Transcription yield of fully 2'-modified RNA can be increased by the addition of thermostabilizing mutations to T7 RNA polymerase mutants.
Transcription yield of fully 2'-modified RNA can be increased by the addition of thermostabilizing mutations to T7 RNA polymerase mutants.
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DOI：
10.1093/nar/gkv734
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发表时间：
2015-09-03
影响因子：
14.9
通讯作者：
Ellington AD
中科院分区：
文献类型：
--
作者：
Meyer AJ;Garry DJ;Hall B;Byrom MM;McDonald HG;Yang X;Yin YW;Ellington AD
平均而言，突变对蛋白质是有害的。赋予蛋白质新功能的突变往往以蛋白质折叠或稳定性为代价，从而降低整体活性。多年来，已经设计或进化了一组T7RNA聚合酶来接受带有修饰核糖部分的核苷酸。这些修饰的RNA已被证明是有用的，特别是在体内，但转录产量往往相当低。在这里，我们证明了以前被证明增加T7RNA聚合酶耐热性的突变可以增加底物范围扩大的突变体的活性。由此产生的聚合酶突变株可用于产生2‘-O-甲基修饰的RNA，其产量比目前使用的酶高得多。
On average, mutations are deleterious to proteins. Mutations conferring new function to a protein often come at the expense of protein folding or stability, reducing overall activity. Over the years, a panel of T7 RNA polymerases have been designed or evolved to accept nucleotides with modified ribose moieties. These modified RNAs have proven useful, especially in vivo, but the transcriptional yields tend to be quite low. Here we show that mutations previously shown to increase the thermal tolerance of T7 RNA polymerase can increase the activity of mutants with expanded substrate range. The resulting polymerase mutants can be used to generate 2′-O-methyl modified RNA with yields much higher than enzymes currently employed.