The protocol presents in vitro transcription (IVT) of chemically modified mRNA, cationic liposome preparation, and functional analysis of liposome enabled mRNA transfections in mammalian cells.
In recent years, chemically modified messenger RNA (mRNA) has emerged as a potent nucleic acid molecule for developing a wide range of therapeutic applications, including a novel class of vaccines, protein replacement therapies, and immune therapies. Among delivery vectors, lipid nanoparticles are found to be safer and more effective in delivering RNA molecules (e.g., siRNA, miRNA, mRNA) and a few products are already in clinical use. To demonstrate lipid nanoparticle-mediated mRNA delivery, we present an optimized protocol for the synthesis of functional me1Ψ-UTP modified eGFP mRNA, the preparation of cationic liposomes, the electrostatic complex formation of mRNA with cationic liposomes, and the evaluation of transfection efficiencies in mammalian cells. The results demonstrate that these modifications efficiently improved the stability of mRNA when delivered with cationic liposomes and increased the eGFP mRNA translation efficiency and stability in mammalian cells. This protocol can be used to synthesize the desired mRNA and transfect with cationic liposomes for target gene expression in mammalian cells.
As a therapeutic molecule, mRNA offers several advantages due to its non-integrative nature and its ability to transfect non-mitotic cells when compared to plasmid DNA (pDNA)1. Although mRNA delivery was demonstrated in the early 1990s, therapeutic applications were limited due to its lack of stability, its lack of immune activation, and poor translational efficiency2. Recently identified chemical modifications, such as pseudouridine 5'-triphosphate (Ψ-UTP) and methyl pseudouridine 5'-triphosphate (me1Ψ-UTP) on mRNA, helped to overcome these limitations, revolutionized mRNA research, and in turn, made mRNA a promising tool in both basic and applied research. The range of applications covers the generation of iPSCs to vaccination and gene therapy3,4.
In parallel to advancement in mRNA technology, significant advances in non-viral delivery systems made the delivery of mRNA effective, making this technology feasible for multiple therapeutic applications5. Among the non-viral vectors, lipid nanoparticles have been found to be effective in delivering nucleic acids6,7. Recently, Alnylam has received FDA approval of lipid-based siRNA drugs for treating liver diseases, including Patisiran for hereditary transthyretin-mediated amyloidosis (hATTR amyloidosis) and Givosiran for acute hepatic porphyrias (AHP)8. During the COVID19 pandemic, lipid encapsulated mRNA based vaccines from Pfizer-BioNtech and Moderna demonstrated their efficacy and received FDA approvals9,10. Thus, lipid enabled mRNA delivery has a great therapeutic potential.
Here, we describe a detailed protocol for the production of chemically modified, in vitro transcribed eGFP mRNA, cationic liposome preparation, mRNA-lipid complex optimization and transfections into mammalian cells (Figure 1).
1. Production of me1 Ψ-UTP modified mRNA
2. Preparation of cationic liposomes and evaluation of in vitro mRNA transfection properties
We optimized the protocol for me1Ψ-UTP modified mRNA production, liposome preparation, and mRNA transfection experiments with cationic liposomes into multiple mammalian cells (Figure 1). To synthesize mRNA, the mammalian codon-optimized eGFP IVT template was amplified from the mEGFP-N1 mammalian expression vector and purified by organic extraction/ethanol precipitation method (Figure 2). Later, me1Ψ-UTP modified RNA and mRNA were produced by the IVT process. The denaturing RNA agarose gel electrophoresis data showed that these synthesized RNAs had good integrity and the correct length (750 base RNA, and ~1000 base mRNA with respect to the RNA ladder) (Figure 3).
