Experimental details for mRNA sequences can be found in the Supplemental Information . mRNA imaging probe sequences were designed and synthesized as listed in Supplemental Table 1 . AmpliScribe™ T7-Flash™ Transcription Kit (Lucigen, Middleton, WI) was used to synthesize mRNAs through in vitro transcription, and then used Vaccinia Capping System and mRNA Cap 2´-O-Methyltransferase (New England Biolabs, Ipswich, MA) to add a Cap1 structure to the mRNAs. Pseudouridine-5’-Triphosphate (TriLink Biotechnologies, San Diego, CA) was used to synthesize ψ modified mRNA for the in vivo studies. Lastly, RNA Clean & Concentrator Kits (Zymo Research, Irvine, CA) was used to purify mRNAs.
Pseudouridine 5 triphosphate
Manufactured by TriLink
Sourced in United States
Pseudouridine-5'-triphosphate is a nucleotide analog that contains the modified nucleoside pseudouridine. It is used as a laboratory reagent for various research applications.
Automatically generated - may contain errors
Lab products found in correlation
5 methylcytidine 5 triphosphate, by TriLink (7 mentions)
Megascript t7 kit, by Thermo Fisher Scientific (5 mentions)
Favorprep blood cultured cells total rna extraction column, by Favorgen Biotech (4 mentions)
Antarctic phosphatase, by New England Biolabs (4 mentions)
Turbo dnase, by Thermo Fisher Scientific (4 mentions)
N1 methylpseudouridine 5 triphosphate, by TriLink (4 mentions)
Vaccinia capping system, by New England Biolabs (3 mentions)
Rneasy minelute cleanup kit, by Qiagen (3 mentions)
Dspe peg, by NOF corporation (3 mentions)
Mrna cap 2 o methyltransferase, by New England Biolabs (2 mentions)
Rna clean concentrator kit, by Zymo Research (2 mentions)
Anti reverse cap analog, by New England Biolabs (2 mentions)
Hotstar hifidelity polymerase kit, by Qiagen (2 mentions)
Antarctic phosphatase kit, by New England Biolabs (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Qiaquick pcr purification kit, by Qiagen (2 mentions)
Transcriptaid t7 high yield transcription kit, by Thermo Fisher Scientific (2 mentions)
Ampure xp bead, by Beckman Coulter (2 mentions)
Anti reverse cap analog, by TriLink (2 mentions)
Megaclear transcription clean up kit, by Thermo Fisher Scientific (2 mentions)
17 protocols using pseudouridine 5 triphosphate
1
In vitro mRNA Synthesis and Modification
2
Transcription and Modification of SINEUP-GFP RNA
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Transcribed SINEUP-GFP RNA was synthesized in vitro as described by Toki et al.76 (link) by using a mMESSAGE mMACHINE SP6 Transcription Kit (Thermo Fisher Scientific) and was then modified by following the protocol by Mandal and Rossi77 (link). For the 20% Ψ modification, UTP was replaced with 20% pseudouridine-5′-triphosphate (TriLink, USA; final concentration, 7.5 mM) by mixing the reagents with 40 ng/µL of linearized SINEUP RNA. To add a poly-A tail, 1–10 µg of RNA transcribed in vitro was treated with Escherichia coli poly(A) polymerase (5000 U/mL, New England Biolabs, M0276) at 37 °C for 30 min followed by clean-up using an RNeasy Mini kit (Qiagen) and the modified transcripts of SINEUP-GFP RNA were recovered.
3
Post-transcriptional Regulation of Reporter via miRNA Switches
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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.
The reporter was translationally repressed when the mature target miRNA binds to its completely complementary sequence in the miRNA switch.
4
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.
5
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).
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).
6
Synthetic RNA Production with Pseudouridine and Inosine
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Double-stranded DNA templates with T7 promoter for synthetic RNA were commercially purchased from IDT as blocks, which also has poly-A tail for nanopore adapter binding. IVT reactions were performed using MEGA script™ T7 Transcription Kit (AM1334), for overnight at 37°C followed by DNase treatment and purified using Quick Spin Columns for radiolabelled RNA purification (Roche, 11274015001). For Ψ and I modified RNAs synthesis, Pseudouridine-5'-Triphosphate (N-1019, Trilink) and Inosine-5'-Triphosphate (N-1020, Trilink) were used in place of uridine and guanosine, respectively.
7
In vitro Transcription of Modified RNA
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In vitro transcription reactions were carried out in 40 µL volumes with 10 pmol of DNA template, using the TranscriptAid T7 High Yield Transcription Kit (Thermo Fisher, K0441). In some syntheses, pseudouridine-5′-triphosphate (TriLink Biotechnologies, N-1019) or N1-methylpseudouridine-5’-triphosphate (TriLink Biotechnologies, N-1081) were used to replace regular UTP. Reactions were incubated for 3 h at 37 °C, followed by degradation of DNA template with 2 µL of DNase I at 37 °C for 30 min. RNA samples were purified with 1.8x volume of AMPure XP beads (Beckman Coulter) mixed with 40% PEG-8000 (ratio of 7:3), following the manufacturer’s instructions. Concentrations were measured by absorbance at 260 nm on Nanodrop 100 or 8000 spectrophotometers.
8
CCL5 mRNA Nanoparticle Synthesis
9
Synthesis and Purification of Modified mRNA
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DOPE, DMG-PEG2000, and C18-CONH-PEG2000 and other lipid materials were acquired from Avanti Polar Lipids Inc. (Alabaster, AL). Eagle’s minimum essential medium (EMEM) and other cell culture supplies were purchased from Corning Incorporated (Corning, NY). Quant-iT RiboGreen RNA reagent and Gibco heat-inactivated fetal bovine serum (FBS) were acquired from Thermo Fisher Scientific (Waltham, MA). All the other chemical reagents were obtained from Sigma-Aldrich or Alfa Aesar and used without further purifications.
mRNAs were synthesized through IVT using the AmpliScribe T7-Flash Transcription Kit (Lucigen Corporation, Middleton, WI) and followed by the addition of a Cap1 structure using the Vaccinia Capping System and mRNA Cap 2′-O-methyltransferase (New England Biolabs, Ipswich, MA). Pseudouridine-5′-triphosphate (TriLink BioTechnologies, San Diego, CA) was used for synthesizing ψ modified mRNA. mRNAs were purified through RNA Clean & Concentrator Kits (Zymo Research, Irvine, CA) before diluted in Tris-EDTA (TE) buffer for applications.
mRNAs were synthesized through IVT using the AmpliScribe T7-Flash Transcription Kit (Lucigen Corporation, Middleton, WI) and followed by the addition of a Cap1 structure using the Vaccinia Capping System and mRNA Cap 2′-O-methyltransferase (New England Biolabs, Ipswich, MA). Pseudouridine-5′-triphosphate (TriLink BioTechnologies, San Diego, CA) was used for synthesizing ψ modified mRNA. mRNAs were purified through RNA Clean & Concentrator Kits (Zymo Research, Irvine, CA) before diluted in Tris-EDTA (TE) buffer for applications.
10
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.
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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.
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