Cleavage of influenza RNA by using a human PUF-based artificial RNA-binding protein-staphylococcal nuclease hybrid.
Various viruses infect animals and humans and cause a variety of diseases, including cancer. However, effective methodologies to prevent virus infection have not yet been established. Therefore, development of technologies to inactivate viruses is highly desired. We have already demonstrated that cleavage of a DNA virus genome was effective to prevent its replication. Here, we expanded this methodology to RNA viruses. In the present study, we used staphylococcal nuclease (SNase) instead of the PIN domain (PilT N-terminus) of human SMG6 as an RNA-cleavage domain and fused the SNase to a human Pumilio/fem-3 binding factor (PUF)-based artificial RNA-binding protein to construct an artificial RNA restriction enzyme with enhanced RNA-cleavage rates for influenzavirus. The resulting SNase-fusion nuclease cleaved influenza RNA at rates 120-fold greater than the corresponding PIN-fusion nuclease. The cleaving ability of the PIN-fusion nuclease was not improved even though the linker moiety between the PUF and RNA-cleavage domain was changed. Gel shift assays revealed that the RNA-binding properties of the PUF derivative used was not as good as wild type PUF. Improvement of the binding properties or the design method will allow the SNase-fusion nuclease to cleave an RNA target in mammalian animal cells and/or organisms.
Department of Applied Chemistry and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Tsushima-Naka, Kita-ku, Okayama 700-8530, Japan.
Tomoaki
T
Mori
Department of Applied Chemistry and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Tsushima-Naka, Kita-ku, Okayama 700-8530, Japan.
Kento
K
Nakamura
Department of Applied Chemistry and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Tsushima-Naka, Kita-ku, Okayama 700-8530, Japan.
Keisuke
K
Masaoka
Department of Applied Chemistry and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Tsushima-Naka, Kita-ku, Okayama 700-8530, Japan.
Yusuke
Y
Fujita
Department of Applied Chemistry and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Tsushima-Naka, Kita-ku, Okayama 700-8530, Japan.
Ryosuke
R
Morisada
Department of Applied Chemistry and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Tsushima-Naka, Kita-ku, Okayama 700-8530, Japan.
Koichi
K
Mori
Department of Applied Chemistry and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Tsushima-Naka, Kita-ku, Okayama 700-8530, Japan.
Takamasa
T
Tobimatsu
Department of Applied Chemistry and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Tsushima-Naka, Kita-ku, Okayama 700-8530, Japan. Electronic address: sera@cc.okayama-u.ac.jp.
Takashi
T
Sera
D000818:Animals; D005240:Feasibility Studies; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008836:Micrococcal Nuclease / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D038301:Virus Inactivation
C471242:PUM1 protein, human; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D008836:Micrococcal Nuclease
Artificial RNA restriction enzyme; RNA cleavage; RNA virus