Soler Bistué Alfonso J C, Martín Fernando A, Vozza Nicolás, Ha Hongphuc, Joaquín Jonathan C, Zorreguieta Angeles, Tolmasky Marcelo E
Fundación Instituto Leloir, Instituto de Investigaciones Bioquímicas de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas and Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Patricias Argentinas 435, C1405BWE Buenos Aires, Argentina.
Proc Natl Acad Sci U S A. 2009 Aug 11;106(32):13230-5. doi: 10.1073/pnas.0906529106. Epub 2009 Jul 28.
Inhibition of bacterial gene expression by RNase P-directed cleavage is a promising strategy for the development of antibiotics and pharmacological agents that prevent expression of antibiotic resistance. The rise in multiresistant bacteria harboring AAC(6')-Ib has seriously limited the effectiveness of amikacin and other aminoglycosides. We have recently shown that recombinant plasmids coding for external guide sequences (EGS), short antisense oligoribonucleotides (ORN) that elicit RNase P-mediated cleavage of a target mRNA, induce inhibition of expression of aac(6')-Ib and concomitantly induce a significant decrease in the levels of resistance to amikacin. However, since ORN are rapidly degraded by nucleases, development of a viable RNase P-based antisense technology requires the design of nuclease-resistant RNA analog EGSs. We have assayed a variety of ORN analogs of which selected LNA/DNA co-oligomers elicited RNase P-mediated cleavage of mRNA in vitro. Although we found an ideal configuration of LNA/DNA residues, there seems not to be a correlation between number of LNA substitutions and level of activity. Exogenous administration of as low as 50 nM of an LNA/DNA co-oligomer to the hyperpermeable E. coli AS19 harboring the aac(6')-Ib inhibited growth in the presence of amikacin. Our experiments strongly suggest an RNase P-mediated mechanism in the observed antisense effect.
通过核糖核酸酶P介导的切割来抑制细菌基因表达，对于开发预防抗生素耐药性表达的抗生素和药理剂而言，是一种很有前景的策略。携带AAC(6')-Ib的多重耐药细菌的增加，严重限制了阿米卡星和其他氨基糖苷类药物的有效性。我们最近发现，编码外部引导序列（EGS）的重组质粒，即能引发核糖核酸酶P介导的靶mRNA切割的短反义寡核糖核苷酸（ORN），可诱导对aac(6')-Ib表达的抑制，并同时导致对阿米卡星的耐药水平显著降低。然而，由于ORN会被核酸酶迅速降解，因此要开发可行的基于核糖核酸酶P的反义技术，就需要设计抗核酸酶的RNA类似物EGS。我们检测了多种ORN类似物，其中选定的锁核酸/DNA共聚物在体外能引发核糖核酸酶P介导的mRNA切割。虽然我们找到了锁核酸/DNA残基的理想构型，但锁核酸取代的数量与活性水平之间似乎并无关联。向携带aac(6')-Ib的高通透性大肠杆菌AS19外源给予低至50 nM的锁核酸/DNA共聚物，在有阿米卡星存在的情况下会抑制其生长。我们的实验有力地表明，在所观察到的反义效应中存在一种核糖核酸酶P介导的机制。
