Log In Sign up
The largest database of trusted experimental protocols
> Chemicals & Drugs > Biologically Active Substance > Pseudouridine

Pseudouridine

Pseudouridine is a naturally occurring ribonucleotide found in various types of RNA, including transfer RNA (tRNA), ribosomal RNA (rRNA), and small nuclear RNA (snRNA).
It is formed by the isomerization of uridine, resulting in a carbon-carbon bond between the base and the sugar moiety.
Pseudouridine plays important roles in RNA structure, stability, and function, and its presence is often used as a marker for post-transcriptional modifications.
Reserach in this field aims to elucidate the biosynthesis, distribution, and functional significance of pseudouridine in different biological systems.
Accurate and reproducible experimental protocols are crucial for advancing the understanding of this key RNA modification.

Most cited protocols related to «Pseudouridine»

1
Synthetic mRNA Reprogramming Cocktail

Protocol full text hidden due to copyright restrictions

Open the protocol to access the free full text link

Publication 2010
5-methylcytidine Adenosine Triphosphate austin Deoxyribonucleases Edetic Acid Guanosine Triphosphate KLF4 protein, human Molar Nucleotides Oncogenes, myc Phosphoric Monoester Hydrolases POU5F1 protein, human Pseudouridine Ribonucleosides SOX2 protein, human Tail triphosphate Tromethamine
2
mRNA Transcription and Modification Protocol
mRNAs were transcribed as previously described (5 (link)), using linearized plasmids encoding firefly luciferase (pT7TSLuc and pTEVLuc), codon-optimized murine erythropoietin (pTEVmEPO), enhanced green fluorescent protein (pTEVeGFP), Metridia luciferase (pT7TSMetluc) or Renilla luciferase (pT7TSRen and pTEVRen) and T7 RNA polymerase (Megascript, Ambion). All mRNAs were transcribed to contain 30 or 51-nt long poly(A) tails. Additional poly(A) tail was added with yeast poly(A) polymerase (USB) and noted as An. Triphosphate-derivatives of pseudouridine (Ψ) and 5-methylcytidine (m5C) (TriLink) were used to generate modified nucleoside containing RNA. All RNAs were capped using the m7G capping kit with or without 2′-O-methyltransferase (ScriptCap, CellScript) to obtain cap1 or cap0. We did not observed differences in the immunogenicity of cap0- and cap1-containing nucleoside-modified RNAs. All RNAs were analyzed by denaturing or native agarose gel electrophoresis. Pseudouridine-modified mRNAs encoding KLF4, LIN28, cMYC, NANOG, OCT4 and SOX2 were a kind gift of CellScript, Inc.
Karikó K., Muramatsu H., Ludwig J, & Weissman D. (2011). Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA. Nucleic Acids Research, 39(21), e142.
Publication 2011
5-methylcytidine Antigens bacteriophage T7 RNA polymerase Codon derivatives Electrophoresis, Agar Gel enhanced green fluorescent protein Erythropoietin KLF4 protein, human Luciferases Luciferases, Firefly Luciferases, Renilla methylcobalamin-coenzyme M methyltransferase Mus Nucleosides Plasmids Poly(A) Tail Polynucleotide Adenylyltransferase POU5F1 protein, human Pseudouridine RNA RNA, Messenger Saccharomyces cerevisiae SOX2 protein, human triphosphate
3
Streamlined in vitro mRNA Production
Production of in vitro transcription (IVT) template constructs and subsequent RNA synthesis have been described previously3 (link). All oligonucleotide reagents were synthesized by Integrated DNA Technologies (Coralville). ORFs were amplified by PCR from plasmids encoding GFP, mCherry, firefly luciferase, Cre recombinase, and human VEGF-A (165) (Addgene, see Supplementary Table 4 for ORF sequences). PCR reactions were performed with HiFi Hotstart (KAPA Biosystems) according to the manufacturer's instructions. Splint-mediated ligations were carried out with Ampligase Thermostable DNA Ligase (Epicenter Biotechnologies). UTR ligations were conducted in the presence of 200 nM UTR oligos and 100 nM splint oligos. All intermediate PCR and ligation products were purified with QIAquick spin columns (Qiagen) before further processing. Template PCR amplicons were subcloned with the pcDNA 3.3-TOPO TA cloning kit (Invitrogen). Plasmid inserts were excised by restriction digest and recovered with SizeSelect gels (Invitrogen) before being used to template Poly A tail PCRs. RNA was synthesized with the MEGAscript T7 kit (Ambion), with 1.6 μg of purified tail PCR product to template each 40 μL reaction. A custom ribonucleoside blend was used comprising 3’-O-Me-m7G(5’)ppp(5’)G cap analog (New England Biolabs), adenosine triphosphate and guanosine triphosphate (USB), 5-methylcytidine triphosphate and pseudouridine triphosphate (TriLink Biotechnologies). Final nucleotide concentrations in the reaction mixture were 6 mM for the cap analog, 1.5 mM for guanosine triphosphate, and 7.5 mM for the other nucleotides. RNA was purified with Ambion MEGAclear spin columns and then treated with Antarctic Phosphatase (New England Biolabs) for 30 min at 37°C to remove residual 5’-phosphates. Treated RNA was repurified, quantitated by Nanodrop (Thermo Scientific) and precipitated with 5 M Ammonium Acetate according to the manufacturer's instructions. modRNA was resuspended in 10 mM Tris HCl, 1 mM EDTA at 100 ng/μl for in vitro use or 20-30 μg/μl for in vivo use.
Zangi L., Lui K.O., von Gise A., Ma Q., Ebina W., Ptaszek L.M., Später D., Xu H., Tabebordbar M., Gorbatov R., Sena B., Nahrendorf M., Briscoe D.M., Li R.A., Wagers A.J., Rossi D.J., Pu W.T, & Chien K.R. (2013). Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction. Nature biotechnology, 31(10), 898-907.
Publication 2013
2',5'-oligoadenylate 5-methylcytidine Adenosine Triphosphate ammonium acetate Ampligase Anabolism Cre recombinase DNA Ligases Edetic Acid Gels Guanosine Triphosphate Homo sapiens Ligation Luciferases, Firefly Nucleotides Oligonucleotides Open Reading Frames Phosphates Phosphoric Monoester Hydrolases Plasmids Poly(A) Tail Pseudouridine Ribonucleosides Splints Tail Transcription, Genetic trioctyl phosphine oxide triphosphate Tromethamine Vascular Endothelial Growth Factors
4
XGBoost Algorithm for Biological Tasks

