Contributions of individual nucleotides to tertiary binding of substrate by a Pneumocystis carinii group I intron
Contributions of individual nucleotides to tertiary binding of substrate by a Pneumocystis carinii group I intron
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DOI：
10.1021/bi001345x
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发表时间：
2000-11-21
期刊：
影响因子：
2.9
通讯作者：
Turner, DH
中科院分区：
文献类型：
--
作者：
Disney, MD;Gryaznov, SM;Turner, DH
卡氏肺孢子虫是一种哺乳动物病原体，感染并杀死免疫功能低下的宿主，如癌症和艾滋病患者。卡氏疟原虫的LSU rRNA前体含有保守的I组内含子，其是有吸引力的药物靶标，因为人类不含有I组内含子。寡核苷酸r（AUGACU），其序列模拟5 '外显子的3'末端，以5.2nM的K-d与源自内含子的核酶结合，其比单独的碱基配对所预期的紧密61000倍[Testa，S. M.，Haidaris、G. C.的方法，Gigliotti，F.，Turner，D. H.（1997）Biochemistry，36，9379-9385]。因此，通过三级相互作用增强寡核苷酸结合。为了定位产生这种三级稳定性的相互作用，已测量了与核酶的结合作为寡核苷酸长度和序列的函数。结果表明，4.3千卡/摩尔的三级稳定性是由于在内含子的剪接点处形成的GU对。消除r（AUGACU）5 '端的核苷酸不会比预期的更影响内含子结合，因为碱基配对的差异直到r（_ACU），其结合比预期的更紧密。在5 '-或3'-末端添加可能与靶标形成C-G对的C对结合亲和力几乎没有影响。测试截短的寡核苷酸通过自杀抑制机制抑制内含子自剪接的能力。四聚体r（_ _GACU）保留与六聚体r（AUGACU）相似的结合亲和力和反应性。因此，短至四聚体的寡核苷酸可以用作可以使用自杀抑制机制来抑制自我剪接的治疗剂。氨基磷酸酯四聚体和硫代氨基磷酸酯六聚体的结果表明，具有对核酸酶消化稳定的骨架的寡核苷酸保留了有利的结合和反应特性。
Pneumocystis carinii is a mammalian pathogen that infects and kills immunocompromised hosts such as cancer and AIDS patients. The LSU rRNA precursor of P. carinii contains a conserved group I intron that is an attractive drug target because humans do not contain group I introns. The oligonucleotide r(AUGACU), whose sequence mimics the 3'-end of the 5'-exon, binds to a ribozyme derived from the intron with a K-d Of 5.2 nM, which is 61000-fold tighter than expected from base-pairing alone [Testa, S. M., Haidaris, G. C., Gigliotti, F., and Turner, D. H. (1997) Biochemistry, 36, 9379-9385]. Thus, oligonucleotide binding is enhanced by tertiary interactions. To localize interactions that give rise to this tertiary stability, binding to the ribozyme has been measured as a function of oligonucleotide length and sequence. The results indicate that 4.3 kcal/mol of tertiary stability is due to a GU pair that forms at the intron's splice junction. Eliminating nucleotides at the 5'-end of r(AUGACU) does not affect intron binding more than expected from differences in base-pairing until r(_ _ _ACU), which binds much more tightly than expected. Adding a C at the 5'- or 3'-end that can potentially form a C-G pair with the target has little effect on binding affinity. Truncated oligonucleotides were tested for their ability to inhibit intron self-splicing via a suicide inhibition mechanism. The tetramer, r(_ _GACU), retains similar binding affinity and reactivity as the hexamer, r(AUGACU). Thus oligonucleotides as short as tetramers might serve as therapeutics that can use a suicide inhibition mechanism to inhibit self-splicing. Results with a phosphoramidate tetramer and thiophosphoramidate hexamer indicate that oligonucleotides with backbones stable to nuclease digestion retain favorable binding and reactivity properties.