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Comprehensive evaluation of T7 promoter for enhanced yield and quality in mRNA production – Scientific Reports

Unless otherwise stated, all chemicals and reagents were purchased from ThermoFisher Scientific (UK).

Template construction for mRNA synthesis

Template design

The mRNA template consists of the EGFP gene (GenBank Accession #AAB02572.1) flanked by two untranslated regions (5’-UTR and 3’-UTR) and followed by a poly-A sequence. The 5’-UTR comprises three elements: the wildtype or mutant promoter of T7 RNA polymerase, a binding site of eukaryotic translation initiation factor eIF4G, and a Kozak consensus sequence16,23. The 3’-UTR utilises two tandem repeats of 3’-UTR from the human β-globin gene. The poly-A sequence (120 bp) is segmented with a 6 bp spacer46. Additional templates were assembled by fusing the EGFP gene with the Klebsiella pneumoniae transaminase gene (GenBank Accession #AF074934.1), and the EGFP gene with the T7 RNA polymerase gene (GenBank Accession #NP_041960.1). Sequences used in this study are presented in Supporting Information Table S1. All the mRNA templates are inserted in a pUC57 plasmid vector with kanamycin resistance.

Promoter modification and plasmid construction

Site-directed mutagenesis

Sited-directed mutagenesis was performed to mutate or add downstream and upstream insertion in the promoter region using Q5® Site-Directed Mutagenesis Kit (New England Biolabs, UK). A plasmid control, comprising of mRNA template with wildtype T7 promoter adapted from Rosa et al.23 was used as the template for mutagenesis. The amplification of reaction mix (1 × Q5® Hot Start High-Fidelity Master Mix, 0.5 μM forward primer, 0.5 μM reverse primer, and 25 ng plasmid template, and Gibco™ Water for Injection, WFI) was performed through touchdown polymerase chain reaction (TD-PCR) using Applied Biosystems™ Veriti™ 96-Well Thermal Cycler (ThermoFisher Scientific, UK) with the following detailed cycle conditions: an initial denaturation step at 98 °C for 30 s, 10 cycles of 10 s at 98 °C and annealing at 66–57 °C (for samples: T7#4, T7c62, T7c62_T7#4, and T7DI_1 to T7DI_11) or 70–61 °C (for samples: T7Max and T7Max_T7#4) for 30 s. The annealing temperature decreased 1 °C per cycle and an extension step was performed at 72 °C for 30 s per kb. This was followed by 20 cycles of 10 s at 98 °C; annealing at 57.5 °C (for samples: T7#4, T7c62, T7c62_T7#4, and T7DI_1 to T7DI_11) or 61.5 °C (for samples: T7Max and T7Max_T7#4) for 30 s; and extension at 72 °C for 30 s per kb. The final extension was executed at 72 °C for 2 min. Afterwards, 1 μL of TD-PCR products were treated with 1 × Kinase, Ligase, and DpnI (KLD) enzyme mix and buffer (New England Biolabs, UK), and adjusted with WFI to a final volume of 10 μL. The KLD mix was incubated for 5 min at 21 °C e and 5 μL of the mix was used for transformation using a heat shock method. All plasmids and primers used in this study are presented in Supporting Information Table S2 and S3, respectively.

Gibson assembly

Using the Gibson assembly method, two plasmids, pT7wt_TA_EGFP and pT7wt_T7 RNAP_EGFP, were obtained. This was achieved by separately integrating the genes for K. pneumoniae transaminase (TA) and T7 RNA polymerase (T7 RNAP) into the pT7wt_EGFP template. TA and T7 RNAP genes were amplified and isolated from pET29A_TA and pET29A_T7 RNAP using PCR with specific primers. The linearisation of vector pT7wt_EGFP also performed through PCR. All the PCR reactions were performed using high-fidelity VeriFi™ DNA Polymerase, VeriFi™ Buffer, and VeriMax Enhancer (PCR Biosystems, UK). The PCR products of linear vector pT7wt_EGFP, and isolated TA and T7 RNAP genes were analysed using agarose-gel electrophoresis and the gel containing correct sizes of DNA bands were further isolated and purified. The purified linear vector pT7wt_EGFP, and the insert (TA gene or T7 RNAP gene) were assembled using Gibson Assembly Master Mix (New England Bioscience, UK). For each reaction, the same mass (0.1 pmol) of insert and vector were mixed with 10 μL of 2 × Gibson Assembly Master Mix and adjusted with WFI to a total volume of 20 μL. The reactions were performed at 50 °C for 2 h. Following the incubation, 2 μL of the assembly products were subsequently transformed to E. coli NEB 10-beta (New England Biolabs, UK).

Molecular cloning

Chemically competent E. coli NEB 10-beta cells (New England Biolabs, UK) were prepared by the calcium chloride method and used for routine transformation. Transformation of plasmids was performed using the heat-shock method. Transformed cells were plated on Luria–Bertani agar media (25 g/L Miller LB broth (Sigma-Aldrich) with 15 g/L culture media agar (MP Biomedicals, USA)) with kanamycin (50 µg/mL) and overnight incubated at 37 °C. Colony PCR was performed using high-fidelity VeriFi™ DNA Polymerase with VeriFi™ Buffer and VeriMax Enhancer (PCR Biosystems, UK). Plasmid DNA was purified using GeneJET Plasmid Miniprep Kit, following the protocol by the manufacturer.

