NUCLEASE-RESISTANT CHIMERIC RIBOZYMES CONTAINING DEOXYRIBONUCLEOTIDES AND PHOSPHOROTHIOATE LINKAGES
NUCLEASE-RESISTANT CHIMERIC RIBOZYMES CONTAINING DEOXYRIBONUCLEOTIDES AND PHOSPHOROTHIOATE LINKAGES
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DOI：
10.1093/nar/21.11.2605
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发表时间：
1993-06-11
影响因子：
14.9
通讯作者：
TAIRA, K
中科院分区：
文献类型：
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作者：
SHIMAYAMA, T;NISHIKAWA, F;TAIRA, K
锤头状核酶因其具有位点特异性的RNA切割活性而被认为是潜在的HIV病毒治疗剂。为了阐明结构与功能的关系，并希望赋予核酶对核糖核酸酶的抗性，我们首先合成了嵌合DNA/RNA核酶，其中脱氧核糖核苷酸取代了非催化残基（茎I，II和III）的核糖核苷酸。动力学分析表明，（i）杂交臂（茎I和III）中的DNA增强了化学切割步骤。(ii)茎II及其环不影响其酶活性。其次，我们将具有硫代磷酸酯键的脱氧核糖核苷酸引入相同的区域（茎I、II和III），以测试这种硫代键是否进一步提高它们对核酸酶的抗性。动力学测定表明，该嵌合thio-DNA/RNA核酶的切割活性比全RNA核酶高7倍（k（cat）= 27 min ~（-1））。在血清中的稳定性方面，DNA武装的核酶在人血清中获得了约10倍的稳定性，但在牛血清中没有增加稳定性，这可能是因为牛血清主要含有攻击这些核酶的未修饰的催化环区域的内切核糖核酸酶。第三，为了保护它们免受内切核糖核酸酶的侵害，在内部催化环区域内的位置U 7、U4和C3处进行了三个额外的修饰，这成功地在两种血清中获得了超过百倍的对核酸酶的抗性。更重要的是，这些催化环修饰的核酶具有与野生型核酶相当的切割活性（k（cat））。由于这些嵌合硫代-DNA/RNA核酶比野生型全RNA核酶在体内更能抵抗核酸外切酶和核糖核酸内切酶的攻击，并且由于它们的切割活性没有被牺牲，因此它们似乎是比野生型更好的抗病毒治疗剂的候选物。
Hammerhead ribozymes are considered to be potential therapeutic agents for HIV virus because of their site-specific RNA cleavage activities. In order to elucidate structure - function relationship and also to hopefully endow ribozymes with resistance to ribonucleases, we firstly synthesized chimeric DNA/RNA ribozymes in which deoxyribonucleotides were substituted for ribonucleotides st noncatalytic residues (stems I, II, and III). Kinetic analysis revealed that (i) DNA in the hybridizing arms (stems I and III) enhanced the chemical cleavage step. (ii) stem II and its loop do not affect its enzymatic activity. Secondly, we introduced deoxyribonucleotides with phosphorothioate linkages to the same regions (stems I, II, and III) in order to test whether such thio-linkages further improve their resistance to nucleases. Kinetic measurements revealed that this chimeric thio-DNA/RNA ribozyme had seven-fold higher cleavage activity (k(cat) = 27 min-1) than that of the all-RNA ribozyme. In terms of stability in serum, DNA-armed ribozymes gained about 10-fold higher stability in human serum but no increase in stability was recognized in bovine serum, probably because the latter serum mainly contained endoribonucleases that attacked unmodified catalytic-loop regions of these ribozymes. Thirdly, in order to protect them from endoribonucleases, three additional modifications were made at positions U7, U4 and C3 within the internal catalytic-loop region, that succeeded in gaining more than a hundred times greater resistance to nucleases in both serums. More importantly, these catalytic-loop modified ribozymes had the comparable cleavage activity (k(cat)) to the wild-type ribozyme. Since these chimeric thio-DNA/RNA ribozymes are more resistant to attack by both exonucleases and endoribonucleases than the wild-type all-RNA ribozymes in vivo and since their cleavage activities are not sacrificed, they appear to be better candidates than the wild type for antiviral therapeutic agents.