PCR amplification of DNA containing non-standard base pairs by variants of reverse transcriptase from Human Immunodeficiency Virus-1
PCR amplification of DNA containing non-standard base pairs by variants of reverse transcriptase from Human Immunodeficiency Virus-1
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DOI：
10.1093/nar/gkh241
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发表时间：
2004-01-01
影响因子：
14.9
通讯作者：
Benner, SA
中科院分区：
文献类型：
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作者：
Sismour, AM;Lutz, S;Benner, SA
作为从人工遗传信息系统生成合成生物学的下一步，我们检查了 HIV 逆转录酶 (RT) 变体合成双链 DNA 的能力，其中包含 2,4-二氨基嘧啶 (pyDAD)（一种与互补碱基呈现氢键“供体-受体-供体”模式的嘧啶）和黄嘌呤 (puADA)（一种呈现氢键的嘌呤）之间的非标准碱基对。 “接受者-捐赠者-接受者”模式。该碱基对符合 Watson-Crick 几何结构，但由氢键供体和受体基团的模式连接，与连接 GC 和 AT 对的氢键供体和受体基团的模式不同。 HIV-RT 的一种变体，其中 Tyr 188 被 Leu 取代，是从实验中出现的，在这些实验中，HIV 在针对 RT 的药物（如 L-697639、TIBO 和奈韦拉平）的存在下生长受到挑战。这些药物结合在活性位点附近但不在活性位点内的位点。该变体接受 pyDAD-puADA 碱基对的能力明显优于野生型 HIV-RT，我们以此为起点。如果观察到的残留核酸酶活性是由于 RT 而不是污染物造成的，则将第二个突变 E478Q 引入 Y188L 变体中。双突变 RT 以足够的保真度整合了非标准碱基对，使得该变体可用于通过多轮聚合酶链式反应 (PCR) 扩增含有 pyDAD 和 puADA 的寡核苷酸，而不会丢失非标准碱基对。这是第一次通过完整的 PCR 扩增含有具有替代氢键模式的非标准碱基对的 DNA。这项工作还说明了一种研究策略，该策略结合了蛋白质的临床预进化，然后进行合理设计以获得满足特定技术规范的酶。
As the next step towards generating a synthetic biology from artificial genetic information systems, we have examined variants of HIV reverse transcriptase (RT) for their ability to synthesize duplex DNA incorporating the non-standard base pair between 2,4-diaminopyrimidine (pyDAD), a pyrimidine presenting a hydrogen bond 'donor-acceptor-donor' pattern to the complementary base, and xanthine (puADA), a purine presenting a hydrogen bond 'acceptor-donor-acceptor' pattern. This base pair fits the Watson-Crick geometry, but is joined by a pattern of hydrogen bond donor and acceptor groups different from those joining the GC and AT pairs. A variant of HIV-RT where Tyr 188 is replaced by Leu, has emerged from experiments where HIV was challenged to grow in the presence of drugs targeted against the RT, such as L-697639, TIBO and nevirapine. These drugs bind at a site near, but not in, the active site. This variant accepts the pyDAD-puADA base pair significantly better than wild type HIV-RT, and we used this as a starting point. A second mutation, E478Q, was introduced into the Y188L variant, in the event that the residual nuclease activity observed is due to the RT, and not a contaminant. The doubly mutated RT incorporated the non-standard pair with sufficient fidelity that the variant could be used to amplify oligonucleotides containing pyDAD and puADA through several rounds of a polymerase chain reaction (PCR) without losing the non-standard base pair. This is the first time where DNA containing non-standard base pairs with alternative hydrogen bonding patterns has been amplified by a full PCR. This work also illustrates a research strategy that combines in clinico pre-evolution of proteins followed by rational design to obtain an enzyme that meets a particular technological specification.