The sequence required for linear saRNA was first cloned by PCR. For this purpose, 1 ng of purified plasmid, 0.5 μM primers (forward: 5′- GTGAATGATGATGGCGTC -3′ and reverse: 5′- TTTTTAACATCAAATTAA-3′) and 2 U of Mastermix PFU DNA Polymerase (Thermo Fisher Scientific) were added and amplified under the following conditions: 35 cycles of 95 °C for 10 s, 55 °C for 30 s, and 72 °C for 10 s. The cloned linear DNA was purified by a Plasmid Plus MaxiPrep kit (QIAGEN, UK) and its purity was determined by a NanoDrop (ThermoFisher, UK). Next, 10 ng purified DNA was first reacted with MEGAScript™ (Ambion, UK) for 1 h at 37 °C and then it was reacted with ScriptCap™ (CellScript, WI, USA) for 1 h at 37 °C. Then, synthesized linear saRNA was purified by LiCl precipitation, re-suspended in RNA storage buffer, and stored at − 80 °C. The purity of synthesized saRNA was finally checked by NanoDrop (ThermoFisher, UK) 18 (link).
Plasmid plus maxiprep kit
Manufactured by Qiagen
Sourced in United Kingdom
The Plasmid Plus MaxiPrep kit is a laboratory equipment designed for the purification of high-quality plasmid DNA from bacterial cultures. It utilizes a specialized column-based method to efficiently isolate plasmid DNA from large-scale samples.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop one, by Thermo Fisher Scientific (12 mentions)
Carbenicillin, by Merck Group (6 mentions)
Scriptcap, by CellScript (2 mentions)
Megascript reaction, by Thermo Fisher Scientific (2 mentions)
Scriptcap m7g capping system, by CellScript (2 mentions)
Scriptcap 2 o methyltransferase kit, by CellScript (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Megascript, by Thermo Fisher Scientific (1 mentions)
Gibson assembly master mix, by New England Biolabs (1 mentions)
Soc media, by Thermo Fisher Scientific (1 mentions)
Lenticrispr v2, by Addgene (1 mentions)
E coli dh5α, by New England Biolabs (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Ultrapure h2o, by Thermo Fisher Scientific (1 mentions)
Vswp01300, by Merck Group (1 mentions)
Ampicillin, by Thermo Fisher Scientific (1 mentions)
Tris edta buffer, by Merck Group (1 mentions)
12 protocols using plasmid plus maxiprep kit
1
Linear saRNA Synthesis and Purification
2
Lentiviral CRISPR Library Construction
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A lentiviral vector containing Cas9 and a human U6 promoter for sgRNA expression (LentiCRISPRv2: Addgene 52961) was digested with BsmBI (NEB R0580) for 3 h at 55 °C. The digested vector was then purified using a Qiaquick PCR purification column (Qiagen 28104). Gibson Assembly reactions containing 200 ng of digested vector, 36 ng of insert (containing pooled library), and 10 μL of Gibson Assembly Master Mix (NEB E2611S) were then incubated at 50 °C for 1 h, and subsequently transformed into 200μL of Stbl4 electrocompetent bacteria (Thermo 11635018). Transformed cells were resuspended in 8 mL of SOC media (Invitrogen 15544034) and allowed to recover for 1 h shaking before being used to inoculate 150 mL of LB media supplemented with carbenicillin. After 16 h of further growth, plasmid DNA containing the sgRNA library was isolated via a Qiagen Plasmid Plus MaxiPrep kit (Qiagen 12963).
3
Transient Transfection of Glycation Mutants
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All Glycation mutants were assembled individually using PCR by overlap extension with High Fidelity PCR Supermix (Invitrogen) and cloned into a mammalian expression vector with unique restriction sites. DNA for transfection was prepared using the Qiagen Plasmid Plus Maxi Prep Kit. Chinese hamster ovary (CHO) cells were transiently transfected at a density of 2.1 × 106/mL with DNA constructs using PEI (Polyethyleneimine) diluted in 150 mM NaCl and following standard transfection protocol. Transfected CHO cells were fed every 48 hours with enriched media. Cells were harvested and supernatant was collected 14 days post transfection for analysis.