Protocol full text hidden due to copyright restrictions

Open the protocol to access the free full text link

Publication 2020
Cloning Vectors Internal Ribosome Entry Sites Pseudouridine RNA, Single Guide Trees
5
Detecting RNA Modifications by Reverse Transcription
Reverse transcription (RT) methods were applied to detect the presence of pseudouridine (Ψ), dihydrouridine (D) and 2′-O-methylations according to Motorin et al. (23 (link)) and Wintermeyer et al. (24 (link)). To detect pseudouridine (23 (link)) (Supplementary Figure S6), two 4 μg aliquots of the same total small tRNA sample were differently treated. One aliquot (sample Ψ) was added to 30 μl of 1-cyclohexyl-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate (CMCT) solution (50 mM Bicine, pH 8.0, 7 M urea, 4 mM EDTA and 330 mM CMCT) and incubated at 37°C for 20 min. The RNA was precipitated with ethanol, dissolved in 50 mM Na2CO3 (aq) and incubated at 37°C for 3 h. The second aliquot (control C1) was treated exactly as sample Ψ, but omitting CMCT in the first buffer. The RNA was precipitated with ethanol and resuspended in nuclease-free water. 10 pmol of pre-treated in vitro transcribed tRNA or 1 μg total small tRNA in a volume of 10 μl were mixed with 20 pmol 5′-32P-labeled RT-primer (TGGCGGAAACCCCG for both tRNAPyl* and tRNAM15, TGGCGGGAGAGGGG for tRNAC15), incubated at 65°C for 5 min and cooled on ice. Five units Avian Myeloblastosis Virus (AMV) Reverse Transcriptase (New England Biolabs) in AMV reaction buffer containing dNTPs to 1 mM were added. Reverse transcription was performed for 30 min at 42°C. Products were precipitated with ethanol, resuspended in 10 μl 2× colorless loading dye and resolved on 15% denaturing polyacrylamide gels. Sequencing ladders were generated via RT reactions, each containing an additional ddNTP in a ratio of 20:1 (ddNTP:dNTP). Radioactive signals were detected with storage phosphor screens and analyzed with a Typhoon 9410 Phosphor imager (GE Healthcare).
Serfling R., Lorenz C., Etzel M., Schicht G., Böttke T., Mörl M, & Coin I. (2017). Designer tRNAs for efficient incorporation of non-canonical amino acids by the pyrrolysine system in mammalian cells. Nucleic Acids Research, 46(1), 1-10.
Full text: Click here
Publication 2017
1-cyclohexyl-3-(2-(4-morpholinyl)ethyl)carbodiimide tosylate Alkanesulfonates Avian Myeloblastosis Virus Buffers Carbodiimides Edetic Acid Ethanol Methylation N,N-bis(2-hydroxyethyl)glycine Oligonucleotide Primers Phosphorus polyacrylamide gels Pseudouridine Radioactivity Reverse Transcription RNA-Directed DNA Polymerase Toluene Transfer RNA Typhoons Urea

