Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Department of Bioorganic Chemistry, Sienkiewicza 112, 90-363 Lodz, Poland.
Org Biomol Chem. 2015 Feb 28;13(8):2375-84. doi: 10.1039/c4ob02364a.
Homopurine phosphorothioate analogs of DNA, possessing all phosphorus atoms of RP configuration ([All-RP-PS]-DNA), when interact with appropriate complementary RNA or (2'-OMe)-RNA templates, form parallel triplexes or parallel duplexes of very high thermodynamic stability. The present results show that T-LNA or 5-Me-C-LNA units introduced into the parallel Hoogsteen-paired (2'-OMe)-RNA strands (up to four units in the oligomers of 9 or 12 nt in length) stabilize these parallel complexes. At neutral pH, dodecameric parallel duplexes have Tm values of 62-68 °C, which are by 4-10 °C higher than Tm for the reference duplex (with no LNA units present), while for the corresponding triplexes, Tm values exceeded 85 °C. For nonameric parallel duplexes, melting temperatures of 38-62 °C were found and (2'-OMe)-RNA oligomers containing 5-Me-C-LNA units stabilized the complexes more efficiently than the T-LNA containing congeners. In both series the stability of the parallel complexes increased with an increasing number of LNA units present. The same trend was observed in experiments of reverse transcription RNA→DNA (using AMV RT reverse transcriptase) where the formation of parallel triplexes (consisting of an RNA template, [All-RP-PS]-DNA nonamer and Hoogsteen-paired (2'-OMe)-RNA strands containing the LNA units) led to the efficient inhibition of the process. Under the best conditions checked (four 5-Me-C-LNA units, three-fold excess over the RNA template) the inhibition was 94% effective, compared to 71% inhibition observed in the reference system with the Hoogsteen-paired (2'-OMe)-RNA strand carrying no LNA units. This kind of complexation may "arrest" harmful RNA oligomers (e.g., viral RNA or mRNA of unwanted proteins) and, beneficially, exclude them from enzymatic processes, otherwise leading to viral or genetic diseases.
DNA 的同嘌呤硫代磷酸酯类似物，其所有磷原子均具有 RP 构型([全-RP-PS]-DNA)，当与适当的互补 RNA 或(2'-OMe)-RNA 模板相互作用时，形成热力学稳定性非常高的平行三聚体或平行双链体。本研究结果表明，T-LNA 或 5-Me-C-LNA 单元引入到平行 Hoogsteen 配对的(2'-OMe)-RNA 链中(在 9 或 12 个核苷酸长的寡聚物中最多有四个单元)稳定了这些平行复合物。在中性 pH 下，十二聚体平行双链体的 Tm 值为 62-68°C，比参考双链体(Tm 值为 62-68°C)(不存在 LNA 单元)的 Tm 值高 4-10°C，而相应的三聚体的 Tm 值超过 85°C。对于非九聚体平行双链体，发现熔解温度为 38-62°C，并且含有 5-Me-C-LNA 单元的(2'-OMe)-RNA 寡聚物比含有 T-LNA 的同类物更有效地稳定复合物。在这两个系列中，随着存在的 LNA 单元数量的增加，平行复合物的稳定性增加。在 RNA→DNA 的反转录实验(使用 AMV RT 反转录酶)中观察到了相同的趋势，其中平行三聚体的形成(由 RNA 模板、[全-RP-PS]-DNA 九聚体和包含 LNA 单元的 Hoogsteen 配对的(2'-OMe)-RNA 链组成)导致该过程的有效抑制。在检查的最佳条件下(四个 5-Me-C-LNA 单元，比 RNA 模板多三倍)，抑制率为 94%，而在参考体系中观察到的抑制率为 71%，该参考体系中的 Hoogsteen 配对(2'-OMe)-RNA 链不含 LNA 单元。这种络合作用可以“阻止”有害的 RNA 寡聚物(例如，病毒 RNA 或不需要的蛋白质的 mRNA)，并有益地将其排除在酶促过程之外，否则会导致病毒或遗传疾病。