4
VEEV Self-Amplifying RNA Synthesis and Formulation
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saRNA was synthesized from a backbone plasmid vector based on a Trinidad donkey Venezuelan equine encephalitis strain (VEEV) alphavirus genome as previously described (Fig. 1 A) [21 (link)]. The gene of interest (GOI) for in vivo protein quantification studies was firefly luciferase (fLuc) and either hemagglutinin from the H1N1 A/California/07/2009 strain [4 ] or the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [21 (link)] for in vivo immunogenicity studies. Plasmid DNA (pDNA) was transformed into DH5α E. coli (New England BioLabs, UK), cultured in 100 mL of Luria Broth (LB) with 100 μg/mL carbenicillin (SigmaAldrich, UK) and isolated using a Plasmid Plus MaxiPrep™ kit (QIAGEN, UK). The concentration of pDNA was measured on a NanoDrop One™ (ThermoFisher, UK).Fig. 1 ![]()
Schematic illustration of VEEV self-amplifying RNA (A), polymeric and lipid nanoparticle formulations (B) and pABOL chemical structure (C).
5
Plasmid DNA Isolation and Purification
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pDNA was transformed into Escherichia coli and grown in 50 mL cultures in LB with 100 μg mL−1 carbenicillin [Sigma Aldrich, UK, (NSP splitzicon)] or 100 μg mL−1 kanamycin [Sigma Aldrich, UK, (positive and negative strand splitzicons)]. pDNA was isolated and purified using a Plasmid Plus Maxiprep kit (QIAGEN, UK). pDNA concentration and purity were measured on a NanoDrop One (ThermoFisher, UK) prior to use with in vitro transcription reactions.
6
Plasmid-based saRNA Production and Purification
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The plasmid encoding saRNA construct was transformed into E. coli (institute Pasteur, Iran), cultured in Luria Broth with 100 μg/mL carbenicillin (Sigma Aldrich, UK). Plasmids were purified using a Plasmid Plus MaxiPrep kit (QIAGEN, UK) and their concentration and purity were measured on a NanoDrop spectrophotometer (ThermoFisher, UK). Then, cloned plasmids were linearized using MluI for 3 h at 37 °C. Then, saRNA transcripts were produced using 1 μg of linearized DNA template in a MEGAScript™ reaction (Ambion, UK) for 1 h at 37 °C. One μg linear saRNA was mixed with 1 μM ScriptCap™ (CellScript, WI, USA) for 1 h at 37 °C. Synthesized saRNA was purified by LiCl precipitation, re-suspended in RNA storage buffer, and stored at − 80 °C. To evaluate the purity of synthesized saRNA, the A260/A280 ratio was measured using the NanoDrop spectrophotometer (ThermoFisher, UK) [21] (link).
7
SARS-CoV-2 Stabilized Pre-Fusion RNA Production
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Self-amplifying RNA encoding the pre-fusion stabilized SARS-CoV-2 was produced using in vitro transcription. pDNA was transformed into E. coli (New England BioLabs, UK), cultured in 100 mL of Luria Broth (LB) with 100 μg mL−1 carbenicillin (Sigma Aldrich, UK). Plasmid was purified using a Plasmid Plus MaxiPrep kit (QIAGEN, UK) and the concentration and purity was measured on a NanoDrop One (ThermoFisher, UK). pDNA was linearized using MluI for 3 h at 37 °C. Uncapped in vitro RNA transcripts were produced using 1 μg of linearized DNA template in a MEGAScript™ reaction (Ambion, UK) for 2 h at 37 °C, according to the manufacturer’s protocol. Transcripts were then purified by overnight LiCl precipitation at −20 °C, centrifuged at 14,000 RPM for 20 min at 4 °C to pellet, washed with 70% EtOH, centrifuged at 14,000 RPM for 5 min at 4 °C and resuspended in UltraPure H2O (Ambion, UK). Purified transcripts were capped using the ScriptCap™ Cap 1 Capping System Kit (CellScript, WI, USA) for 2 h at 37 °C, according to the manufacturer’s protocol. Capped transcripts were purified by LiCl precipitation as described above, resuspended in RNA storage buffer (10 mM HEPES, 0.1 mM EDTA, and 100 mg mL−1 trehalose) and stored at −80 °C until further use.
