Compartmentalization in a Water-in-Oil Emulsion Repressed the Spontaneous Amplification of RNA by Qβ Replicase

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• 作者：Hiroya Urabe ; Norikazu Ichihashi ; Tomoaki Matsuura ; Kazufumi Hosoda ; Yasuaki Kazuta ; Hiroshi Kita ; Tetsuya Yomo
• 刊名：Biochemistry
• 出版年：2010
• 出版时间：March 9, 2010
• 年：2010
• 卷：49
• 期：9
• 页码：1809-1813
• 全文大小：783K
• 年卷期：v.49,no.9(March 9, 2010)
• ISSN：1520-4995

文摘

During RNA replication mediated by Qβ 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 as genomic RNA of Qβ 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 a method to repress RQ RNA amplification by compartmentalization of the reaction using a water-in-oil emulsion but maintaining the activity of Qβ replicase. This method allowed us to amplify the phage Qβ 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.