Plasmid verification and sequencing

NanoDrop™ One Microvolume UV–Vis Spectrophotometer (ThermoFisher Scientific, UK) was used to measure the concentration of purified plasmids. Purified plasmids were digested using EcoRI (New England Biolabs, UK) and LguI/SapI with CutSmart® buffer (New England Biolabs, UK) for one hour incubation at 37 °C, followed by inactivation at 65 °C for 20 min. Approximately 100 ng/µL of the purified plasmids were Sanger sequenced (Eurofins Genomics, UK).

Agarose gel electrophoresis

To analyse the PCR and digestion products, 1% (w/v) of agarose (Sigma-Aldrich, USA) was prepared using 0.5 × TBE buffer (45 mM Tris–borate and 1 mM EDTA), Invitrogen SYBR® Safe DNA Gel Stain (1:10,000 dilution), and run at 100 V for one hour. Purple Gel Loading Dye (New England Biolabs, UK) was used to load the samples into the gel and 1 kb Plus DNA Ladder (New England Biolabs, UK) for analysis.

mRNA and dsRNA synthesis

Template production

DNA templates for IVT reactions were produced through touchdown polymerase chain reaction (TD-PCR). The TD-PCR reaction mixture contained between 200 and 250 ng mL−1 of plasmid, 0.4 μM of forward and reverse primers, 1 × VeriFi™ Buffer, 1 × VeriMax Enhancer, and 0.02 U μL−1 high-fidelity VeriFi™ DNA Polymerase (PCR Biosystems, UK). The reaction mixture was prepared to a total volume of 500 μL and split into 50 μL reaction per tube. Detail of plasmids (as templates) and primers are found in S2 and S3, respectively. Supportive Information Table S. The TD-PCR was performed using a Applied Biosystems™ Veriti™ 96-Well Thermal Cycler (ThermoFisher Scientific, UK) with an initial denaturation step at 95 °C for 1 min, 10 cycles of 15 s at 95 °C; annealing at 60–51 °C (for samples: T7#4 and T7DI_1 to T7DI_11) or 66–57 °C (for samples: T7wt, T7Max, T7c62, T7Max_T7#4, and T7c62_T7#4) for 30 s with annealing temperature decreased 1 °C per cycle. Extension was performed at 72 °C for 30 s per kb, followed by 20 cycles of 15 s at 95 °C; annealing at 51.5 °C (for samples: T7#4 and T7DI_1 to T7DI_11) or 58 °C (for samples: T7wt, T7Max, T7c62, T7Max_T7#4, and T7c62_T7#4) for 30 s and extension at 72 °C for 30 s per kb. The final extension was executed at 72 °C for 2 min. The TD-PCR product was purified using GeneJET PCR Purification Kit (ThermoFisher Scientific, UK) following the manufacturer’s instructions. A 10 × concentrated TD-PCR product was obtained from the purification step and further quantified using NanoDrop™ One Microvolume UV–Vis Spectrophotometer (ThermoFisher Scientific, UK).

In-vitro transcription (IVT) reactions

The IVT reaction conditions were adapted from Rosa et al. (2022). The IVT reaction mixture contained 89 nM of linear DNA template (purified TD-PCR product), 7.75 mM of each NTP (ATP, GTP, CTP, and UTP), 5.3 mM of DTT, 49 mM of Mg-acetate (Santa Cruz Biotechnology, USA), 40 mM pH 6.5 Tris buffer, 2.3 mM of spermidine, 0.008 U μL−1 of Saccharomyces cerevisiae inorganic pyrophosphatase, 1.48 U μL−1 of RiboShield™ RNase Inhibitor (PCR Biosystems, UK), 7.7 U μL−1 of bacteriophage T7 RNA polymerase, and was made up to a final volume of 20 μL (for sample measurements) or 100 μL (for the calibration curve) with water for injection (WFI). The IVT was performed at 43 °C for 2 h. The mRNA produced from IVT was quantified using reverse‐phase high‐performance liquid chromatography (RP‐HPLC) described in Section “mRNA quantification”.

To produce dsRNA for the calibration curve, a subsequent incubation was performed after IVT to facilitate the dsRNA hybridisation. The 100 μL of dsRNA IVT product was diluted to 200 μL with water for Injection (WFI) and incubated at gradient temperature 85 °C to 23 °C for 2 min at each temperature.

