Gruegelsiepe Heike, Brandt Ole, Hartmann Roland K
Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marbacher Weg 6, D-35037 Marburg, Germany.
J Biol Chem. 2006 Oct 13;281(41):30613-20. doi: 10.1074/jbc.M603346200. Epub 2006 Aug 10.
We explored bacterial RNase P as a drug target using antisense oligomers against the P15 loop region of Escherichia coli RNase P RNA. An RNA 14-mer, or locked nucleic acid (LNA) and peptide nucleic acid (PNA) versions thereof, disrupted local secondary structure in the catalytic core, forming hybrid duplexes over their entire length. Binding of the PNA and LNA 14-mers to RNase P RNA in vitro was essentially irreversible and even resisted denaturing PAGE. Association rates for the RNA, LNA, and PNA 14-mers were approximately 10(5) m(-1) s(-1) with a rate advantage for PNA and were thus rather fast despite the need to disrupt local structure. Conjugates in which the PNA 14-mer was coupled to an invasive peptide via a novel monoglycine linker showed RNase P RNA-specific growth inhibition of E. coli cells. Cell growth could be rescued when expressing a second bacterial RNase P RNA with an unrelated sequence in the target region. We report here for the first time specific and growth-inhibitory drug targeting of RNase P in live bacteria. This is also the first example of a duplex-forming oligomer that invades a structured catalytic RNA and inactivates the RNA by (i) trapping it in a state in which the catalytic core is partially unfolded, (ii) sterically interfering with substrate binding, and (iii) perturbing the coordination of catalytically relevant Mg2+ ions.
我们利用针对大肠杆菌核糖核酸酶P RNA的P15环区域的反义寡聚物，将细菌核糖核酸酶P作为药物靶点进行了研究。一种14聚体RNA，或其锁定核酸（LNA）和肽核酸（PNA）版本，破坏了催化核心中的局部二级结构，在其全长范围内形成杂交双链体。PNA和LNA 14聚体在体外与核糖核酸酶P RNA的结合基本上是不可逆的，甚至能抵抗变性聚丙烯酰胺凝胶电泳。RNA、LNA和PNA 14聚体的缔合速率约为10⁵ m⁻¹ s⁻¹，PNA具有速率优势，因此尽管需要破坏局部结构，但缔合速率相当快。其中PNA 14聚体通过新型单甘氨酸接头与侵入性肽偶联的缀合物，对大肠杆菌细胞表现出核糖核酸酶P RNA特异性生长抑制作用。当在靶区域表达具有不相关序列的第二种细菌核糖核酸酶P RNA时，细胞生长可以恢复。我们在此首次报道了在活细菌中对核糖核酸酶P进行特异性和生长抑制性药物靶向。这也是双链体形成寡聚物侵入结构化催化RNA并通过以下方式使RNA失活的第一个例子：（i）将其捕获在催化核心部分展开的状态；（ii）在空间上干扰底物结合；（iii）扰乱催化相关Mg²⁺离子的配位。
