5 methylcytidine 5 triphosphate

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5-methylcytidine-5′-triphosphate is a chemical compound that serves as a modified nucleotide. It is the 5′-triphosphate form of the nucleoside 5-methylcytidine.

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Lab products found in correlation

Pseudouridine 5 triphosphate, by TriLink (7 mentions) Megascript t7 kit, by Thermo Fisher Scientific (5 mentions) Turbo dnase, by Thermo Fisher Scientific (4 mentions) Favorprep blood cultured cells total rna extraction column, by Favorgen Biotech (4 mentions) Antarctic phosphatase, by New England Biolabs (4 mentions) Rneasy minelute cleanup kit, by Qiagen (3 mentions) Anti reverse cap analog, by New England Biolabs (2 mentions) Rneasy kit, by Qiagen (2 mentions) Qiaquick pcr purification kit, by Qiagen (2 mentions) Hotstar hifidelity polymerase kit, by Qiagen (2 mentions) Antarctic phosphatase kit, by New England Biolabs (2 mentions) Anti reverse cap analog, by TriLink (2 mentions) Rnase inhibitor, by Thermo Fisher Scientific (1 mentions) 3 0 me m7g 5 ppp 5 g rna cap structure analog arca, by New England Biolabs (1 mentions) Rnaclean xp beads, by Beckman Coulter (1 mentions) Agilent rna 6000 nano kit, by Agilent Technologies (1 mentions) Megascript t7 transcription kit, by Thermo Fisher Scientific (1 mentions) Biotin 11 ctp, by PerkinElmer (1 mentions) Pcdna3.1 gfp1 10, by Addgene (1 mentions) Megascript kit, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

5-methylcytidine triphosphate (9 citations) 5-methylcytidine (2 citations) 5-methylcytidine triphosphate and pseudouridine triphosphate (2 citations)

7 protocols using 5 methylcytidine 5 triphosphate

Post-transcriptional Regulation of Reporter via miRNA Switches

Cited 3 times

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miRNA-responsive mRNAs (miRNA switches) were encoded on modified mRNA (Miki et al., 2015 (link); Endo et al., 2016 (link)) to post-transcriptionally regulate a fluorescent reporter (blue fluorescent protein, BFP) in response to the activity of a miRNA expressed in living cells (see schematic in Figure 3). miRNA switches were generated using a MEGAScript T7 kit (Ambion) and a modified protocol (Matsuura et al., 2018 (link); Warren et al., 2013 (link)) In the reaction, pseudouridine-5′-triphosphate and 5-methylcytidine-5′-triphosphate (TriLink BioTechnologies) were used instead of uridine triphosphate and cytosine triphosphate, respectively. Guanosine-5′-triphosphate was 5-fold diluted with Anti-Reverse Cap Analog (New England Biolabs) before the IVT reaction. Reaction mixtures were incubated at 37°C for 4 hr, mixed with TURBO DNase (Ambion), and further incubated at 37°C for 30 min. The resulting mRNAs were purified using a FavorPrep Blood/Cultured Cells total RNA extraction column (Favorgen Biotech), incubated with Antarctic Phosphatase (New England Biolabs) at 37°C for 30 min, and then purified again using an RNeasy MinElute Cleanup Kit (Qiagen).
The reporter was translationally repressed when the mature target miRNA binds to its completely complementary sequence in the miRNA switch.

Sunohara T., Morizane A., Matsuura S., Miyamoto S., Saito H, & Takahashi J. (2019). MicroRNA-Based Separation of Cortico-Fugal Projection Neuron-Like Cells Derived From Embryonic Stem Cells. Frontiers in Neuroscience, 13, 1141.

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In Vitro Synthesis of Modified mRNA

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The synthesis of modified mRNA via in vitro transcription (IVT) was described earlier.53 (link), 54 (link) Briefly, the plasmid DNA sequences of EGFP and AAT (Eurofins Medigenomix) were multiplied by using HotStar HiFidelity Polymerase Kit (QIAGEN), as well as a forward primer (5′-TTG GAC CCT CGT ACA GAA GCTA ATA CG-3′; Ella Biotech) and a reverse primer (poly T-tail of 120 thymidines [T₁₂₀] 5′-CTT CCT ACT CAG GCT TTA TTC AAA GAC CA-3′; Ella Biotech). After subsequent purification (QIAquick PCR purification kit; QIAGEN) and gel electrophoresis, the DNA was used as a template for IVT using the MEGAscript T7 kit (Ambion). The following mRNA modifications were implemented: 3′-0-Me-m⁷G(5′)ppp(5′)G RNA cap structure analog (ARCA; New England Biolabs), pseudouridine-5′-triphosphate (TriLink Biotech), and 5-methylcytidine-5′-triphosphate (TriLink Biotech). For the fluorescent labeling of the mRNA, cy3-cytidine-triphosphate (PerkinElmer) was used. For RNase inhibition, an RNase inhibitor (Thermo Scientific) was added. The reaction mix was incubated for 4 hr at 37°C. Afterward, the mRNA was purified with RNeasy kit (QIAGEN), dephosphorylated using the Antarctic Phosphatase kit (New England Biolabs), purified again, and controlled with 1% agarose gel.

