Characterizing exogenous mRNA delivery, trafficking, cytoplasmic release and RNA-protein correlations at the level of single cells.

Autor: Kirschman JL; Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, UA Whitaker Building, 313 Ferst Drive, Atlanta, GA 30332, USA., Bhosle S; Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, UA Whitaker Building, 313 Ferst Drive, Atlanta, GA 30332, USA., Vanover D; Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, UA Whitaker Building, 313 Ferst Drive, Atlanta, GA 30332, USA., Blanchard EL; Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, UA Whitaker Building, 313 Ferst Drive, Atlanta, GA 30332, USA., Loomis KH; Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, UA Whitaker Building, 313 Ferst Drive, Atlanta, GA 30332, USA., Zurla C; Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, UA Whitaker Building, 313 Ferst Drive, Atlanta, GA 30332, USA., Murray K; Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, UA Whitaker Building, 313 Ferst Drive, Atlanta, GA 30332, USA., Lam BC; Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, UA Whitaker Building, 313 Ferst Drive, Atlanta, GA 30332, USA., Santangelo PJ; Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, UA Whitaker Building, 313 Ferst Drive, Atlanta, GA 30332, USA.
Jazyk: angličtina
Zdroj: Nucleic acids research [Nucleic Acids Res] 2017 Jul 07; Vol. 45 (12), pp. e113.
DOI: 10.1093/nar/gkx290
Abstrakt: The use of synthetic messenger ribonucleic acid (mRNA) to express specific proteins is a highly promising therapeutic and vaccine approach that avoids many safety issues associated with viral or DNA-based systems. However, in order to optimize mRNA designs and delivery, technology advancements are required to study fundamental mechanisms of mRNA uptake and localization at the single-cell and tissue level. Here, we present a single RNA sensitive fluorescent labeling method which allows us to label and visualize synthetic mRNA without significantly affecting function. This approach enabled single cell characterization of mRNA uptake and release kinetics from endocytic compartments, the measurement of mRNA/protein correlations, and motivated the investigation of mRNA induced cellular stress, all important mechanisms influencing protein production. In addition, we demonstrated this approach can facilitate near-infrared imaging of mRNA localization in vivo and in ex-vivo tissue sections, which will facilitate mRNA trafficking studies in pre-clinical models. Overall, we demonstrate the ability to study fundamental mechanisms necessary to optimize delivery and therapeutic strategies, in order to design the next generation of novel mRNA therapeutics and vaccines.
(© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)
Databáze: MEDLINE