trm4 antisense oligo  (Integrated DNA Technologies)

Journal: Methods in enzymology

Article Title: Pseudouridine in mRNA: Incorporation, Detection, and Recoding

doi: 10.1016/bs.mie.2015.03.009

Figure Lengend Snippet: Schematic representation of pTRM4-TAA expression plasmid. The wild-type TRM4 gene is mutated at F602 position (TTT converted to TAA) to generate a premature termination codon (PTC) (indicated). The plasmid-borne TRM4 gene is fused with a multicomponent C-terminal tag to facilitate the detection and purification of full-length Trm4 product. The tag contains a 3C protease cleavage site that can be used to release the purified protein from the tag. The expression of TRM4 gene is under the control of Gal promoter, which is galactose-inducible. URA3 is an auxotroph selective marker in yeast. This is a high copy 2 µ plasmid.

Article Snippet: Biotin-streptavidin binding buffer: 0.1 M phosphate, 0.15 M NaCl, 0.1% SDS, 1% NP-40, pH 7.2 Biotinylated- TRM4 antisense oligo (IDT): biotin-5′-CCACTCTTGTTGGTTCACCAGTGGC-3′ Streptavidin agarose bead (Pierce) Elution buffer: 0.1 M glycine–HCl pH 7.0

Techniques: Expressing, Plasmid Preparation, Purification, Marker

Journal: Methods in enzymology

Article Title: Pseudouridine in mRNA: Incorporation, Detection, and Recoding

doi: 10.1016/bs.mie.2015.03.009

Figure Lengend Snippet: The strategy for cloning artificial snR81 using four primers overlapping PCR (also see Fig. 2). The construction is based on naturally occurring yeast snR81 box H/ACA RNA. The 5′-most primer is sense-strand sequence (forward), while the other primers have antisense sequences (reverse). The guide sequences (guide1, guide1′, guide2, and guide2′) are changed to base pair with substrate mRNAs, while the rest of the snR81 sequence remain unchanged. The amplified artificial snR81 is digested with BamHI and HindIII and subsequently cloned into pSEC plasmid under the control of the Gal promoter. LEU2 is an auxotroph selective marker in yeast.

Article Snippet: Biotin-streptavidin binding buffer: 0.1 M phosphate, 0.15 M NaCl, 0.1% SDS, 1% NP-40, pH 7.2 Biotinylated- TRM4 antisense oligo (IDT): biotin-5′-CCACTCTTGTTGGTTCACCAGTGGC-3′ Streptavidin agarose bead (Pierce) Elution buffer: 0.1 M glycine–HCl pH 7.0

Techniques: Clone Assay, Sequencing, Amplification, Plasmid Preparation, Marker

Journal: Methods in enzymology

Article Title: Pseudouridine in mRNA: Incorporation, Detection, and Recoding

doi: 10.1016/bs.mie.2015.03.009

Figure Lengend Snippet: Schematic representation of site-specific RNase H cleavage of TRM4 mRNA directed by 2′-O-methyl RNA–DNA chimera. The target sequence of TRM4 mRNA is shown and the PTC (UAA) is indicated. The antisense TRM4 2′-O-methyl RNA–DNA chimera is also shown. d represents deoxy, and m stands for 2′-O-methyl. The arrow indicates the RNase H cleavage site.

Article Snippet: Biotin-streptavidin binding buffer: 0.1 M phosphate, 0.15 M NaCl, 0.1% SDS, 1% NP-40, pH 7.2 Biotinylated- TRM4 antisense oligo (IDT): biotin-5′-CCACTCTTGTTGGTTCACCAGTGGC-3′ Streptavidin agarose bead (Pierce) Elution buffer: 0.1 M glycine–HCl pH 7.0

Techniques: Sequencing

Journal: Methods in enzymology

Article Title: Pseudouridine in mRNA: Incorporation, Detection, and Recoding

doi: 10.1016/bs.mie.2015.03.009

Figure Lengend Snippet: TLC analysis of uridine-to-Ψ conversion at the PTC within the TRM4 mRNA transcript. The PTC-containing TRM4 mRNA, coexpressed with a PTC-specific guide RNA, is purified by oligonucleotide affinity chromatography. The RNA is cleaved by RNase H (directed by a specific 2′-O-methyl RNA–DNA chimera) at the site 5′ of the PTC (UAA). The resulting 3′-half fragment is 5′ radiolabeled with 32P through dephosphorylation and rephosphorylation (see text). The labeled RNA is digested with nuclease P1 to completion. The digested sample is mixed with an equal amount of 5′-32P-adenosine-monophosphate, 5′–32P-cytidine-monophosphate, and 5′–32P-guanosine-monophosphate, and analyzed by two-dimensional TLC. The first and second dimensions are indicated. The origin and the positions of each 5′-phosphorylated nucleotide are also indicated.

