Compartmentalization in a water-in-oil emulsion repressed the spontaneous amplification of RNA by Q beta replicase.
During RNA replication mediated by Qbeta 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 Qbeta 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 Qbeta replicase. This method allowed us to amplify the phage Qbeta 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.
Urabe
Hiroya
H
Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, Japan.
Ichihashi
Norikazu
N
Matsuura
Tomoaki
T
Hosoda
Kazufumi
K
Kazuta
Yasuaki
Y
Kita
Hiroshi
H
Yomo
Tetsuya
T
D017908:Allolevivirus / Q000201:enzymology*; D004655:Emulsions; D035001:Host Factor 1 Protein / Q000737:chemistry / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D009821:Oils; D011777:Q beta Replicase / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000737:chemistry; D012367:RNA, Viral / Q000037:antagonists & inhibitors* / Q000096:biosynthesis* / Q000737:chemistry; D013698:Templates, Genetic; D038301:Virus Inactivation; D014779:Virus Replication / Q000235:genetics; D014867:Water
D004655:Emulsions; D035001:Host Factor 1 Protein; D009821:Oils; D012367:RNA, Viral; D014867:Water; D011777:Q beta Replicase