To prepare cationic liposomes, a thin-film hydration method and sonication were used to form small unilamellar vesicles (SUVs). Physico-chemical characterization of the liposomes revealed that the hydrodynamic diameters were observed around 65 nm and the surface potentials were around +20 meV (Figure 4). A gel retardation assay was performed with liposome-mRNA complexes at varying charge ratios of lipid/base from 1:1 to 8:1. The cationic liposomes showed a high binding efficiency to mRNAs even at a 1:1 charge ratio (Figure 5). Hence, we used a 1:1 charge ratio for mRNA transfection experiments. Liposome mediated eGFP mRNA transfection experiments were performed in HEK-293T and NIH/3T3 cell lines. eGFP expression was analyzed with flow cytometry. me1Ψ-UTP modified mRNA showed superior and stable eGFP protein expression when compared to unmodified mRNA in HEK-293T and NIH/3T3 cells on the 3rd-day post-transfection (Figure 6, 7).
Figure 1: Schematic presentation of me1Ψ-UTP modified mRNA production, liposome preparation and transfection protocol. The IVT DNA Template (T7 promoter-Gene ORF) is amplified by PCR and purified. me1Ψ-UTP modified mRNAs are generated by the IVT process using the IVT DNA template and purified. The cationic liposome is prepared and complexed with me1Ψ-UTP modified mRNA (Lipoplex) and can be transfected into mammalian cells. Please click here to view a larger version of this figure.
Figure 2: Determination of IVT DNA template quality in agarose gel electrophoresis. The purified eGFP IVT DNA template was run on a 1% agarose gel and visualized by gel. Please click here to view a larger version of this figure.
Figure 3: Analysis of size and quality of me1Ψ-UTP modified IVT RNA by denaturing RNA electrophoresis in agarose gel. The purified me1Ψ-UTP modified RNAs were denatured and loaded on a 1% TAE-agarose gel, and the size and quality of RNAs were determined by gel. Lane 1: ssRNA ladder, Lane 2: me1Ψ-UTP modified IVT RNA, Lane 3: me1Ψ-UTP modified IVT RNA with Cap1 and Poly A tail.Please click here to view a larger version of this figure.
Figure 4: Physico-chemical characterization of the liposomes: Surface potentials (A) and hydrodynamic diameters (B). Please click here to view a larger version of this figure.
Figure 5: mRNA binding ability of liposome was determined by denaturing agarose gel retardation assay. Liposome-mRNA complexes (Lipoplexes) were prepared at different lipid/base charge ratios and loaded on a 1% agarose gel and gel documented. Please click here to view a larger version of this figure.
Figure 6: Protein expression efficiency of me1Ψ-UTP modified eGFP mRNA in human HEK-293T cells. (A) Fluorescence images of unmodified eGFP mRNA and me1Ψ-UTP-modified eGFP mRNA transfected with cationic liposomes into HEK-293T cells obtained on 3rd-day post-transfection (100x Magnification). % of eGFP protein expression (B) and mean fluorescent intensity (MFI). (C) of the transfected cellswere analyzed using flow cytometry. (N=3) Please click here to view a larger version of this figure.
Figure 7: Translation efficiency of me1Ψ-UTP modified eGFP mRNA in mouse NIH/3T3 cells. (A) Fluorescence images of unmodified eGFP mRNA and me1Ψ-UTP-modified eGFP mRNA transfected into NIH/3T3 cells obtained on the 3rd-day post-transfection (100x Magnification). % of eGFP protein expression (B) and mean fluorescent intensity (MFI). (C) of the transfected cellswere analyzed using flow cytometry. (N=3) Please click here to view a larger version of this figure.
