Compartmentalization in a Water-in-Oil Emulsion Repressed the Spontaneous Amplification of RNA by Qβ Replicase
Compartmentalization in a Water-in-Oil Emulsion Repressed the Spontaneous Amplification of RNA by Qβ Replicase
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DOI：
10.1021/bi901805u
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发表时间：
2010-03-09
期刊：
影响因子：
2.9
通讯作者：
Yomo, Tetsuya
中科院分区：
文献类型：
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作者：
Urabe, Hiroya;Ichihashi, Norikazu;Yomo, Tetsuya
在Q β复制酶介导的RNA复制过程中，自我复制RNA（RQ RNA）在不添加模板RNA的情况下扩增。这种不希望的扩增使得研究的目标。RNA复制困难，特别是对于长RNA如其基因组RNA的Q β噬菌体。这可能是为什么在宿主因子Hfq存在下，基因组RNA复制的精确速率在体外没有报道的原因之一。在这里，我们报告了一种抑制RQ RNA扩增的方法，该方法通过使用油包水乳液将反应区室化，但保持Q β复制酶的活性。该方法允许LIS指数地扩增噬菌体Q β基因组RNA，而没有可检测到的RQ RNA扩增。此外，我们发现，在指数阶段的基因组RNA复制的速率常数在最佳Hfq浓度是约4.6倍，比以前的报告，接近体内数据。该结果表明，体内复制率在很大程度上是由Hfq的存在来解释的。这种简单的方法为研究基因组RNA复制铺平了道路，而无需特别注意不需要的RQ RNA扩增。
During RNA replication mediated by Q beta replicase, self-replicating RNAs (RQ RNAs) are amplified without the addition of template RNA. This undesired amplification makes the study of target. RNA replication difficult, especially for long RNA Such its genomic RNA of Q beta phage. This perhaps is one of the reasons why the precise rate of genomic RNA replication In the presence of host factor Hfq has not been reported in vitro, Here, we report I method to repress RQ RNA amplification by compartmentalization of the reaction using a water-in-oil emulsion but Maintaining the activity of Q beta replicase. This method allowed LIS to amplify the phage Q beta genome RNA exponentially Without detectable amplification of RQ RNA. Furthermore, we found that the rate constant of genome RNA replication in the exponential phase at the optimum Hfq concentration was approximately 4.6 times larger than that of a previous report, close to in vivo data. This result indicates that the replication rate in vivo is largely explained by the presence of Hfq. This easy method paves the way for the Study of genomic RNA replication Without Special Care for the Undesired RQ RNA amplification.