RNA purification

The mRNA purification for the calibration curve was performed using MEGAclear™ Transcription Clean-Up Kit as instructed by the manufacturer with slight modifications. The 100 μL of IVT product was treated with 2 μL of TURBO™ DNase and incubated for 15 min at 37 °C. For dsRNA purification, after the hybridisation incubation step, 2 μL of TURBO™ DNase and 2 uL of RNase T1 were added to 200 μL of diluted dsRNA IVT product and incubated at 37 °C for 15 min. The 350 μL of binding solution and 250 μL of 100% v/v ethanol were added to the samples and then loaded into the filter cartridge for centrifugation (15,000 × g, 1 min, 21 °C). The filter was washed with 500 μL of wash solution and centrifuged under the same condition in the previous step twice. The mRNA was eluted with 50 μL of elution buffer, followed by 5 min incubation at 65 °C and centrifugation at 15,000 × g for 1 min at 21 °C. The elution step was repeated in the same previous condition. The 100 μL of purified mRNA was further precipitated with 10 μL of 5 M ammonium acetate and 275 μL of 100% v/v ethanol, and then overnight incubated at − 20 °C. Samples were centrifuged (top speed) at 4 °C for 15 min. The supernatant was discarded, and the obtained pellet was air-dried to remove the remaining ethanol. The pellet was resuspended in 40 μL of elution buffer. The concentrated purified mRNA sample was then quantified using NanoDrop™ One Microvolume UV–Vis Spectrophotometer (ThermoFisher Scientific, UK) and RP-HPLC (Section “mRNA quantification”).

mRNA capping for expression studies

One-pot cap-1 reactions were performed using Faustovirus Capping Enzyme (New England Bioloabs, UK) and Cap 2’-O-methyltransferase (New England Bioloabs, UK). Briefly, 50 µg of purified mRNA was added to a reaction containing 1X FCE capping buffer, 0.5 mM GTP, 2 mM S-adenosylmethionine, 1 µL of Rnase inhibitor, 1 U µL−1 of Faustovirus Capping Enzyme, 4 U µL−1 of Cap 2’-O-methyltransferase, and WFI water to a final volume of 50 µL. The samples were incubated at 37ºC without shaking for 2 h. Afterwards, the samples were purified as described in Section “rna purification”. The pellets were resuspended in 10 µL of WFI water, and quantified using NanoDrop™ One Microvolume UV–Vis Spectrophotometer (ThermoFisher Scientific, UK).

Analytical methods

mRNA quantification

RP-HPLC

The total mRNA concentration was quantified using the established RP‐HPLC gradient method adapted from Issa and Packer47 and Rosa et al.23. An UltiMate™ 3000 UHPLC System with a 2.1 × 100 mm DNAPac™ RP column and a 3 × 10 mm guard column was used. 5 µL of each sample, diluted 6 times, was run in the pre‐equilibrated column with TAE buffer absorbance measured at 260 nm. Elution was achieved by a gradient elution using TAE buffer with 25% acetonitrile. The runs were performed at 80 °C with the following conditions: After injection, the column is washed for 1 min and 0.2 mL × min−1. The flow is gradually increased to 0.25 mL × min−1 for 30 s. A gradient to 6% of elution buffer and 0.35 mL × min−1 is applied for 30 s, followed by a gradient to 76.5% of elution buffer at 0.4 mL × min−1 for 4 min, and a final gradient to 100% elution buffer for 1 min. The column is then washed with 100% elution buffer for 3 min, and re-equilibrated with the binding buffer for 6 min.

Agarose gel electrophoresis

A 2% (w/v) of agarose with 0.5 × TBE buffer (45 mM Tris–borate and 1 mM EDTA) and 5.5 mM of magnesium chloride was prepared and pre-stained with Invitrogen SYBR® Safe DNA Gel Stain (1:10,000 dilution). The gel was loaded with 1.5 μL of mRNA sample diluted in WFI into a final volume of 10 μL and 2 μL of 6 × purple Loading Dye (New England Biolabs, UK). A 5 μL of 1 kb Plus DNA Ladder (New England Biolabs, UK) was used as the molecular marker. The electrophoresis was run at 100 V for 75 min using 0.5 × TBE buffer containing 5.5 mM of MgCl2. The gel was visualised using Amersham™ Imager 600 (GE Healthcare, UK).

dsRNA quantification

The dsRNA concentration was measured using the RP‐HPLC method adapted from Issa et al.47 described in Section “mRNA quantification”. Samples of 10 μL were treated with 0.5 μL of RNase T1 and incubated at 37 °C for 15 min to digest the ssRNA (Supplementary Information Fig. S2).

Protein expression

EGFP expression was performed using the 1-Step Human Coupled IVT kit. Capped mRNA (Section “mRNA capping for expression studies”) was diluted to a final concentration of 1 g L−1 and 2 µL were added to the reaction mixture. Positive and negative controls were the kit GFP control and WFI water, respectively. The samples were incubated for 6 h at 30 °C without agitation. The samples were diluted 1:2 with WFI water to a final volume of 50 µL and the EGFP fluorescence was measured using Infinite Pro 200 (Tecan, USA).

Statistical analysis

Statistical analyses were conducted using GraphPad Prism (version 10.0.2): one-way ANOVA with the Brown-Forsythe test (determine the standard deviation for duplicate IVT experiments for mRNA concentration and dsRNA level across promoter variants), Dunnett’s and Tukey’s multiple comparisons test (compare the total mRNA concentration and the dsRNA level across promoter variants) and two-way ANOVA followed by Tukey’s multiple comparisons test (analysing and comparing the mRNA concentration and dsRNA level in T7 promoter variants over a range of template sizes).