Most recents protocols related to «Pseudouridine»

1
miRNA-Mediated Silencing of Modified mRNAs
Not available on PMC !

Example 10

To test whether this miRNA dependent silencing of modified mRNAs is not limited to miR-126, GFP coding mRNA containing target sites for miR-21 or miR-145 was designed. Ad-HEH293 cells were transfected with miR-21, miR-145 or miR-143 mimics 24 hr prior to transfection with unmodified or pseudouridine and 5-methylcystidine modified GFP, GFP4x21TS or GFP4x145TS mRNAs to ensure for RISC assembly and the GFP expression levels were assessed after 24 hr. Cells transfected with 100% Pseudouridine and 5-methylcystidine substituted GFP4x21TS mRNA, the over-expression of miR-21 and not miR-145, miR-21 or moR-143 reduced the expression GFP to the same extent as cells transfected with unmodified GFP4x21TS mRNA (FIGS. 45A-45C). Cells transfected with 100% Pseudouridine and 5-methylcystidine substituted GFP4x154TS mRNA, showed the same reduction in GFP expression as unmodified mRNA when miR-145 mimic was added and not when miR-21, miR-126 or miR-143 mimics were added (FIGS. 45A-45C). In the control transfected cells with unmofdified or 100% substitution with Pseudouridine and 5-methylcystidine GFP mRNA (with no miRNA target sites), the over-expression of miR-21, miR-145 or miR-143 did not have an effect on GFP expression (FIGS. 45A-45C).

US11857598B2. Self-replicating cell selective gene delivery compositions, methods, and uses thereof (2024-01-02). University of South Florida [US]. Inventors: Hana Totary-Jain [US].
Full text: Click here
Patent 2024
Cells Figs Genes, Duplicate MicroRNAs Obstetric Delivery Pseudouridine RNA, Messenger RNA-Induced Silencing Complex Transfection
2
Flag-Tagged p27 Encoding mRNA Expression
Not available on PMC !

Example 2

A Flag tagged p27 encoding plasmids can be engineered to facilitate in vitro transcription of p27 encoding mRNA (FIG. 17): A) Flag-tagged p27); B) Flag-tagged p27 followed by two 2 fully complementary target sequences for the mature miR-126-3p strand at its 3′-UTR (p27-2x126TS). A Flag-tag can be incorporated to distinguish between endogenous and exogenous p27 expression.

To reduce innate immune responses and toxicity and at the same time maximize the efficiency and duration of expression of the mRNA encoding p27 described in FIG. 17, the following modified nucleotide substitutions or combinations thereof can be used: 1) Pseudouridine; 2) N-1-methylpseudouridine; 3) 5-methoxy-U; 4) 5-hydroxymethyl-C; 5) 5-methyl-C and 6) combination of Pseudouridine and 5-methyl-C. mRNAs can be in vitro transcribed using T7 RNA polymerase followed by 5′ capping and poly(A) tail addition using a Vaccinia Capping Enzyme and E. coli Poly(A)Polymerase (New England BioLabs Inc.), respectively.

US11857598B2. Self-replicating cell selective gene delivery compositions, methods, and uses thereof (2024-01-02). University of South Florida [US]. Inventors: Hana Totary-Jain [US].
Full text: Click here
Patent 2024
bacteriophage T7 RNA polymerase Cells Enzymes Escherichia coli Genes, Duplicate Immunity, Innate Nucleotides Obstetric Delivery Plasmids Poly(A) Tail Polynucleotide Adenylyltransferase Pseudouridine RNA, Messenger Transcription, Genetic Vaccinia virus
3
Pseudouridine and 5-methylcystidine Modified mRNA Targeting
Not available on PMC !

