Improved design of hammerhead ribozyme for selective digestion of target RNA through recognition of site-specific adenosine-to-inosine RNA editing.
RNA
; 20(3): 392-405, 2014 Mar.
Article
em En
| MEDLINE
| ID: mdl-24448449
ABSTRACT
Adenosine-to-inosine (A-to-I) RNA editing is an endogenous regulatory mechanism involved in various biological processes. Site-specific, editing-state-dependent degradation of target RNA may be a powerful tool both for analyzing the mechanism of RNA editing and for regulating biological processes. Previously, we designed an artificial hammerhead ribozyme (HHR) for selective, site-specific RNA cleavage dependent on the A-to-I RNA editing state. In the present work, we developed an improved strategy for constructing a trans-acting HHR that specifically cleaves target editing sites in the adenosine but not the inosine state. Specificity for unedited sites was achieved by utilizing a sequence encoding the intrinsic cleavage specificity of a natural HHR. We used in vitro selection methods in an HHR library to select for an extended HHR containing a tertiary stabilization motif that facilitates HHR folding into an active conformation. By using this method, we successfully constructed highly active HHRs with unedited-specific cleavage. Moreover, using HHR cleavage followed by direct sequencing, we demonstrated that this ribozyme could cleave serotonin 2C receptor (HTR2C) mRNA extracted from mouse brain, depending on the site-specific editing state. This unedited-specific cleavage also enabled us to analyze the effect of editing state at the E and C sites on editing at other sites by using direct sequencing for the simultaneous quantification of the editing ratio at multiple sites. Our approach has the potential to elucidate the mechanism underlying the interdependencies of different editing states in substrate RNA with multiple editing sites.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Encéfalo
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RNA
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Adenosina
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RNA Catalítico
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Edição de RNA
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Receptor 5-HT2C de Serotonina
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Inosina
Limite:
Animals
Idioma:
En
Revista:
RNA
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2014
Tipo de documento:
Article