A Pneumocystis carinii group I intron ribozyme that does not require 2' OH groups on its 5' exon mimic for binding to the catalytic core.
A Pneumocystis carinii group I intron ribozyme that does not require 2' OH groups on its 5' exon mimic for binding to the catalytic core.
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卡氏肺囊虫 I 组内含子核酶，其 5 外显子模拟物上不需要 2 OH 基团即可与催化核心结合。
DOI：
10.1021/bi9713097
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Turner,DH
中科院分区：
文献类型：
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作者：
Testa,SM;Haidaris,CG;Gigliotti,F;Turner,DH
近年来免疫功能低下患者的增加导致了人类机会性真菌感染的激增。由于缺乏针对这些病原体的有效治疗方法，因此开发新的治疗策略非常重要。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发展，用于研究真菌病原体中I组自剪接内含子的反义靶向潜力。从鼠源性肺孢子虫核糖体大亚基RNA中分离出Ⅰ组内含子。这个内含子在体外自我剪接。一个催化活性的核酶，P-8/4x，已被构建从这个内含子，允许潜在的反义试剂的解离常数的测量。在37 °C下，在pH 7.5的50 mM Hepes（25 mM Na+）、15 mM MgCl 2和135 mM KCl中，外源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）的结合紧密约60000倍。这种增强的结合是由于三级相互作用。这种三级稳定性通过在外显子模拟物中的每个位置处的单脱氧核苷酸取代而增加，除了内部A，其基本上不变。因此，与四膜虫L-21 ScaI核酶相反，5'外显子模拟物的2' OH基团不与P-8/4x核酶形成稳定的三级相互作用。此外，在37 °C下，外源5'外显子模拟物d（ATGACT）与P-8/4x核酶的结合比与r（GGUCAU）的结合紧密近32000倍。因此，不具有2' OH基团的寡核苷酸可以利用三级稳定化来显著地更紧密地结合，并且具有比来自碱基配对的可能性更高的特异性。这些结果提示了一种新的反义靶向模式：靶向结构RNA与短反义寡核苷酸的三级相互作用。
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-derivedPneumocystiscariniihas been isolated and characterized. This intron self-splicesin 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 °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 theTetrahymenaL-21ScaI ribozyme. Furthermore, at 37 °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.