Example 9

It was also tested whether 100% substitution of both uridine and cytosine with pseudouridine and 5-methylcystidine nucleotides would affect miRNA-dependent gene silencing. To that end, GFP or GFP-4x126TS mRNA were in vitro transcribed with 100% pseudouridine and 100% 5-methylcystidine and transfected into Ad-HEK293 cells that were priory (24 hr) transfected with miR-126 or miR-143 mimics. The inhibitory effect of miR-126 on the expression of the double-modified GFP-4x126TS mRNA were compared to the unmodified mRNA after 24 hr. Cells transfected with pseudouridine and 5-methylcystidine modified mRNA, miR-126 and not miR-143 reduced the percentage of GFP positive cells and inhibited GFP expression to the same extent as the unmodified mRNA (FIGS. 44A-44C). Thus, our data show that complete substitution of pseudouridine, or combination of pseudouridine and 5-methylcystidine modified mRNA, can still be targeted by microRNA-dependent silencing.

US11857598B2. Self-replicating cell selective gene delivery compositions, methods, and uses thereof (2024-01-02). University of South Florida [US]. Inventors: Hana Totary-Jain [US].
Full text: Click here
Patent 2024
Cytosine Figs Genes, Duplicate HEK293 Cells MicroRNAs Nucleotides Obstetric Delivery Pseudouridine Psychological Inhibition RNA, Messenger Uridine
4
Pseudouridine Impacts on miRNA-Mediated Gene Silencing
Not available on PMC !

Example 8

To test whether modified nucleic acids affect the efficiency of miRNA-target site recognition and miRNA-dependent gene silencing, GFP or GFP4x126TS mRNA was in vitro transcribed with substitutions of uridine with pseudouridine (0%, 25%, 50% or 100%). Since ad-HEK293 cells do not express miR-126 or miR-143, we transfected with miR-126 mimic or miR-143 mimic 24 hr prior to transfection with GFP or GFP4x126TS mRNAs to ensure for miRNA/RISC assembly and GFP expression levels were assessed after 24 hr. In the control, unmodified (0% Pseudouridine) GFP transfected cells, the over-expression of miR-126-3p or miR-143 did not have any effect on GFP expression (FIGS. 43A-43C). This was also observed in cells transfected with GFP-mRNA containing increasing percentage of Pseudouridine (FIGS. 43A-43C). However, in cells transfected with GFP4x126TS mRNA, the over-expression of miR-126 and not miR-143 dramatically reduced the percentage of GFP positive cells and inhibited GFP expression. The miR-126-specific inhibition was not affected by increasing the percentage of pseudouridine substitution (FIGS. 43A-43C).

US11857598B2. Self-replicating cell selective gene delivery compositions, methods, and uses thereof (2024-01-02). University of South Florida [US]. Inventors: Hana Totary-Jain [US].
Full text: Click here
Patent 2024
Figs Genes, Duplicate HEK293 Cells MicroRNAs Nucleic Acids Obstetric Delivery Pseudouridine Psychological Inhibition RNA, Messenger RNA-Induced Silencing Complex Transfection Uridine
5
Efficient In Vitro Synthesis of Immune-Evading modRNA

Protocol full text hidden due to copyright restrictions

Open the protocol to access the free full text link

Publication 2023
3' Untranslated Regions 5' Untranslated Regions 5-methylcytidine Anabolism bacteriophage T7 RNA polymerase Buffers Cellular Immune Response Cloning Vectors Deoxyribonuclease I DNA, A-Form DNA, Complementary IL10 protein, human Immunity, Innate Ligase Mus Nucleotides Phocidae Phosphodiesterase I Phosphoric Monoester Hydrolases Plasmids Poly(A) Tail Pseudouridine Saline Solution Transcription, Genetic triphosphate

Top products related to «Pseudouridine»