Components | 25 µL reaction | Final concentration |
5x Q5 buffer | 5 µL | 1x |
10 mM dNTP | 0.5 µL | 200 µM |
10 µM Forward primer | 1.25 µL | 0.5 µM |
10 µM Reverse primer | 1.25 µL | 0.5 µM |
Q5 polymerase | 0.25 µL | 0.02 U/µL |
Gene of interest in plasmid (template) | 1-5 ng | variable |
Nuclease free water | To 25 µL |
Table 1: PCR reaction mixture preparation
Steps | Duration | Temperature | Cycle number |
Initial denaturation | 30 seconds | 98 °C | 1 |
Denaturation | 10 seconds | 98 °C | |
Annealing | 20 seconds | variable | 18-25 |
Extension | variable | 72 °C | |
Final extension | 2 minutes | 72 °C | 1 |
Hold | ∞ | 1 5°C |
Table 2: PCR cycling conditions
Components | Unmodified RNA | me1Ψ-UTPmodified RNA |
RNase-Free Water | variable | variable |
Linearized template DNA with T7 RNAP promoter | variable (1 µg) | variable (1 µg) |
10x T7 TranscriptionBuffer | 2 µL | 2 µL |
100 mM N1-methyl Pseudouridine | – | 1.5 µL |
100 mM ATP | 1.8 µL | 1.8 µL |
100 mM UTP | 1.8 µL | – |
100 mM CTP | 1.8 µL | 1.8 µL |
100 mM GTP | 1.8 µL | 1.8 µL |
100 mM DTT | 2 µL | 2 µL |
40 U/µL RNase Inhibitor | 0.5 µL | 0.5 µL |
T7 Enzyme Solution | 2 µL | 2 µL |
Total Reaction Volume | 20 µL | 20 µL |
Table 3: IVT reaction mixture preparation
Components | RNA ladder | RNA sample |
2x RNA loading dye (NEB) | 6 µL | 6 µL |
RNA | 2 µL | 1 µL (0.5-1 µg ) |
DEPC treated water | 4 µL | 5 µL |
Total volume | 12 µL | 12 µL |
Table 4: RNA loading dye preparation
Components | Quantity |
10x CappingBuffer | 10 µL |
20 mM GTP | 5 µL |
20 mM SAM | 2.5 µL |
RNase Inhibitor | 2.5 µL |
2'-O-Methyltransferase | 4 µL |
Total Volume | 24 µL |
Table 5: Enzymatic Cap-1 synthesis reaction mixture
Components | Quantity |
5’-Capped IVT RNA | 100 µL |
RNase Inhibitor | 0.5 µL |
10x A-PlusTailingBuffer | 12 µL |
20 mM ATP | 6 µL |
4 U/µL A-Plus Poly(A) Polymerase | 5 µL |
Total Volume | 123.5 µL |
Table 6: Poly A tailing reaction mixture
Charge ratio | Liposome | DI water | mRNA(500ng) | DI water |
1:1 | 1.5 μL | 8.5 μL | 1 μL | 9 μL |
2:1 | 3 μL | 7 μL | 1 μL | 9 μL |
4:1 | 6 μL | 4 μL | 1 μL | 9 μL |
8:1 | 12 μL | – | 1 μL | 9 μL |
Total volume (20 μL) | 10 μL | 10 μL |
Table 7: Preparation of lipoplex based on charge ratios
Name | Components |
50x TAE buffer | Dissolve 50 mM EDTA sodium salt, 2 M Tris, 1 M glacial acetic acid in 1 L of water |
HEK-293T and NIH/3T3 cell culture medium | DMEM with 4.5 g/L glucose, L-glutamine, 1% penicillin/streptomycin and 10% FBS |
Table 8: Preparation of buffer and media
Therapeutic applications of unmodified mRNAs have been limited due to their shorter half-life and their ability to activate intracellular innate immune responses, which in turn lead to poor protein expression in transfected cells11. Katalin et al. demonstrated that RNA containing modified nucleosides such as m5C, m6A, ΨU, and me1Ψ-UTP could avoid TLR activation12. More importantly, incorporation of ΨU or me1Ψ-UTP in IVT mRNA showed superior translational efficiency of target proteins, improved stability at room temperature, and prevented degradation from nucleases13, 14.
