Testa S M, Haidaris C G, Gigliotti F, Turner D H
Department of Chemistry, University of Rochester, Rochester, New York 14627-0216, USA.
Biochemistry. 1997 Dec 9;36(49):15303-14. doi: 10.1021/bi9713097.
The recent increase in the population of immunocompromised patients has led to an insurgence of opportunistic human fungal infections. The lack of effective treatments against some of these pathogens makes it important to develop new therapeutic strategies. One such strategy is to target key RNAs with antisense compounds. We report the development of a model system for studying the potential for antisense targeting of group I self-splicing introns in fungal pathogens. The group I intron from the large ribosomal subunit RNA of mouse-derived Pneumocystis carinii has been isolated and characterized. This intron self-splices in vitro. A catalytically active ribozyme, P-8/4x, has been constructed from this intron to allow measurement of dissociation constants for potential antisense agents. At 37 degrees C, in 50 mM Hepes (25 mM Na+), 15 mM MgCl2, and 135 mM KCl at pH 7.5, the exogenous 5' exon mimic r(AUGACU) binds about 60 000 times more tightly to this ribozyme than to r(GGUCAU), a mimic of its complementary binding site on the ribozyme. This enhanced binding is due to tertiary interactions. This tertiary stabilization is increased by single deoxynucleotide substitutions in the exon mimic at every position except for the internal A, which is essentially unchanged. Thus 2' OH groups of the 5' exon mimic do not form stabilizing tertiary interactions with the P-8/4x ribozyme, in contrast to the Tetrahymena L-21 ScaI ribozyme. Furthermore, at 37 degrees C, the exogenous 5' exon mimic d(ATGACT) binds nearly 32 000 times more tightly to the P-8/4x ribozyme than to r(GGUCAU). Therefore, oligonucleotides without 2' OH groups can exploit tertiary stabilization to bind dramatically more tightly and with more specificity than possible from base pairing. These results suggest a new paradigm for antisense targeting: targeting the tertiary interactions of structural RNAs with short antisense oligonucleotides.
免疫功能低下患者群体最近有所增加，导致机会性人类真菌感染激增。针对其中一些病原体缺乏有效的治疗方法，因此开发新的治疗策略很重要。一种这样的策略是用反义化合物靶向关键RNA。我们报告了一个模型系统的开发，用于研究在真菌病原体中对I组自我剪接内含子进行反义靶向的潜力。从小鼠来源的卡氏肺孢子虫的大核糖体亚基RNA中分离并鉴定了I组内含子。该内含子在体外进行自我剪接。已从该内含子构建了具有催化活性的核酶P-8/4x，以测量潜在反义剂的解离常数。在37℃、pH 7.5的50 mM Hepes（25 mM Na+）、15 mM MgCl2和135 mM KCl中，外源性5'外显子模拟物r（AUGACU）与该核酶的结合比与r（GGUCAU）（其在核酶上的互补结合位点的模拟物）紧密约60000倍。这种增强的结合是由于三级相互作用。除了内部的A基本不变外，外显子模拟物中每个位置的单个脱氧核苷酸取代都会增加这种三级稳定性。因此，与嗜热四膜虫L-21 ScaI核酶相反，5'外显子模拟物的2'羟基基团不会与P-8/4x核酶形成稳定的三级相互作用。此外，在37℃时，外源性5'外显子模拟物d（ATGACT）与P-8/4x核酶的结合比与r（GGUCAU）紧密近32000倍。因此，没有2'羟基基团的寡核苷酸可以利用三级稳定性，比通过碱基配对更紧密、更特异地结合。这些结果为反义靶向提出了一种新的模式：用短反义寡核苷酸靶向结构RNA的三级相互作用。
