，都具有相同的序列，这些都被进行了比较。两种不同的模型被用于修饰 PNA：一种是手性和非手性单体交替的模型，另一种是 N 末端具有一段手性单体的模型。这些衍生物的熔点非常高，需要使用 5 M 尿素来评估不同结构之间的差异。FACS 分析和 qRT-PCR 对 K562 慢性髓性白血病细胞的研究表明，精氨酸修饰的和骨架修饰的 PNA 具有良好的细胞摄取能力，其中 C2 修饰的系列表现最佳。骨架修饰的 PNA 的酶降解抗性更高，因此在血清中的细胞中使用这些衍生物而不是共轭 PNA 具有更大的优势，并且这种效应在存在肽酶（如胰蛋白酶）时会放大。miR-210 活性的抑制导致了红细胞分化途径的变化，在用米托蒽醌处理的细胞中变化更为明显。有趣的是，不同的 PNA 具有不同的抗 miR 活性，这表明取代基不仅在细胞摄取中起作用，而且在 miR 识别和失活的机制中也起作用。这是第一个关于将骨架修饰的 PNA 用作抗 miR 剂的报告。结果清楚地表明，由于增强了生物利用度，骨架修饰的 PNA 是基于抗 miR 活性开发非常有效药物的良好候选物，并且整体抗 miR 性能是细胞摄取和 RNA 结合的结合。