In this video, we demonstrated the protocol for lipid enabled me1Ψ-UTP modified mRNA delivery into multiple cultured cells. The protocol includes production of me1Ψ-UTP modified mRNA, cationic liposome preparation, transfection into cells, and evaluation of protein expression. We used the mammalian codon-optimized eGFP reporter gene for transfection experiments to analyze protein expression levels by measuring fluorescent intensity. Cationic liposomes were prepared to complex mRNA, and their electrostatic complexation was analyzed at varying lipid/base charge ratios from 1:1 to 8:1. Since at 1:1, the cationic liposomes could completely complex mRNA, we used a 1:1 charge ratio for transfection. We demonstrated that transfection of mRNA with cationic liposomes could efficiently deliver both modified and unmodified eGFP mRNAs with 90% transfection efficiency in HEK-293T cells, whereas there were 80% efficiency with modified and 60% efficiency with unmodified mRNAs in NIH/373 cells. More importantly, me1Ψ-UTP modified mRNA showed superior eGFP protein expression for 3 days in mammalian cells compared to unmodified mRNA (>6 fold in HEK-293T and >2 fold in NIH/3T3 cells). These studies demonstrated that modification of me1Ψ-UTP on mRNA could improve translation and stability of mRNA in mammalian cells.
The transfection efficiency of cationic liposome and translation efficiency of synthesized mRNA vary with different cell types. Hence, it is important to optimize mRNA concentration for each different cell type. Using the protocol, we synthesized functional me1Ψ-UTP modified mRNA, size up to 6 kb but the IVT DNA template concentration and time could be optimized to get good mRNA yield and correct length.
The authors have nothing to disclose.
MS thanks the Department of Biotechnology, India, for the financial support (BT/PR25841/GET/119/162/2017), Dr Alok Srivastava, Head, CSCR, Vellore, for his support and Dr Sandhya, CSCR core facilities for imaging and FACS experiments. We thank R. Harikrishna Reddy and Rajkumar Banerjee, Applied Biology Division, CSIR-Indian Institute of Chemical Technology Uppal Road, Tarnaka, Hyderabad, 500 007, TS, India, for their help in analyzing physico-chemical data of the liposomes. Vigneshwaran V, and Joshua A, CSCR for their help in video making.
Agarose | Lonza | 50004 | |
Bath sonicator | DNMANM Industries | USC-100 | |
Cationic lipid | Synthesized in the lab | ||
Chlorofrom | MP biomedicals | 67-66-3 | "Caution" |
Cholesterol | Himedia | GRM335 | |
DEPC water | SRL BioLit | 66886 | |
DMEM | Lonza | 12-604F | |
DNA Ladder | GeneDireX | DM010-R50C | |
DOPE | TCI | D4251 | |
EDTA sodium salt | MP biomedicals | 194822 | |
Ethanol | Hayman | F204325 | "Caution" |
Fetal bovine serum | Gibco | 10270 | |
Flow cytometry | BD | FACS Celesta | |
Fluroscence Microscope | Leica | MI6000B | |
Gel documentation system | Cell Biosciences | Flurochem E | |
Glacial acetic acid | Fisher Scientific | 85801 | "Caution" |
mEGFP-N1, Mammalian expression vector | Addgene | 54767 | |
N1-Methylpseudo-UTP | Jena Bioscience | NU-890 | |
Phenol:chloroform:isoamyl alchol (25:24:1), pH 8.0 | SRL BioLit | 136112-00-0 | "Caution" |
Phosphate Buffer Saline (PBS), pH 7.4 | CellClone | CC3041 | |
Probe sonicator | Sonics Vibra Cells | VCX130 | |
RNA ladder | NEB | N0362S | |
RNase inhibitor | Thermo Scientific | N8080119 | |
SafeView dye | abm | G108 | |
Sodium acetate | Sigma | S7545 | |
Thermocycler | Applied biosystems | 4375786 | |
Thermomixer | Eppendrof | 22331 | |
Tris buffer | SRL BioLit | 71033 | |
Trypsin | Gibco | 25200056 |
.