title: Optimal terminal sequences for continuous or serial isothermal amplification of dsRNA with norovirus RNA replicase.
abstract: The norovirus RNA replicase (NV3D(pol), 56 kDa, single chain monomeric protein) can amplify double-stranded (ds) RNA isothermally. It will play an alternative role in the in vitro evolution against traditional Qβ RNA replicase, which cannot amplify dsRNA and consists of four subunits, three of which are borrowed from host E.coli. In order to identify the optimal 3'-terminal sequence of the RNA template for NV3D(pol), an in vitro selection using the serial transfer was performed for a random library having the 3'-terminal sequence of ---UUUUUUNNNN-3'. The population landscape on the 4-dimensional sequence space of the 17(th) round of transfer gave a main peak around ---CAAC-3'. In the preceding studies on the batch amplification reaction starting from a single-stranded RNA, a template with 3'-terminal C-stretch was amplified effectively. It was confirmed that in the batch amplification the ---CCC-3' was much more effective than the ---CAAC-3', but in the serial transfer condition in which the ----CAAC-3' was sustained stably, the ---CCC-3' was washed out. Based on these results we proposed the existence of the "shuttle mode" replication of dsRNA. We also proposed the optimal terminal sequences of RNA for in vitro evolution with NV3D(pol).
authors: Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan.
authors: Hidenao
authors: H
authors: Arai
authors: Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan.
authors: Koichi
authors: K
authors: Nishigaki
authors: Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan.
authors: Naoto
authors: N
authors: Nemoto
authors: Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan.
authors: Miho
authors: M
authors: Suzuki
authors: Innovative Research Organization, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan.
authors: Yuzuru
authors: Y
authors: Husimi
keywords: RNA-dependent RNA polymerase; breathing of dsRNA terminus; in vitro selection; initiation of RNA replication; shuttle mode replication