An RNA oligonucleotide (5′-UUUUCAUGCUACGCGUAGUUUUCUACGCG-3′; 4N) with Cyanine 5.5 at the 5′-end was obtained from Millipore Sigma (USA); this RNA hairpin serves as both the primer and the template (15 (link)). The RNA scaffold was annealed in 20 mM HEPES, pH 7.5, 50 mM KCl by heating to 75°C and then gradually cooling to 4°C. To test RdRp activity, reactions were carried out at 37°C with 500 nM nsp12 variants, 1 μM nsp7, 1.5 μM nsp8, 250 nM RNA and 250 μM NTPs in the transcription buffer (20 mM HEPES, pH 7.5, 15 mM KCl, 5% glycerol, 1 mM MgCl2, 2 mM DTT) for 20 min at 37°C. For pyrophosphorolysis, holo RdRp was preincubated with the RNA scaffold at 37°C for 5 min in the transcription buffer; then the indicated combinations of PPi and NTPs were added. Reactions were stopped by adding 2× stop buffer (8 M Urea, 20 mM EDTA, 1× TBE, 0.2% bromophenol blue).
Cyanine 5
Manufactured by Merck Group
Sourced in United States
Cyanine 5.5 is a fluorescent dye used in various laboratory applications. It has an excitation maximum at 675 nm and an emission maximum at 694 nm. Cyanine 5.5 is commonly used for labeling and detection purposes in techniques such as flow cytometry, microarray analysis, and fluorescence microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Rna oligonucleotide, by Merck Group (2 mentions)
Typhoon 9400 imager, by Cytiva (2 mentions)
Typhoon fla 9000, by GE Healthcare (1 mentions)
Originpro 2021, by OriginLab (1 mentions)
Ppgpp, by TriLink (1 mentions)
Anti egr2, by Thermo Fisher Scientific (1 mentions)
Typhoon 9400 imager, by GE Healthcare (1 mentions)
8 protocols using cyanine 5
1
SARS-CoV-2 RNA-Dependent RNA Polymerase Assay
2
SARS-CoV-2 RdRp In Vitro Assay
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An RNA oligonucleotide GA1 (5′ -AAAAGAAAAGACGCGUAGUUUUCUACGCG- 3′) with Cyanine 5.5 at the 5′-end was obtained from Millipore Sigma. Prior to the reaction, the RNA was annealed in 25 mM HEPES, pH 7.5, 50 mM KCl by heating to 75 °C and then gradually cooling to 4 °C. Reactions were carried out at 37 °C. 500 nM nsp12 variant, 1 μM Nsp7, 1.5 μM Nsp8, and inhibitors were incubated in the transcription buffer (25 mM HEPES, pH 7.5, 15 mM KCl, 5% glycerol, 1 mM MgCl2, 1 mM DTT) for 3 min at 37 °C. Following the addition of the RNA scaffold (100 nM final) and NTPs (UTP and CTP, 150 μM final), reactions were incubated for 15 min and quenched with an equal volume of 2× stop buffer (8 M Urea, 20 mM EDTA, 1× TBE, 0.2% bromophenol blue).
3
RNA Polymerization Assay for SARS-CoV-2 Nsp12
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An RNA oligonucleotide (5’ -UUUUCAUGCUACGCGUAGUUUUCUACGCG- 3’) with Cyanine 5.5 at the 5’-end was obtained from Millipore Sigma (USA). Prior to the reaction, the RNA was annealed in 20 mM HEPES, pH 7.5, 50 mM KCl by heating to 75 °C and then gradually cooling to 4 °C. Reactions were carried out at 37 °C with 500 nM Nsp12 variant, 1 μM Nsp7, 1.5 μM Nsp8, 200 nM RNA, and 250 μM NTPs in the transcription buffer (20 mM HEPES, pH 7.5, 15 mM KCl, 5% glycerol, 2 mM MgCl2, 1 mM DTT). RNA extension reactions were stopped at the desired times by adding 2 × stop buffer (8 M Urea, 20 mM EDTA, 1X TBE, 0.2 % bromophenol blue). Samples were heated for 2 min at 95 °C and separated by electrophoresis in denaturing 9 % acrylamide (19:1) gels (7 M Urea, 0.5X TBE). The RNA products were visualized and quantified using Typhoon FLA9000 (GE Healthcare) and ImageQuant Software. RNA extension assays were carried out in triplicates. Means and standard error of the mean (s.e.m.) were calculated by OriginPro 2021 (OriginLab), and unpaired two-tailed t-test was performed using Excel (Microsoft).
