Biology:RNA transfection

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Short description: Deliberately introducing RNA to living cells

RNA transfection is the process of deliberately introducing RNA into a living cell. RNA can be purified from cells after lysis or synthesized from free nucleotides either chemically, or enzymatically using an RNA polymerase to transcribe a DNA template. As with DNA, RNA can be delivered to cells by a variety of means including microinjection, electroporation, and lipid-mediated transfection. If the RNA encodes a protein, transfected cells may translate the RNA into the encoded protein.[1] If the RNA is a regulatory RNA (such as a miRNA), the RNA may cause other changes in the cell (such as RNAi-mediated knockdown).

Encapsulating the RNA molecule in lipid nanoparticles was a breakthrough for producing viable RNA vaccines, solving a number of key technical barriers in delivering the RNA molecule into the human cell.[2][3]

Terminology

RNA molecules shorter than about 25nt (nucleotides) largely evade detection by the innate immune system, which is triggered by longer RNA molecules. Most cells of the body express proteins of the innate immune system, and upon exposure to exogenous long RNA molecules, these proteins initiate signaling cascades that result in inflammation. This inflammation hypersensitizes the exposed cell and nearby cells to subsequent exposure. As a result, while a cell can be repeatedly transfected with short RNA with few non-specific effects, repeatedly transfecting cells with even a small amount of long RNA can cause cell death unless measures are taken to suppress or evade the innate immune system (see "Long-RNA transfection" below).

Short-RNA transfection

Short-RNA transfection is routinely used in biological research to knock down the expression of a protein of interest (using siRNA) or to express or block the activity of a miRNA (using short RNA that acts independently of the cell's RNAi machinery, and therefore is not referred to as siRNA). While DNA-based vectors (viruses, plasmids) that encode a short RNA molecule can also be used, short-RNA transfection does not risk modification of the cell's DNA, a characteristic that has led to the development of short RNA as a new class of macromolecular drugs.[4]

Long-RNA transfection

Long-RNA transfection is the process of deliberately introducing RNA molecules longer than about 25nt into living cells. A distinction is made between short- and long-RNA transfection because exogenous long RNA molecules elicit an innate immune response in cells that can cause a variety of nonspecific effects including translation block, cell-cycle arrest, and apoptosis.

Endogenous vs. exogenous long RNA

The innate immune system has evolved to protect against infection by detecting pathogen-associated molecular patterns (PAMPs), and triggering a complex set of responses collectively known as “inflammation”. Many cells express specific pattern recognition receptors (PRRs) for exogenous RNA including toll-like receptor 3,7,8 (TLR3, TLR7, TLR8),[5][6][7][8] the RNA helicase RIG1 (RARRES3),[9] protein kinase R (PKR, a.k.a. EIF2AK2),[10][11] members of the oligoadenylate synthetase family of proteins (OAS1, OAS2, OAS3), and others. All of these proteins can specifically bind to exogenous RNA molecules and trigger an immune response. The specific chemical, structural or other characteristics of long RNA molecules that are required for recognition by PRRs remain largely unknown despite intense study. At any given time, a typical mammalian cell may contain several hundred thousand mRNA and other, regulatory long RNA molecules. How cells distinguish exogenous long RNA from the large amount of endogenous long RNA is an important open question in cell biology. Several reports suggest that phosphorylation of the 5'-end of a long RNA molecule can influence its immunogenicity, and specifically that 5'-triphosphate RNA, which can be produced during viral infection, is more immunogenic than 5'-diphosphate RNA, 5'-monophosphate RNA or RNA containing no 5' phosphate.[12][13][14][15][16][17] However, in vitro-transcribed (ivT) long RNA containing a 7-methylguanosine cap (present in eukaryotic mRNA) is also highly immunogenic despite having no 5' phosphate,[18] suggesting that characteristics other than 5'-phosphorylation can influence the immunogenicity of an RNA molecule.

Eukaryotic mRNA contains chemically modified nucleotides such as N6-methyladenosine, 5-methylcytidine, and 2'-O-methylated nucleotides. Although only a very small number of these modified nucleotides are present in a typical mRNA molecule, they may help prevent mRNA from activating the innate immune system by disrupting secondary structure that would resemble double-stranded RNA (dsRNA),[19][7] a type of RNA thought to be present in cells only during viral infection. The immunogenicity of long RNA has been used to study both innate and adaptive immunity.