Article Snippet: Biotin-streptavidin binding buffer: 0.1 M phosphate, 0.15 M NaCl, 0.1% SDS, 1% NP-40, pH 7.2 Biotinylated- TRM4 antisense oligo (IDT): biotin-5′-CCACTCTTGTTGGTTCACCAGTGGC-3′ Streptavidin agarose bead (Pierce) Elution buffer: 0.1 M glycine–HCl pH 7.0

Techniques: Purification, Affinity Chromatography, De-Phosphorylation Assay, Labeling

Journal: Methods in enzymology

Article Title: Pseudouridine in mRNA: Incorporation, Detection, and Recoding

doi: 10.1016/bs.mie.2015.03.009

Figure Lengend Snippet: Ψ-Mediated nonsense suppression detected by Western blotting. Equal amounts of total protein are loaded (lanes 1–4), and anti-Protein A and anti-GAPDH (loading control) are used for blotting. Readthrough of PTC is visualized (lane 4), where a cognate (PTC-specific) guide RNA designed to target the PTC for pseudouridylation is coexpressed. In contrast, no significant suppression is observed when no guide RNA (lane 1) or a guide RNA-containing random guide sequences is coexpressed (lane 3). Lane 2 is a positive control where the wild-type TRM4 mRNA (with no PTC) is expressed.

Article Snippet: Biotin-streptavidin binding buffer: 0.1 M phosphate, 0.15 M NaCl, 0.1% SDS, 1% NP-40, pH 7.2 Biotinylated- TRM4 antisense oligo (IDT): biotin-5′-CCACTCTTGTTGGTTCACCAGTGGC-3′ Streptavidin agarose bead (Pierce) Elution buffer: 0.1 M glycine–HCl pH 7.0

Techniques: Western Blot, Positive Control

Journal: Methods in enzymology

Article Title: Pseudouridine in mRNA: Incorporation, Detection, and Recoding

doi: 10.1016/bs.mie.2015.03.009

Figure Lengend Snippet:

Article Snippet: Biotin-streptavidin binding buffer: 0.1 M phosphate, 0.15 M NaCl, 0.1% SDS, 1% NP-40, pH 7.2 Biotinylated- TRM4 antisense oligo (IDT): biotin-5′-CCACTCTTGTTGGTTCACCAGTGGC-3′ Streptavidin agarose bead (Pierce) Elution buffer: 0.1 M glycine–HCl pH 7.0

Techniques: Plasmid Preparation

Journal: Methods in enzymology

Article Title: Pseudouridine in mRNA: Incorporation, Detection, and Recoding

doi: 10.1016/bs.mie.2015.03.009

Figure Lengend Snippet:

Article Snippet: Biotin-streptavidin binding buffer: 0.1 M phosphate, 0.15 M NaCl, 0.1% SDS, 1% NP-40, pH 7.2 Biotinylated- TRM4 antisense oligo (IDT): biotin-5′-CCACTCTTGTTGGTTCACCAGTGGC-3′ Streptavidin agarose bead (Pierce) Elution buffer: 0.1 M glycine–HCl pH 7.0

Techniques:

Journal: Methods in enzymology

Article Title: Pseudouridine in mRNA: Incorporation, Detection, and Recoding

doi: 10.1016/bs.mie.2015.03.009

Figure Lengend Snippet:

Article Snippet: Biotin-streptavidin binding buffer: 0.1 M phosphate, 0.15 M NaCl, 0.1% SDS, 1% NP-40, pH 7.2 Biotinylated- TRM4 antisense oligo (IDT): biotin-5′-CCACTCTTGTTGGTTCACCAGTGGC-3′ Streptavidin agarose bead (Pierce) Elution buffer: 0.1 M glycine–HCl pH 7.0

Techniques:

Journal: Methods in enzymology

Article Title: Pseudouridine in mRNA: Incorporation, Detection, and Recoding

doi: 10.1016/bs.mie.2015.03.009

Figure Lengend Snippet:

Article Snippet: Biotin-streptavidin binding buffer: 0.1 M phosphate, 0.15 M NaCl, 0.1% SDS, 1% NP-40, pH 7.2 Biotinylated- TRM4 antisense oligo (IDT): biotin-5′-CCACTCTTGTTGGTTCACCAGTGGC-3′ Streptavidin agarose bead (Pierce) Elution buffer: 0.1 M glycine–HCl pH 7.0

Techniques:

Journal: Methods in enzymology

Article Title: Pseudouridine in mRNA: Incorporation, Detection, and Recoding

doi: 10.1016/bs.mie.2015.03.009

Figure Lengend Snippet:

Article Snippet: Biotin-streptavidin binding buffer: 0.1 M phosphate, 0.15 M NaCl, 0.1% SDS, 1% NP-40, pH 7.2 Biotinylated- TRM4 antisense oligo (IDT): biotin-5′-CCACTCTTGTTGGTTCACCAGTGGC-3′ Streptavidin agarose bead (Pierce) Elution buffer: 0.1 M glycine–HCl pH 7.0

Techniques: Plasmid Preparation

Journal: Methods in enzymology

Article Title: Pseudouridine in mRNA: Incorporation, Detection, and Recoding

doi: 10.1016/bs.mie.2015.03.009

Figure Lengend Snippet:

Article Snippet: Biotin-streptavidin binding buffer: 0.1 M phosphate, 0.15 M NaCl, 0.1% SDS, 1% NP-40, pH 7.2 Biotinylated- TRM4 antisense oligo (IDT): biotin-5′-CCACTCTTGTTGGTTCACCAGTGGC-3′ Streptavidin agarose bead (Pierce) Elution buffer: 0.1 M glycine–HCl pH 7.0

Techniques: Plasmid Preparation