The RLuc-PepMV-3UTR plasmid was obtained by insertion of a DNA fragment covering the PepMV 3′UTR (Baseclear, Leiden, The Netherlands) into a Renilla luciferase reporter plasmid previously described29 (link). Templates for transcription were obtained by PCR using the forward primer SP6FLU and reverse primer (pep1, pA2, or pA6—Supplementary Table S1 ). Transcription reactions were carried out as described above, but with the kit enzyme mix substituted with a 7:2:1 mixture of SP6 polymerase, recombinant RNAse inhibitor, and inorganic pyrophosphatase (all New England Biolabs). 5 × 105 N. benthamiana protoplasts were transfected with 3 µg of RNA and after 16 h incubation at 25 °C under constant light, were freeze-dried and sent by airmail for analysis at Leiden University. Upon arrival material was resuspended in 100ul Tris (10 mM pH 8) and luciferase activity in 50 µl samples was measured using a GloMax multi system (Promega).
Inorganic pyrophosphatase
Manufactured by New England Biolabs
Sourced in United States
Inorganic pyrophosphatase is an enzyme that catalyzes the hydrolysis of inorganic pyrophosphate (PPi) into two orthophosphate (Pi) molecules. This enzymatic activity helps maintain low intracellular PPi concentrations, which is important for various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Glomax multi system, by Promega (1 mentions)
Recombinant rnase inhibitor, by New England Biolabs (1 mentions)
Sp6 polymerase, by New England Biolabs (1 mentions)
Ubiquitinylation kit, by Enzo Life Sciences (1 mentions)
Gel loading buffer 2, by Thermo Fisher Scientific (1 mentions)
Micro bio spin p 30 columns, by Bio-Rad (1 mentions)
Superase in, by Thermo Fisher Scientific (1 mentions)
Rq1 dnase, by Promega (1 mentions)
6 protocols using inorganic pyrophosphatase
1
Renilla Luciferase Assay in Nicotiana benthamiana
2
Ubiquitination Assay for E3 Ligase
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Ubiquitination assays were carried out using a ubiquitinylation kit (ENZO Life Sciences, Farmingdale, NY, USA). We used a previously described protocol (Zhang et al. 2017 (link)) with some modifications. In brief, Escherichia coli-purified His-PUB8 (E3) and GST-ABI3 (substrate), and His-PUB8 (E3) and GST-ABI5 (substrate) were incubated with 1× ubiquitinylation buffer, 5 mM Mg-ATP solution, 1 mM dithiothreitol, 100 nM human E1, 2.5 mM human E2 (ubiquitin conjugating enzyme UBCH8), 2.5 mM biotin-ubiquitin, and 1 unit inorganic pyrophosphatase (New England Biolabs, Beverley, MA, USA) at 37°C for 4 h. Proteins were separated on a 4% to 20% (w/v) SDS–PAGE (catalog no. F11420Gel, ACE Biotechnology, Nanjing, China) under nonreducing conditions. Immunodetection was carried out using the HRP-Streptavidin detection system as described in the product manual. Experiments were repeated at least 4 times. The primers used for the vector construction are listed in Supplemental Table S1 .
3
In Vitro Lysidine Modification of tRNA
4
In Vitro Transcription of Single-Stranded DNA Templates
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Single-stranded DNA templates (Table S2 ), each containing a T7 promoter sequence, were synthesized (IDT), purified on a 12% or 15% urea-polyacrylamide gel, and resuspended in water. Each template DNA was annealed with an oligonucleotide complementary to the T7 promoter sequence (Table S2 ), and then used in an in vitro transcription reaction containing 0.5 μM annealed DNA template, 5 mM ATP, 2 mM UTP, 5 mM CTP, 8 mM GTP, 5 mM DTT, 40 mM Tris pH 7.9, 2.5 mM spermidine, 26 mM MgCl2, 0.01% (v./v.) Triton X-100, 5 mM DTT, SUPERase•In (1 U/μL, Invitrogen, AM2694), thermostable Inorganic Pyrophosphatase (0.0083 U/μL, New England Biolabs, M0296), and T7 RNA Polymerase (purified in-house). The reaction was incubated at 37°C for 3–4 h, before RQ1 DNase (0.037 U/μL, Promega, M6101) was added, followed by 30 min of incubation at 37°C. The final reaction mix was desalted using Micro Bio-Spin P-30 columns (Bio-Rad, 7326250), then denatured by incubating with 0.5X volume of gel-loading buffer (8 M urea, 25 mM EDTA, 0.025% [w./v.] xylene cyan, 0.025% [w./v.] bromophenol blue) or Gel Loading Buffer II (Invitrogen, AM8547) at 90°C for 1 min. The full-length RNA product was then purified on a 12% or 15% ureapolyacrylamide gel and resuspended in water.
5
In vitro transcription of labeled RNA
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DNA fragments including the optimized T7 class II promoter Ф2.5 with ATP as initial nucleotide and 5′-ACCCCCCCCCCCCCCCCCCC-3′ were used as a template for in vitro transcription. We used the nsp12 and nsp13 and [α-32P]-GTP or [α-32P]-UTP to label the 5′ terminus of the RNA to G∗pppA-RNA. After reaction, the reaction systems were centrifuged at 14,000 rpm for 10 min, and the supernatant were spotted on polyethylenimine cellulose plates. To improve the efficiency of the reaction, 0.05 U inorganic pyrophosphatase (New England Biolabs, USA) was added in the reaction system. The marker G∗pppA-RNA was prepared with the Vaccinia capping system (except for SAM) (New England Biolabs, USA).
6
High-Yield In Vitro Transcription
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High-yield transcription reaction contained 1 μM of the indicated DNA, 1 μM T7 RNA polymerase, and 7.5 mM each of guanosine triphosphate (GTP), cytidine triphosphate (CTP), adenosine triphosphate (ATP) and uridine triphosphate (UTP). Reactions were carried out at 37°C for 4 h in a buffer containing 40 mM magnesium acetate, 30 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 25 mM potassium glutamate, 0.25 mM ethylenediaminetetraacetic acid (EDTA) and 0.05% Tween 20 and were supplemented with 2000 U/ml of RNase Inhibitor, Murine (New England Biolabs) and 5 U/ml of inorganic pyrophosphatase (yeast, New England Biolabs).
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