1
Megascript t7 kit by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany, China, Lithuania
The MEGAscript T7 kit is a powerful tool for in vitro transcription of RNA. It utilizes the T7 RNA polymerase to efficiently generate large quantities of RNA from DNA templates. The kit provides the necessary reagents and instructions to perform this process.
2
Antarctic phosphatase by New England Biolabs
Sourced in United States, Germany, United Kingdom, Morocco
Antarctic Phosphatase is a thermolabile enzyme that catalyzes the hydrolysis of phosphate groups from various substrates, including nucleic acids and proteins. It is derived from Antarctic bacterial sources and exhibits optimal activity at lower temperatures compared to other phosphatases.
3
Pseudouridine 5 triphosphate 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.
4
Pseudouridine triphosphate by TriLink
Sourced in United States
Pseudouridine triphosphate is a nucleotide analog that contains the isomer pseudouridine in place of uridine. It is used in biochemical and molecular biology research applications.
5
Lipofectamine 2000 by Thermo Fisher Scientific
Sourced in United States, China, Germany, United Kingdom, Canada, Japan, France, Italy, Switzerland, Australia, Spain, Belgium, Denmark, Singapore, India, Netherlands, Sweden, New Zealand, Portugal, Poland, Lithuania, Hong Kong, Argentina, Ireland, Austria, Israel, Czechia, Cameroon, Taiwan, Province of China, Morocco
Lipofectamine 2000 is a cationic lipid-based transfection reagent designed for efficient and reliable delivery of nucleic acids, such as plasmid DNA and small interfering RNA (siRNA), into a wide range of eukaryotic cell types. It facilitates the formation of complexes between the nucleic acid and the lipid components, which can then be introduced into cells to enable gene expression or gene silencing studies.
6
Turbo dnase by Thermo Fisher Scientific
Sourced in United States, Germany, United Kingdom, Japan, France, Lithuania, Canada, Australia, Netherlands, Denmark
Turbo DNase is a laboratory equipment product designed for the efficient degradation of DNA molecules. It functions by rapidly and effectively removing any unwanted DNA from samples, ensuring the integrity and purity of subsequent analyses.
7
5 methylcytidine 5 triphosphate by TriLink
Sourced in United States
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.
8
5 methylcytidine triphosphate by TriLink
5-methylcytidine triphosphate is a modified nucleotide that can be incorporated into DNA or RNA molecules. It serves as a substrate for various enzymatic processes.
9
E coli poly a polymerase by New England Biolabs
Sourced in United States, Canada, United Kingdom
E. coli Poly(A) Polymerase is an enzyme isolated from Escherichia coli that catalyzes the addition of a poly(A) tail to the 3' end of RNA molecules.
10
Vaccinia capping system by New England Biolabs
Sourced in United States
The Vaccinia Capping System is a laboratory tool used to add a 5' cap structure to RNA, a process known as capping. Capping is a crucial step in the synthesis of mRNA and helps protect the RNA from degradation and enhance translation efficiency. The system utilizes enzymes derived from the vaccinia virus to perform the capping reaction.

More about "Pseudouridine"

Pseudouridine, also known as 5-ribosyluracil, is a naturally occurring ribonucleotide found in various types of RNA, including transfer RNA (tRNA), ribosomal RNA (rRNA), and small nuclear RNA (snRNA).
It is formed by the isomerization of uridine, resulting in a carbon-carbon bond between the base and the sugar moiety.
This unique structural feature of pseudouridine plays important roles in RNA structure, stability, and function, and its presence is often used as a marker for post-transcriptional modifications.
Researchers in this field aim to elucidate the biosynthesis, distribution, and functional significance of pseudouridine in different biological systems.
Accurate and reproducible experimental protocols are crucial for advancing the understanding of this key RNA modification.
Tools such as the MEGAscript T7 kit, Antarctic Phosphatase, Pseudouridine-5′-triphosphate, and Pseudouridine triphosphate are commonly used in the study of pseudouridine and its incorporation into RNA.
Additionally, transfection reagents like Lipofectamine 2000 and enzymes such as Turbo DNase, 5-methylcytidine-5′-triphosphate, 5-methylcytidine triphosphate, and E. coli Poly(A) Polymerase are often employed in the research and analysis of pseudouridine and its role in various biological processes.
The Vaccinia Capping System may also be utilized to study the capping and modification of RNA, which can provide insights into the function of pseudouridine.
By leveraging these tools and techniques, researchers can optimize their pseudouridine research protocols, enhance reproducibility, and gain a deeper understanding of this important RNA modification and its impact on biological systems.
The AI-driven platform PubCompare.ai can assist in this process by helping researchers locate the best protocols from literature, pre-prints, and patents, using intelligent comparisons to streamline their pseudouridie research.