Willkomm Dagmar K, Gruegelsiepe Heike, Goudinakis Olga, Kretschmer-Kazemi Far Rosel, Bald Rolf, Erdmann Volker A, Hartmann Roland K
Philipps-Universität Marburg, Institut für Pharmazeutische Chemie, Marbacher Weg 6, 35037 Marburg, Germany.
Chembiochem. 2003 Oct 6;4(10):1041-8. doi: 10.1002/cbic.200300674.
RNA has gained increasing importance as a therapeutic target. However, so far mRNAs rather than stable cellular RNAs have been considered in such studies. In bacteria, the tRNA-processing enzyme RNase P has a catalytic RNA subunit. Fundamental differences in structure and function between bacterial and eukaryotic RNase P, and its indispensability for cell viability make the bacterial enzyme an attractive drug target candidate. Herein we describe two approaches utilized to evaluate whether the catalytic RNA subunit of bacterial RNase P is amenable to inactivation by antisense-based strategies. In the first approach, we rationally designed RNA hairpin oligonucleotides targeted at the tRNA 3'-CCA binding site (P15 loop region) of bacterial RNase P RNA by attempting to include principles derived from the natural CopA-CopT antisense system. Substantial inactivation of RNase P RNA was observed for Type A RNase P RNA (such as that in Escherichia coli) but not for Type B (as in Mycoplasma hyopneumoniae). Moreover, only an RNA oligonucleotide (Eco 3') complementary to the CCA binding site and its 3' flanking sequences was shown to be an efficient inhibitor. Mutation of Eco 3' and analysis of other natural RNase P RNAs with sequence deviations in the P15 loop region showed that inhibition is due to interaction of Eco 3' with this region and occurs in a highly sequence-specific manner. A DNA version of Eco 3' was a less potent inhibitor. The potential of Eco 3' to form an initial kissing complex with the P15 loop did not prove advantageous. In a second approach, we tested a set of oligonucleotides against E. coli RNase P RNA which were designed by algorithms developed for the selection of suitable mRNA targets. This approach identified the P10/11-J11/12 region of bacterial RNase P RNA as another accessible region. In conclusion, both the P15 loop and P10/11-J11/12 regions of Type A RNase P RNAs seem to be promising antisense target sites since they are easily accessible and sufficiently interspersed with nonhelical sequence elements, and oligonucleotide binding directly interferes with substrate docking to these two regions.
RNA作为一种治疗靶点正变得越来越重要。然而，到目前为止，此类研究中考虑的是信使核糖核酸（mRNA）而非稳定的细胞RNA。在细菌中，tRNA加工酶核糖核酸酶P（RNase P）有一个催化性RNA亚基。细菌和真核生物RNase P在结构和功能上的根本差异，以及它对细胞生存力的不可或缺性，使得细菌的这种酶成为一个有吸引力的药物靶点候选对象。在此我们描述了两种用于评估细菌RNase P的催化性RNA亚基是否适合通过基于反义的策略使其失活的方法。在第一种方法中，我们通过尝试纳入源自天然CopA - CopT反义系统的原理，合理设计了靶向细菌RNase P RNA的tRNA 3'-CCA结合位点（P15环区域）的RNA发夹寡核苷酸。对于A型RNase P RNA（如大肠杆菌中的）观察到RNase P RNA有显著失活，但对于B型（如猪肺炎支原体中的）则没有。此外，只有与CCA结合位点及其3'侧翼序列互补的RNA寡核苷酸（Eco 3'）被证明是一种有效的抑制剂。Eco 3'的突变以及对P15环区域有序列偏差的其他天然RNase P RNAs的分析表明，抑制是由于Eco 3'与该区域的相互作用，并且以高度序列特异性的方式发生。Eco 3'的DNA版本是一种效力较弱的抑制剂。Eco 3'与P15环形成初始亲吻复合物（kissing complex）的可能性并未显示出优势。在第二种方法中，我们针对大肠杆菌RNase P RNA测试了一组寡核苷酸，这些寡核苷酸是通过为选择合适的mRNA靶点而开发的算法设计的。这种方法确定细菌RNase P RNA的P10/11 - J11/12区域是另一个可接近的区域。总之，A型RNase P RNAs的P15环和P10/11 - J11/12区域似乎都是有前景的反义靶点位点，因为它们易于接近且充分散布有非螺旋序列元件，并且寡核苷酸结合直接干扰底物与这两个区域的对接。
