Amino-Functionalized 5' Cap Analogs as Tools for Site-Specific Sequence-Independent Labeling of mRNA.
Bioconjug Chem
; 28(7): 1978-1992, 2017 07 19.
Article
en En
| MEDLINE
| ID: mdl-28613834
ABSTRACT
mRNA is a template for protein biosynthesis, and consequently mRNA transport, translation, and turnover are key elements in the overall regulation of gene expression. Along with growing interest in the mechanisms regulating mRNA decay and localization, there is an increasing need for tools enabling convenient fluorescent labeling or affinity tagging of mRNA. We report new mRNA 5' cap analog-based tools that enable site-specific labeling of RNA within the cap using N-hydroxysuccinimide (NHS) chemistry. We explored two complementary methods:
a co-transcriptional labeling method, in which the label is first attached to a cap analog and then incorporated into RNA by in vitro transcription, and a post-transcriptional labeling method, in which an amino-functionalized cap analog is incorporated into RNA followed by chemical labeling of the resulting transcript. After testing the biochemical properties of RNAs carrying the novel modified cap structures, we demonstrated the utility of fluorescently labeled RNAs in decapping assays, RNA decay assays, and RNA visualization in cells. Finally, we also demonstrated that mRNAs labeled by the reported method are translationally active. We envisage that the novel analogs will provide an alternative to radiolabeling of mRNA caps for in vitro studies and open possibilities for new applications related to the study of mRNA fates in vivo.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Coloración y Etiquetado
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Succinimidas
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Caperuzas de ARN
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ARN Mensajero
Límite:
Animals
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Humans
Idioma:
En
Revista:
Bioconjug Chem
Asunto de la revista:
BIOQUIMICA
Año:
2017
Tipo del documento:
Article
País de afiliación:
Polonia
Pais de publicación:
EEUU
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ESTADOS UNIDOS
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ESTADOS UNIDOS DA AMERICA
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EUA
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UNITED STATES
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UNITED STATES OF AMERICA
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US
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USA