Characterization of Norovirus RNA replicase for in vitro amplification of RNA View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2013-12

AUTHORS

Hidenao Arai, Koichi Nishigaki, Naoto Nemoto, Miho Suzuki, Yuzuru Husimi

ABSTRACT

BACKGROUND: The isothermal amplification of RNA in vitro has been used for the study of in vitro evolution of RNA. Although Qβ replicase has been traditionally used as an enzyme for this purpose, we planned to use norovirus replicase (NV3D(pol)) due to its structural simplicity in the scope of in vitro autonomous evolution of the protein. Characteristics of the enzyme NV3D(pol) in vitro were re-evaluated in this context. RESULTS: NV3D(pol), synthesized by using a cell-free translation system, represented the activities which were reported in the previous several studies and the reports were not fully consistent each other. The efficiency of the initiation of replication was dependent on the 3'-terminal structure of single-stranded RNA template, and especially, NV3D(pol) preferred a self-priming small stem-loop. In the non-self-priming and primer-independent replication reaction, the presence of -CCC residues at the 3'-terminus increased the initiation efficiency and we demonstrated the one-pot isothermal RNA (even dsRNA) amplification by 16-fold. NV3D(pol) also showed a weak activity of elongation-reaction from a long primer. Based on these results, we present a scheme of the primer-independent isothermal amplification of RNA with NV3D(pol) in vitro. CONCLUSIONS: NV3D(pol) can be used as an RNA replicase in in vitro RNA + protein evolution with the RNA of special terminal sequences. More... »

PAGES

85

Identifiers

URI

http://scigraph.springernature.com/pub.10.1186/1472-6750-13-85

DOI

http://dx.doi.org/10.1186/1472-6750-13-85

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1003833736

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/24106810


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