CONTINUOUS IN-VITRO EVOLUTION OF BACTERIOPHAGE RNA-POLYMERASE PROMOTERS
CONTINUOUS IN-VITRO EVOLUTION OF BACTERIOPHAGE RNA-POLYMERASE PROMOTERS
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DOI：
10.1021/bi00205a037
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发表时间：
1994-10-04
期刊：
影响因子：
2.9
通讯作者：
JOYCE, GF
中科院分区：
文献类型：
--
作者：
BREAKER, RR;BANERJI, A;JOYCE, GF
噬菌体T7，T3和SP6 RNA聚合酶启动子的快速体外进化是通过一种方法实现的，该方法允许连续富集含有功能性启动子元件的DNA。这种方法利用了一类特殊的核酸分子在逆转录酶和DNA依赖性RNA聚合酶存在下连续复制的能力。复制涉及RNA和cDNA中间体的合成。cDNA链含有嵌入的启动子序列，其转化为功能性双链启动子元件，导致RNA转录物的产生。合成cDNA，包括含有随机化启动子序列的cDNA，可用于启动扩增循环。然而，只有那些含有功能性启动子序列的cDNA能够产生RNA转录物。此外，每个RNA转录物编码负责启动其自身转录的RNA聚合酶启动子序列。因此，扩增分子的群体迅速富集编码功能性启动子的那些模板。在2小时扩增反应后鉴定噬菌体T7、T3和SP6 RNA聚合酶的最佳启动子序列，在每种情况下用编码大于10（10）个启动子序列变体的合成cDNA库起始。
Rapid in vitro evolution of bacteriophage T7, T3, and SP6 RNA polymerase promoters was achieved by a method that allows continuous enrichment of DNAs that contain functional promoter elements. This method exploits the ability of a special class of nucleic acid molecules to replicate continuously in the presence of both a reverse transcriptase and a DNA-dependent RNA polymerase. Replication involves the synthesis of both RNA and cDNA intermediates. The cDNA strand contains an embedded promoter sequence, which becomes converted to a functional double-stranded promoter element, leading to the production of RNA transcripts. Synthetic cDNAs, including those that contain randomized promoter sequences, can be used to initiate the amplification cycle. However, only those cDNAs that contain functional promoter sequences are able to produce RNA transcripts. Furthermore, each RNA transcript encodes the RNA polymerase promoter sequence that was responsible for initiation of its own transcription. Thus, the population of amplifying molecules quickly becomes enriched for those templates that encode functional promoters. Optimal promoter sequences for phage T7, T3, and SP6 RNA polymerase were identified after a 2-h amplification reaction, initiated in each case with a pool of synthetic cDNAs encoding greater than 10(10) promoter sequence variants.