Specificity of hammerhead ribozyme cleavage
Specificity of hammerhead ribozyme cleavage
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DOI：
10.1002/j.1460-2075.1996.tb00745.x
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发表时间：
1996-07-15
期刊：
影响因子：
11.4
通讯作者：
Uhlenbeck, OC
中科院分区：
文献类型：
--
作者：
Hertel, KJ;Herschlag, D;Uhlenbeck, OC
为了有效地灭活基因，锤头状核酶必须切割互补的RNA靶标，而不会因为切割包含错配和较短互补片段的非靶标RNA而产生有害影响。使用HH16来评估锤头切割的特异性，HH16是一种具有良好特性的核酶，旨在切割一个17个残基的靶标。在标准反应条件下，HH16不能区分其全长底物和3‘-截短底物，即使当HH16和底物之间形成的碱基对减少6个时，这种显著的缺乏特异性也会出现，因为所有的底物都以足够的亲和力与核酶结合，因此在它们的亲和力差异显现之前就发生了切割。相反，HH16确实对改变底物的某些3‘-截断形式表现出高度的特异性，这些改变底物也包含单个碱基错配或在5’端被缩短。此外，HH16的特异性在人类免疫缺陷病毒(HIV)-1的p7核衣壳蛋白的存在下得到提高，这加速了RNA螺旋的结合和解离，这些结果支持了锤头具有识别错误碱基的内在能力的观点，但强调高特异性仅在一定的螺旋长度范围内观察到。
To be effective in gene inactivation, the hammerhead ribozyme must cleave a complementary RNA target without deleterious effects from cleaving non-target RNAs that contain mismatches and shorter stretches of complementarity. The specificity of hammerhead cleavage was evaluated using HH16, a well-characterized ribozyme designed to cleave a target of 17 residues, Under standard reaction conditions, HH16 is unable to discriminate between its full-length substrate and 3'-truncated substrates, even when six fewer base pairs are formed between HH16 and the substrate, This striking lack of specificity arises because all the substrates bind to the ribozyme with sufficient affinity so that cleavage occurs before their affinity differences are manifested. In contrast, HH16 does exhibit high specificity towards certain 3'-truncated versions of altered substrates that either also contain a single base mismatch or are shortened at the 5' end. In addition, the specificity of HH16 is improved in the presence of p7 nucleocapsid protein from human immunodeficiency virus (HIV)-1, which accelerates the association and dissociation of RNA helices, These results support the view that the hammerhead has an intrinsic ability to discriminate against incorrect bases, but emphasizes that the high specificity is only observed in a certain range of helix lengths.