Repeated long-RNA transfection

Inhibiting only three proteins, interferon-β, STAT2, and EIF2AK2 is sufficient to rescue human fibroblasts from the cell death caused by frequent transfection with long, protein-encoding RNA.[18] Inhibiting interferon signaling disrupts the positive-feedback loop that normally hypersensitizes cells exposed to exogenous long RNA. Researchers have recently used this technique to express reprogramming proteins in primary human fibroblasts.[20]

See also

  • RNA vaccines

References

  1. ↑ "Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA". Journal of Visualized Experiments (153). November 2019. doi:10.3791/60143. PMID 31762462. 
  2. ↑ Cooney, Elizabeth (1 December 2020). "How nanotechnology helps mRNA Covid-19 vaccines work". https://www.statnews.com/2020/12/01/how-nanotechnology-helps-mrna-covid19-vaccines-work/. Retrieved 3 December 2020. 
  3. ↑ Foley, Katherine Ellen (22 December 2020). "The first Covid-19 vaccines have changed biotech forever". Quartz (Quartz Media). https://qz.com/1948132/the-first-covid-19-vaccines-have-changed-biotech-forever/. 
  4. ↑ Tansey B (11 August 2006). "Macular degeneration treatment interferes with RNA messages". San Francisco Chronicle. http://www.sfgate.com/cgi-bin/article.cgi?f=/c/a/2006/08/11/BUGPNKFU6T1.DTL. 
  5. ↑ "Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3". Nature 413 (6857): 732–738. 2001. doi:10.1038/35099560. PMID 11607032. Bibcode: 2001Natur.413..732A. 
  6. ↑ "mRNA is an endogenous ligand for Toll-like receptor 3". J Biol Chem 279 (13): 12542–12550. 2004. doi:10.1074/jbc.M310175200. PMID 14729660. 
  7. ↑ 7.0 7.1 "Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA". Immunity 23 (2): 165–175. 2005. doi:10.1016/j.immuni.2005.06.008. PMID 16111635. 
  8. ↑ "Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA". Science 303 (5663): 1529–1531. 2004. doi:10.1126/science.1093616. PMID 14976261. Bibcode: 2004Sci...303.1529D. 
  9. ↑ "The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses". Nat Immunol 5 (7): 730–737. 2004. doi:10.1038/ni1087. PMID 15208624. 
  10. ↑ "Protein synthesis in rabbit reticulocytes. Purification and characterization of a double-stranded RNA-dependent protein synthesis inhibitor from reticulocyte lysates". J Biol Chem 256 (12): 6491–6495. 1981. doi:10.1016/S0021-9258(19)69192-1. PMID 7240221. 
  11. ↑ "Characterization of purified double-stranded RNA-activated eIF-2 alpha kinase from rabbit reticulocytes". J Biol Chem 256 (14): 7638–7641. 1981. doi:10.1016/S0021-9258(19)69008-3. PMID 6265457. 
  12. ↑ "5'-triphosphate RNA is the ligand for RIG-I". Science 314 (5801): 994–997. 2006. doi:10.1126/science.1132505. PMID 17038590. Bibcode: 2006Sci...314..964H. 
  13. ↑ Saito T; Owen DM; Jiang F; Marcotrigiano J; Gale M, Jr. (2008). "Innate immunity induced by composition-dependent RIG-I recognition of Hepatitis C virus RNA". Nature 454 (7203): 523–527. doi:10.1038/nature07106. PMID 18548002. Bibcode: 2008Natur.454..523S. 
  14. ↑ "Nonself RNA-sensing mechanism of RIG-I helicase and activation of antiviral immune responses". Mol Cell 29 (4): 428–440. 2008. doi:10.1016/j.molcel.2007.11.028. PMID 18242112. 
  15. ↑ "Structural mechanism of RNA recognition by the RIG-I-like receptors". Immunity 29 (2): 178–181. 2008. doi:10.1016/j.immuni.2008.07.009. PMID 18701081. 
  16. ↑ "5'-triphosphate RNA requires base-paired structures to activate antiviral signaling via RIG-I". Proc Natl Acad Sci USA 106 (29): 12067–12072. 2009. doi:10.1073/pnas.0900971106. PMID 19574455. Bibcode: 2009PNAS..10612067S. 
  17. ↑ "Recognition of 5'-triphosphate by RIG-I helicase requires short blunt double-stranded RNA as contained in panhandle of negative strand virus". Immunity 31 (1): 25–34. 2009. doi:10.1016/j.immuni.2009.05.008. PMID 19576794. 
  18. ↑ 18.0 18.1 "Innate Immune Suppression Enables Frequent Transfection with RNA Encoding Reprogramming Proteins". PLOS ONE 5 (7): e11756. 2010. doi:10.1371/journal.pone.0011756. PMID 20668695. Bibcode: 2010PLoSO...511756A. 
  19. ↑ "Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA". Journal of Visualized Experiments (153). November 2019. doi:10.3791/60143. PMID 31762462. 
  20. ↑ Trafton A (26 July 2010). "RNA offers a safer way to reprogram cells". MIT News Office. http://web.mit.edu/newsoffice/2010/reprogramming-stem-cells-0726.html. 

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