8
Ligation and Transformation of sgRNA Library
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Both the insert and step 1 sgRNA vector were digested with BsmBI for 3 h at 55 °C and subsequently purified via a Qiaquick PCR Purification column. The ligation reactions were then set up using 100 ng of vector, 100 ng of insert, 2 μL of buffer, 1 μL of T4 ligase (NEB M0202T), and ultra pure H2O up to 20 μL. Each reaction was allowed to proceed overnight at 16 °C. The following morning the ligase was heat inactivated at 65 °C for 20 min. Following this, the reaction was dialyzed into ultrapure water (Millipore VSWP01300) to remove any residual salts from the ligase buffer. Once the DNA was dialyzed, the ligation reaction was split evenly between 300 μL of Stbl4 electrocompetent cells, which were then transformed according to the manufacturer's protocol. The transformed cells were resuspended in 10 mL of SOC media (Invitrogen 15544034) and allowed to recover for 1 h shaking before being used to inoculate 150 mL of LB media supplemented with carbenicillin. After 16 h of further growth, plasmid DNA containing the sgRNA library was isolated via a Qiagen Plasmid Plus MaxiPrep kit (Qiagen 12963).
9
Synthesis and Purification of RNA Transcripts
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RNA transcripts
were synthesized from constructs encoding firefly
luciferase in either mRNA with tobacco mosaic virus (TMV) 5′
and 3′ UTRs22 (link) or in saRNA with the
nonstructural proteins of the Venezuelan equine encephalitis virus
(VEEV), as previously described.20 (link) Plasmid
DNA (pDNA) was transformed into Escherichia coli,
cultured in 100 mL of LB with 50 μg/mL ampicillin (Gibco, Thermo
Fisher Scientific), and purified using a Plasmid Plus Maxiprep Kit
(QIAGEN). pDNA concentrations and purity were measured on a NanoDrop
One (Thermo Scientific). pDNA was linearized using SapI for 2 h at
37 °C and heat inactivated at 65 °C for 20 min. Uncapped
RNA transcripts, used for iteration A, were synthesized using 1 μg
of linearized DNA template in a MEGAScript T7 reaction (Invitrogen,
ThermoFisher Scientific), and capped RNA transcripts, used for iteration
B and validation of the optimized formulations, were synthesized using
750 ng of linearized DNA in a mMessage mMachine T7 reaction (Invitrogen,
ThermoFisher Scientific), according to the manufacturer’s protocol.
Transcripts were purified by overnight precipitation with LiCl at
−20 °C, pelleted by centrifugation at 13 000 × g and 4 °C for 15 min, washed one time with 70% EtOH,
centrifuged at 13 000 × g and 4 °C
for 5 min, and resuspended in Tris-EDTA buffer (Sigma-Aldrich), then
stored at −70 °C.
were synthesized from constructs encoding firefly
luciferase in either mRNA with tobacco mosaic virus (TMV) 5′
and 3′ UTRs22 (link) or in saRNA with the
nonstructural proteins of the Venezuelan equine encephalitis virus
(VEEV), as previously described.20 (link) Plasmid
DNA (pDNA) was transformed into Escherichia coli,
cultured in 100 mL of LB with 50 μg/mL ampicillin (Gibco, Thermo
Fisher Scientific), and purified using a Plasmid Plus Maxiprep Kit
(QIAGEN). pDNA concentrations and purity were measured on a NanoDrop
One (Thermo Scientific). pDNA was linearized using SapI for 2 h at
37 °C and heat inactivated at 65 °C for 20 min. Uncapped
RNA transcripts, used for iteration A, were synthesized using 1 μg
of linearized DNA template in a MEGAScript T7 reaction (Invitrogen,
ThermoFisher Scientific), and capped RNA transcripts, used for iteration
B and validation of the optimized formulations, were synthesized using
750 ng of linearized DNA in a mMessage mMachine T7 reaction (Invitrogen,
ThermoFisher Scientific), according to the manufacturer’s protocol.
