Institute of Bioorganic Chemistry, Polish Academy of Sciences , Noskowskiego 12/14 , 61-704 Poznan , Poland.
Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences , Nanyang Technological University , 21 Nanyang Link , 637371 , Singapore.
Bioconjug Chem. 2019 Mar 20;30(3):931-943. doi: 10.1021/acs.bioconjchem.9b00039. Epub 2019 Feb 15.
RNAs play critical roles in diverse catalytic and regulatory biological processes and are emerging as important disease biomarkers and therapeutic targets. Thus, developing chemical compounds for targeting any desired RNA structures has great potential in biomedical applications. The viral and cellular RNA sequence and structure databases lay the groundwork for developing RNA-binding chemical ligands through the recognition of both RNA sequence and RNA structure. Influenza A virion consists of eight segments of negative-strand viral RNA (vRNA), all of which contain a highly conserved panhandle duplex structure formed between the first 13 nucleotides at the 5' end and the last 12 nucleotides at the 3' end. Here, we report our binding and cell culture anti-influenza assays of a short 10-mer chemically modified double-stranded RNA (dsRNA)-binding peptide nucleic acid (PNA) designed to bind to the panhandle duplex structure through novel major-groove PNA·RNA triplex formation. We demonstrated that incorporation of chemically modified PNA residues thio-pseudoisocytosine (L) and guanidine-modified 5-methyl cytosine (Q) previously developed by us facilitates the sequence-specific recognition of Watson-Crick G-C and C-G pairs, respectively, at physiologically relevant conditions. Significantly, the chemically modified dsRNA-binding PNA (dbPNA) shows selective binding to the dsRNA region in panhandle structure over a single-stranded RNA (ssRNA) and a dsDNA containing the same sequence. The panhandle structure is not accessible to traditional antisense DNA or RNA with a similar length. Conjugation of the dbPNA with an aminosugar neamine enhances the cellular uptake. We observed that 2-5 μM dbPNA-neamine conjugate results in a significant reduction of viral replication. In addition, the 10-mer dbPNA inhibits innate immune receptor RIG-I binding to panhandle structure and thus RIG-I ATPase activity. These findings would provide the foundation for developing novel dbPNAs for the detection of influenza viral RNAs and therapeutics with optimal antiviral and immunomodulatory activities.
RNAs 在各种催化和调节生物过程中发挥着关键作用，并且正在成为重要的疾病生物标志物和治疗靶标。因此，开发针对任何所需 RNA 结构的化学化合物在生物医学应用中具有巨大潜力。病毒和细胞 RNA 序列和结构数据库为通过识别 RNA 序列和 RNA 结构来开发 RNA 结合化学配体奠定了基础。流感 A 病毒粒子由八个负链病毒 RNA (vRNA) 片段组成，所有这些片段都包含一个高度保守的发夹双链结构，该结构由 5' 端的前 13 个核苷酸和 3' 端的最后 12 个核苷酸之间形成。在这里，我们报告了一种短的 10 个碱基化学修饰的双链 RNA (dsRNA) 结合肽核酸 (PNA) 的结合和细胞培养抗流感测定结果，该 PNA 设计用于通过新型主沟 PNA·RNA 三聚体形成结合到手性双链结构。我们证明了我们之前开发的化学修饰的 PNA 残基硫代假胞嘧啶 (L) 和胍基修饰的 5-甲基胞嘧啶 (Q) 的掺入有助于在生理相关条件下分别特异性识别 Watson-Crick G-C 和 C-G 对。重要的是，化学修饰的 dsRNA 结合 PNA (dbPNA) 显示出对发夹结构中 dsRNA 区域的选择性结合，而不是对单链 RNA (ssRNA) 和包含相同序列的双链 DNA 的结合。传统的长度相似的反义 DNA 或 RNA 无法进入发夹结构。将 dbPNA 与氨基糖 neamine 缀合可增强细胞摄取。我们观察到 2-5 μM dbPNA-neamine 缀合物可显著减少病毒复制。此外，10 个碱基的 dbPNA 抑制先天免疫受体 RIG-I 与发夹结构的结合，从而抑制 RIG-I ATP 酶活性。这些发现将为开发用于检测流感病毒 RNA 的新型 dbPNA 以及具有最佳抗病毒和免疫调节活性的治疗药物奠定基础。