Michel T., Luft D., Abraham M.K., Reinhardt S., Salinas Medina M.L., Kurz J., Schaller M., Avci-Adali M., Schlensak C., Peter K., Wendel H.P., Wang X, & Krajewski S. (2017). Cationic Nanoliposomes Meet mRNA: Efficient Delivery of Modified mRNA Using Hemocompatible and Stable Vectors for Therapeutic Applications. Molecular Therapy. Nucleic Acids, 8, 459-468.

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Modified nucleotide RNA for Nanopore sequencing

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The genomic region chr19:45406985–45408892 (on genome build hg19) was cloned from human fibroblast DNA into the pcDNA3.1-GFP(1–10) (Addgene cat# 70219) vector. The clone sequence was as follows:
The sequence includes 691 distinct 5-mers, about 70% [691/(4⁵)] of the 5-mers of the RNA synthesized with just the canonical nucleotides.
We used the MEGAscript T7 transcription kit (ThermoFisher, cat# AM1334) for in vitro transcription (IVT) in the presence of varying amounts of modified nucleotides to prepare RNA for Nanopore sequencing. The modified nucleotides used were the 2′-O-methyl-nucleotide set (TriLink Biotechnologies, cat# K-1012), N6-methyladenosine-5′-triphosphate (TriLink Biotechnologies, cat# N-1013-1), N1-methyladenosine-5′-triphosphate (TriLink Biotechnologies, cat# N-1042-1), 5-methylcytidine-5′-triphosphate (TriLink Biotechnologies, cat# N-1014-1), 5-hydroxymethylcytidine-5′-triphosphate (TriLink Biotechnologies, cat# N-1087-1), pseudouridine-5′-triphosphate (TriLink Biotechnologies, cat# N-1019-1), and biotin-11-CTP (Perkin-Elmer, cat# NEL542001EA). In vitro-transcribed RNAs were purified from their reaction mixes using RNAClean XP beads (Beckman Coulter, cat# A63987), and the integrity of the RNA (∼2 kb, and no evidence of degradation) was verified using the Agilent RNA 6000 Nano Kit (cat# 5067-1511).

Burdick J.T., Comai A., Bruzel A., Sun G., Dedon P.C, & Cheung V.G. (2023). Nanopore-based direct sequencing of RNA transcripts with 10 different modified nucleotides reveals gaps in existing technology. G3: Genes|Genomes|Genetics, 13(11), jkad200.

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Synthesizing Modified mRNA and sgRNA for CRISPR-Cas9

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Cas9 mRNAs (with or without miRNA target sequences and with kink-turn motif), L7Ae mRNAs (with or without miRNA target sequences) and BFP mRNA (without miRNA target sequences) were prepared by using a MEGAscript kit (Ambion). In order to reduce the interferon response caused by long RNA, pseudouridine-5΄-triphosphate and 5-methylcytidine-5΄-triphosphate (TriLink Bio Technologies) were used instead of natural rUTP and rCTP, respectively (18 (link)). Guanosine-5΄-triphosphate was 5-fold diluted with an Anti Reverse Cap Analog (TriLink Bio Technologies) before the IVT reaction. The sgRNA was constructed using a MEGAshortscript kit (Ambion) according to the instruction manual. Because sgRNA with modified bases may cause downregulation of Cas9 activity, natural rNTPs were used for preparing sgRNA. The template DNA was degraded by TURBO DNase (Ambion), and the mRNAs and sgRNA were purified using a FavorPrep Blood/Cultured Cells total RNA extraction column (Favorgen Biotech) incubated with Antarctic Phosphatase (New England Biolabs) at 37°C for 30 min and then purified again using an RNeasy MinElute Cleanup Kit (QIAGEN). For further purification, sgRNA was electrophoresed, extracted from gel (10% polyacrylamide gel, 8.3 M urea), and ethanol-precipitated.

Hirosawa M., Fujita Y., Parr C.J., Hayashi K., Kashida S., Hotta A., Woltjen K, & Saito H. (2017). Cell-type-specific genome editing with a microRNA-responsive CRISPR–Cas9 switch. Nucleic Acids Research, 45(13), e118.