Transcripts were purified by overnight precipitation with LiCl at
−20 °C, pelleted by centrifugation at 13 000 × g and 4 °C for 15 min, washed one time with 70% EtOH,
centrifuged at 13 000 × g and 4 °C
for 5 min, and resuspended in Tris-EDTA buffer (Sigma-Aldrich), then
stored at −70 °C.
10
VEEV-Based Self-Amplifying RNA Production
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Self-amplifying RNA
derived from the Venezuelan Equine Encephalitis Virus (VEEV) encoding
either firefly luciferase (fLuc) or enhanced green fluorescent protein
(eGFP) was prepared using in vitro transcription.
pDNA was transformed in Escherichia coli and cultured
in 50 mL of LB with 1 mg/mL carbenicillin (Sigma–Aldrich, U.K.)
and isolated using a Plasmid Plus Maxiprep kit (QIAGEN, U.K.). pDNA
concentration and purity was measured on a NanoDrop One (ThermoFisher,
U.K.) and then linearized using MluI for 2 h at 37 °C and heat
inactivated at 80 °C for 20 min. Uncapped in vitro RNA transcripts
were synthesized using 1 μg of linearized DNA template in a
MEGAScript reaction (Promega, U.K.), according to the manufacturer’s
protocol. Transcripts were then purified by overnight LiCl precipitation
at −20 °C, pelleted by centrifugation at 14 000
rpm for 20 min, washed once with 70% EtOH, centrifuged at 14 000
rpm for 5 min, and then resuspended in UltraPure H2O. Purified
transcripts were then capped using the ScriptCap m7G Capping System (CellScript, Madison, WI, USA) and ScriptCap 2′-O-Methyltransferase
Kit (CellScript, Madison, WI, USA) simultaneously, according to the
manufacturer’s protocol. Capped transcripts were then purified
again by LiCl precipitation, resuspended in ultraPure H2O, and stored at −80 °C until use.
derived from the Venezuelan Equine Encephalitis Virus (VEEV) encoding
either firefly luciferase (fLuc) or enhanced green fluorescent protein
(eGFP) was prepared using in vitro transcription.
pDNA was transformed in Escherichia coli and cultured
in 50 mL of LB with 1 mg/mL carbenicillin (Sigma–Aldrich, U.K.)
and isolated using a Plasmid Plus Maxiprep kit (QIAGEN, U.K.). pDNA
concentration and purity was measured on a NanoDrop One (ThermoFisher,
U.K.) and then linearized using MluI for 2 h at 37 °C and heat
inactivated at 80 °C for 20 min. Uncapped in vitro RNA transcripts
were synthesized using 1 μg of linearized DNA template in a
MEGAScript reaction (Promega, U.K.), according to the manufacturer’s
protocol. Transcripts were then purified by overnight LiCl precipitation
at −20 °C, pelleted by centrifugation at 14 000
rpm for 20 min, washed once with 70% EtOH, centrifuged at 14 000
rpm for 5 min, and then resuspended in UltraPure H2O. Purified
transcripts were then capped using the ScriptCap m7G Capping System (CellScript, Madison, WI, USA) and ScriptCap 2′-O-Methyltransferase
Kit (CellScript, Madison, WI, USA) simultaneously, according to the
manufacturer’s protocol. Capped transcripts were then purified
again by LiCl precipitation, resuspended in ultraPure H2O, and stored at −80 °C until use.
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