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

• Type of Material: Article
• Authors: Jonathan L. Kirschman, Georgia Institute of Technology and Emory University; Sushma Bhosle, Georgia Institute of Technology and Emory University; Daryll Vanover, Georgia Institute of Technology and Emory University; Emmeline L. Blanchard, Georgia Institute of Technology and Emory University; Kristin H. Loomis, Georgia Institute of Technology and Emory University; Chiara Zurla, Georgia Institute of Technology and Emory University; Kathryn Murray, Georgia Institute of Technology and Emory University; Blaine C. Lam, Georgia Institute of Technology and Emory University; Philip Santangelo, Emory University
• Language: English
• Date: 2017-04-26
• Publisher: Oxford University Press
• Publication Version: Final Published Version
• Copyright Statement: © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
• License: Creative Commons Attribution-NonCommercial 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1093/nar/gkx290
• Title of Journal or Parent Work: Nucleic Acids Research
• ISSN: 0305-1048
• Volume: 45
• Issue: 12
• Grant/Funding Information: Defense Advanced Research Projects Agency ADEPT PROTECT program; National Science Foundation Graduate Research Fellowship Program [DGE-1650044].; Funding for open access charge: DARPA ADEPT PROTECT program.
• Keywords: IMMUNE ACTIVATION; Life Sciences & Biomedicine; PROXIMITY LIGATION ASSAY; INTERACTIONS IN-SITU; KINASE PKR; LIVE CELLS; NUCLEOSIDE MODIFICATIONS; GENE-TRANSFER; Science & Technology; SENSITIVE PROBES; MEDIATED ENDOCYTOSIS; Biochemistry & Molecular Biology; EUKARYOTIC STRESS GRANULES
• Research Categories: Engineering, Biomedical

Abstract

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.