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Construction of armored RNA containing long-size chimeric RNA by increasing the number and affinity of the pac site in exogenous rna and sequence coding coat protein of the MS2 bacteriophage.

ABSTRACT: OBJECTIVES:To construct a one-plasmid expression system of the armored RNA containing long chimeric RNA by increasing the number and affinity of the pac site. METHODS:The plasmid pET-MS2-pac was constructed with one C-variant pac site, and then the plasmid pM-CR-2C containing 1,891-bp chimeric sequences and two C-variant pac sites was produced. Meanwhile, three plasmids (pM-CR-C, pM-CR-2W and pM-CR-W) were obtained as parallel controls with a different number and affinity of the pac site. Finally, the armored RNA was expressed and purified. RESULTS:The armored RNA with 1,891 bases target RNA was expressed successfully by the one-plasmid expression system with two C-variant pac sites, while for one pac site, no matter whether the affinity was changed or not, only the 1,200 bases target RNA was packaged. It was also found that the C-variant pac site could increase the expression efficiency of the armored RNA. The armored RNA with 1,891-bp exogenous RNA in our study showed the characterization of ribonuclease resistance and stability at different time points and temperature conditions. CONCLUSIONS:The armored RNA with 1,891 bases exogenous RNA was constructed and the expression system can be used as a platform for preparation of the armored RNA containing long RNA sequences.

SUBMITTER: Wei B

PROVIDER: S-EPMC7179527 | biostudies-literature | 2008

REPOSITORIES: biostudies-literature

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Construction of armored RNA containing long-size chimeric RNA by increasing the number and affinity of the pac site in exogenous rna and sequence coding coat protein of the MS2 bacteriophage.

Wei Baojun B Wei Yuxiang Y Zhang Kuo K Yang Changmei C Wang Jing J Xu Ruihuan R Zhan Sien S Lin Guigao G Wang Wei W Liu Min M Wang Lunan L Zhang Rui R Li Jinming J

Intervirology 20080701 2

<h4>Objectives</h4>To construct a one-plasmid expression system of the armored RNA containing long chimeric RNA by increasing the number and affinity of the pac site.<h4>Methods</h4>The plasmid pET-MS2-pac was constructed with one C-variant pac site, and then the plasmid pM-CR-2C containing 1,891-bp chimeric sequences and two C-variant pac sites was produced. Meanwhile, three plasmids (pM-CR-C, pM-CR-2W and pM-CR-W) were obtained as parallel controls with a different number and affinity of the p ...[more]

PMID: 18594159

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Project description:The self-assembly of viral capsids is an essential step to the formation of infectious viruses. Elucidating the kinetic mechanisms of how a capsid or virus-like particle assembles could advance our knowledge about the viral lifecycle, as well as the general principles in self-assembly of biomaterials. However, current understanding of capsid assembly remains incomplete for many viruses due to the fact that the transient intermediates along the assembling pathways are experimentally difficult to be detected. In this paper, we constructed a new multiscale computational framework to simulate the self-assembly of virus-like particles. We applied our method to the coat proteins of bacteriophage MS2 as a specific model system. This virus-like particle of bacteriophage MS2 has a unique feature that its 90 sequence-identical dimers can be classified into two structurally various groups: one is the symmetric CC dimer, and the other is the asymmetric AB dimer. The homotypic interactions between AB dimers result in a 5-fold symmetric contact, while the heterotypic interactions between AB and CC dimers result in 6-fold symmetric contact. We found that the assembly can be described as a physical process of phase transition that is regulated by various factors such as concentration and specific stoichiometry between AB and CC dimers. Our simulations also demonstrate that heterotypic and homotypic interfaces play distinctive roles in modulating the assembling kinetics. The interaction between AB and CC dimers is much more dynamic than that between two AB dimers. We therefore suggest that the alternate growth of viral capsid through the heterotypic dimer interactions dominates the assembling pathways. This is, to the best of our knowledge, the first multiscale model to simulate the assembling process of coat proteins in bacteriophage MS2. The generality of this approach opens the door to its further applications in assembly of other viral capsids, virus-like particles, and novel drug delivery systems.

Dataset Information

Construction of armored RNA containing long-size chimeric RNA by increasing the number and affinity of the pac site in exogenous rna and sequence coding coat protein of the MS2 bacteriophage.

Publications

Construction of armored RNA containing long-size chimeric RNA by increasing the number and affinity of the pac site in exogenous rna and sequence coding coat protein of the MS2 bacteriophage.

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