4
SARS-CoV-2 RdRp Elongation Assay
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A CU RNA hairpin (5’-AAAAGAAAAGACGCGUAGUUUUCUACGCG- 3’) labeled with Cyanine 5.5 at the 5’-end (Millipore Sigma) was annealed in 20 mM HEPES, pH 7.5, 50 mM KCl by heating to 75 °C and then gradually cooling to 4 °C. RdRp holoenzymes (500 nM wild-type or mutant Nsp12A, 1 μM Nsp7, 1.5 μM Nsp8; final concentrations) were mixed with ATP, GTP, RTP, or ppGpp at concentrations indicated in Figure 5 in 20 mM HEPES, pH 7.5, 15 mM KCl, 5% glycerol, 1 mM DTT and either 2 or 1 mM MgCl2 (with 1 or 0.5 mM activating nucleotide, respectively). Reactions were incubated for 5 min at 37 °C and RNA chain extension was initiated by the addition of 200 nM RNA and 100 μM CTP and UTP. Following 15 min incubation at 37 °C, reactions were stopped at the desired times by adding 2 × stop buffer (8 M Urea, 20 mM EDTA, 1X TBE, 0.2 % bromophenol blue).
5
Fluorescent RNA Hairpin Assay for SARS-CoV-2 RdRp
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A CU RNA hairpin (5′-AAAAGAAAAGACGCGUAGUUUUCUACGCG -3′; CU) labeled with cyanine 5.5 at the 5′ end (Millipore Sigma) was annealed in 20 mM HEPES, pH 7.5, 50 mM KCl by heating the mixture to 75°C and then gradually cooling it to 4°C. RdRp holoenzymes (500 nM wild-type or mutant Nsp12A, 1 μM Nsp7, 1.5 μM Nsp8 [final concentrations]) were mixed with ATP, GTP, remdesivir triphosphate (RTP; MedChemExpress; catalog no. GS443902), or ppGpp (TriLink BioTechnologies; catalog no. N-6001) at concentrations indicated in Fig. 5 in 20 mM HEPES, pH 7.5, 15 mM KCl, 5% glycerol, 1 mM DTT and either 2 or 1 mM MgCl2 (with 1 or 0.5 mM activating nucleotide, respectively). Reaction mixtures were incubated for 5 min at 37°C, and RNA chain extension was initiated by the addition of 200 nM RNA and 100 μM CTP and UTP. Following 15 min of incubation at 37°C, reactions were stopped at the desired times by adding 2× stop buffer (8 M urea, 20 mM EDTA, 1× TBE, 0.2% bromophenol blue).
6
Egr2 DNA Binding Assay with Supershift
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The consensus probe for Egr2 (5′-TGTAGGGGCGGGGGCGGGGTTA-3′) was labeled with Cyanine5 (Sigma-Aldrich) and used in binding reactions with nuclear extracts from CD4 T cells stimulated with anti-CD3 and anti-CD28 for 16 h. For supershift reactions, anti-Egr2 (eBioscience) was added after 10 min of incubation. The samples were electrophoresed on 5% polyacrylamide gels in 0.5× TBE. The gels were scanned using a Typhoon 9400 imager (GE Healthcare). For competition assays, oligos containing the Egr2 binding sites from each gene locus identified using Mulan (Ovcharenko et al., 2005 (link)) were added into the reaction mixtures before incubation. Probes used for competition assays are as follows: Ascl2 (chr7:142969294-142969318), 5′-CCCTGGCGGAGGAGGCGGGAGCCGG-3′; Bhlhe40 (chr6:108660027-108660047), 5′-GCCAGGCGGGGGAGGAGGAAG-3′; Id3 (chr4:136144859-136144882), 5′-CTTCTCTCCTCCCCCGCCCAGAAC-3′; Lef1 (chr3:131109827-131109848), 5′-GGGGCTAGAGTGGGCGGCGGGA-3′; Myb (chr10:21159419-21159440), 5′-GGGCCCACGCCCACGCTCCTGC-3′; Myc (chr15:61983490-61983511), 5′-GGGGGTGAGGGGGCGGGGAAAG-3′; Rora (chr9:69243892-69243913), 5′-GCTTGTTGTGTGGGAGGTGAGC-3′; Rorc (chr3:94371792-94371812), 5′-GGAGGGAGTGGGCGAGTCACG-3′; Tcf7 (chr11:52274904-52274926), 5′-GGACGTCCTCCCACTCGCCCCTG-3′; and Zeb2 (chr2:44979396-44979420), 5′-CTGCCCTCCTCCACCCCCTCCCCTG-3′.
7
T-bet Binding Assay with Nuclear Extracts
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The consensus probe for T-bet (5′-GACAGCTCACACTGGTGTGGAGCAGGG-3′) was labeled with Cyanine 5 (Sigma-Aldrich) and used in binding reactions with nuclear extracts from CD4 T cells stimulated with anti-CD3 and anti-CD28 for 16 h and then restimulated with PMA and ionomycin for 30 min. For supershift reactions, anti–T-bet (H-210; Santa Cruz Biotechnology) was added after 10 min of incubation. The samples were electrophoresed on 5% polyacrylamide gels in 0.5× TBE, and the gels were scanned using a Typhoon 9400 imager (Amersham Biosciences).
8
T-bet Binding Assay Protocol
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