In vitro production of synthetic viral RNAs and their delivery into mammalian cells and the application of viral RNAs in the study of innate interferon responses

• Published in: Methods (San Diego, Calif.) Calif.), 2020-11, Vol.183, p.21-29
• Main Authors: Jiang, Miao, Österlund, Pamela, Poranen, Minna M., Julkunen, Ilkka
• Format: Article
• Language: English
• Subjects: Dendritic cells (DCs); DsiRNA; In vitro RNA production; Innate immunity; Interferon (IFN) response; RNA; RNA delivery; Virus
• ISSN: 1046-2023; 1095-9130
• DOI: 10.1016/j.ymeth.2019.10.013

•ssRNA and dsRNA can be produced in vitro by T7 and phi6 RNA polymerases.•RNA can be delivered into cells by various transfection methods.•Produced ssRNA and dsRNA can be applied e.g. in studying innate immune responses.•In vitro RNA synthesis system can produce DsiRNA swarms to inhibit virus infection. Mammalian cells express different types of RNA molecules that can be classified as protein coding RNAs (mRNA) and non-coding RNAs (ncRNAs) the latter of which have housekeeping and regulatory functions in cells. Cellular RNAs are not recognized by cellular pattern recognition receptors (PRRs) and innate immunity is not activated. RNA viruses encode and express RNA molecules that usually differ from cell-specific RNAs and they include for instance 5′capped and 5′mono- and triphosphorylated RNAs, small viral RNAs and viral RNA-protein complexes called vRNPs. These molecules are recognized by certain members of Toll-like receptor (TLR) and RIG-I-like receptor (RLR) families leading to activation of innate immune responses and the production of antiviral cytokines, such as type I and type III interferons (IFNs). Virus-specific ssRNA and dsRNA molecules that mimic the viral genomic RNAs or their replication intermediates can efficiently be produced by bacteriophage T7 DNA-dependent RNA polymerase and bacteriophage phi6 RNA-dependent RNA polymerase, respectively. These molecules can then be delivered into mammalian cells and the mechanisms of activation of innate immune responses can be studied. In addition, synthetic viral dsRNAs can be processed to small interfering RNAs (siRNAs) by a Dicer enzyme to produce a swarm of antiviral siRNAs. Here we describe the biology of RNAs, their in vitro production and delivery into mammalian cells as well as how these molecules can be used to inhibit virus replication and to study the mechanisms of activation of the innate immune system.