User:Matthew Angel/RNA transfection

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. If the RNA is a regulatory RNA (such as a miRNA), the RNA may cause other changes in the cell (such as RNAi-mediated kockdown).

Terminology
RNA molecules shorter than about 25nt 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.

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), the RNA helicase RIG1 (RARRES3), protein kinase R (PKR, a.k.a. EIF2AK2), 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. 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, 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 may be present in an mRNA molecule, they may help prevent mRNA from activating the innate immune system by disrupting secondary structure that would resemble double-stranded RNA (dsRNA), 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. 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.