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In Vitro Transcription of Modified mRNAs

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All mRNAs were generated using the above PCR products and MEGAscript T7 Kit (Ambion, USA). In the reaction, pseudouridine-5′-triphosphate and 5-methylcytidine-5′-triphosphate (TriLink BioTechnologies, USA) were used instead of uridine triphosphate and cytosine triphosphate, respectively. For IVT of the MS2CP-responsive mRNA used in Fig. 3, N1-methylpseudouridine-5′-triphosphate (m1pU) (TriLink BioTechnologies) was used instead of uridine-5′-triphosphate. Guanosine-5′-triphosphate was 5-fold diluted with an anti-reverse cap analog (TriLink BioTechnologies) before the IVT reaction. Reaction mixtures were incubated at 37°C for up to 6 h and then mixed with TURBO DNase (Ambion), and further incubated at 37°C for 30 min to remove the template DNA. The resulting mRNAs were purified using a FavorPrep Blood/Cultured Cells total RNA extraction column (Favorgen Biotech, Taiwan), incubated with Antarctic Phosphatase (New England Biolabs) at 37 °C for 30 min, and then purified again using an RNeasy MinElute Cleanup Kit (QIAGEN).

Matsuura S., Ono H., Kawasaki S., Kuang Y., Fujita Y, & Saito H. (2018). Synthetic RNA-based logic computation in mammalian cells. Nature Communications, 9, 4847.

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Synthetic mRNA Preparation and Modification

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The synthetic mRNAs (eGFP, CD39) were prepared as previously described.4 (link),16 (link) The vectors with coding sequences were amplified with a HotStar HiFidelity Polymerase Kit (Qiagen, Germany) using two primers, TTG GAC CCT CGT ACA GAA GCT AAT ACG as the forward primer (Ella Biotech, Germany) and T₁₅₀ CTT CCT ACT CAG GCT TTA TTC AAA GAC CA as the reverse primer (Ella Biotech, Germany). DNA sequences were purified (Qiaquick PCR purification Kit, Qiagen, Germany), and in vitro transcription (IVT) was performed using a MEGAscript® T7 Kit (Ambion, Scotland), according to the manufacturer's instructions. The mRNA was modified using the 3'-0-Me-m⁷G(5') ppp(5')G RNA cap structure analog (New England Biolabs, Germany), pseudouridine-5'-triphosphate (TriLink Biotech, U. S. A.), and 5-methylcytidine-5'-triphosphate (TriLink Biotech, U. S. A.). Additionally, an RNase inhibitor (Themo Scientific, U. S. A.) was added. The IVT was incubated for 4 h at 37 °C.
Afterwards the mRNA was cleaned up with an RNeasy kit (Qiagen, Germany), and eluted in 40 µl nuclease-free water. Next the dephosphorylation of mRNA was implemented with an Antarctic Phosphatase Kit (New England Biolabs, Germany), cleaned up and then eluted in the same amount of nuclease-free water. The concentration of mRNA was confirmed photometrically and the purity of mRNA with 1% agarose gel.

Abraham M.K., Peter K., Michel T., Wendel H.P., Krajewski S, & Wang X. (2017). Nanoliposomes for Safe and Efficient Therapeutic mRNA Delivery: A Step Toward Nanotheranostics in Inflammatory and Cardiovascular Diseases as well as Cancer. Nanotheranostics, 1(2), 154-165.

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mRNA Synthesis with Modified Nucleotides

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All mRNAs were synthesized using a MegaScript T7 kit (Ambion, Carlsbad, CA, USA) with a modified protocol. In brief, uridine triphosphate and cytosine triphosphate were replaced with pseudouridine-5′-triphosphate and 5-methylcytidine-5′-triphosphate (TriLink BioTechnologies, San Diego, CA), respectively. As for guanosine triphosphate, a premix of guanosine triphosphate and Anti Reverse Cap Analog (New England Biolabs, Ipswich, MA) (1:4) was used. The reaction mixtures were incubated at 37 °C for 4 h and further incubated at 37 °C for 30 min in the presence of TURBO DNase (Ambion). RNA products were purified with a FavorPrep Blood/Cultured Cells total RNA extraction column (Favorgen Biotech, Ping-Tung, Taiwan) according to the manufacture’s protocol and then subjected to treatment with Antarctic Phosphatase (New England Biolabs) at 37 °C for 30 min. Finally, the resulting mRNAs were purified again using an RNeasy MiniElute Cleanup Kit (Qiagen).

Endo K., Hayashi K, & Saito H. (2016). High-resolution Identification and Separation of Living Cell Types by Multiple microRNA-responsive Synthetic mRNAs. Scientific Reports, 6, 21991.

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