Journal: Nature Communications

Article Title: In vivo PAR-CLIP (viP-CLIP) of liver TIAL1 unveils targets regulating cholesterol synthesis and secretion

doi: 10.1038/s41467-023-39135-8

Figure Lengend Snippet: a Schematic representation of in vivo PAR-CLIP (viP-CLIP) applied in mice. Mice are injected 6 times with 4SU over the time course of 12.5 h and sacrificed 15 h after the first dose. Intact organs are flash-frozen, grinded, and cross-linked with UV light (365 nm) under liquid nitrogen cooling. After cell lysis of the tissue powder, the RNA-RBP complexes are immunoprecipitated in presence of RNase, radiolabeled and the recovered RNA in the size of 19–35 nt is ligated to sequencing adapter, and used for cDNA library preparation and sequencing. b 4SU incorporation rates in tissues ( n = 3) after injection regime displayed in ( a ). HEK293 ( n = 3) and primary hepatocytes ( n = 3) were cultured in the presence of 100 mM 4SU for 16 h and subjected together with the tissue samples to HPLC analysis of 4SU incorporation rates. Values are mean +/– S.D. c Phosphorimage of urea-PAGE transferred to a nitrocellulose membrane that resolved 32 P-labeled RNA-TIAL1 complexes after immunoprecipitation of TIAL1 in several tissues. d Immunoblot showing tissue distribution of TIAL1 in mouse organs. e Autoradiograph of recovered RNAs as displayed in ( c ), after proteinase K treatment and 15% urea gel electrophoresis. 5’ radiolabeled synthetic RNAs of 19 and 35 nt length served as size markers. c , d , e Representative images of three independent replicates. f Graphic presentation of viP-CLIP cDNA library composition by RNA categories from recovered RNA from ( e ). Source data are provided as a Source Data file.

Article Snippet: HPLC purified ( ≥ 98%) 4-thiouridine (4SU) was purchased from Tocris (Cat No. 7005), protected against light, and dissolved fresh before use.

Techniques: In Vivo, Injection, Lysis, Immunoprecipitation, Sequencing, cDNA Library Assay, Cell Culture, Membrane, Labeling, Western Blot, Autoradiography, Nucleic Acid Electrophoresis

Journal: ACS Nano

Article Title: Direct Nanopore Sequencing of Individual Full Length tRNA Strands

doi: 10.1021/acsnano.1c06488

Figure Lengend Snippet: Biological and canonical tRNA strand reads aligned against reference sequences. (A) tRNA fMet , (B) tRNA Phe , and (C) tRNA Lys . In each panel (i) is base coverage along the reference sequence at each position (coverage plot) and (ii) is a randomly selected subset of individual aligned nanopore reads. The total numbers of aligned reads are shown to the left of the coverage plots. The positions of expected modifications on biological tRNA 3 are indicated above the coverage plots and are abbreviated: 4 = 4-thiouridine; D = Dihydrouridine; B = 2′- O -methylcytidine; 7 = 7-methylguanosine; T = 5-methyluridine; P = pseudouridine; X = 3-(3-amino-3-carboxypropyl)uridine; * = 2-methylthio-N6-isopentenyladenosine; S = 5-methyl-aminomethyl-2-thiouridine; and 6 = N6-threonylcarbamoyl-adenosine. Gray columns in the coverage plots indicate positions along the reference where 80% or more of the quality weighted reads are the expected canonical nucleotide. At positions where the value is under the 80% threshold, the proportion of each nucleotide call is shown in color where U(T) = red, A = green, C = blue, and G = gold. Similarly, the rows of individual aligned reads (A–C, ii) are gray at positions matching the reference and colored (using the previously mentioned convention) at positions with mismatches. The black horizontal bars in the aligned reads indicate a deletion, and purple bars indicate an insertion. The rows of aligned reads are presented as they were displayed on IGV.

Article Snippet: The standards included 5-methyluridine (TCI America), B -pseudouridine (TRC Canada), uridine (Sigma), 4-thiouridine (MP Biomedicals), 2′- O -methylcytidine (Alfa Aesar), and 7-methylguanosine (Sigma).

Techniques: Sequencing

Journal: ACS Nano

Article Title: Direct Nanopore Sequencing of Individual Full Length tRNA Strands

doi: 10.1021/acsnano.1c06488

Figure Lengend Snippet: Systematic base miscalls in purified biological and canonical tRNA fMet , tRNA Lys , and tRNA Phe . The coverage plots (A–C) for biological and canonical synthetic tRNAs were generated from alignments using marginAlign. The number of aligned reads for each tRNA is shown under each coverage plot. Boxes surrounding base positions denote systematic miscalls (posterior probability of ≥30%). No systematic miscalls were identified in the synthetic canonical tRNAs. Colored bars are at positions where <80% of the quality weighted alignments match the reference. At these positions, the proportion of individual bases called are shown in color (U(T) = red, A = green, C = blue, and G = gold). The known modifications for the biological tRNAs 3 are indicated above the coverage plots. Modified nucleotides are indicated above the reference sequence with abbreviations (4 = 4-thiouridine, D = dihydrouridine, B = 2′- O -methylcytidine, 7 = 7-methylguanosine, T = 5-methyluridine, P = pseudouridine, X = 3-(3-amino-3-carboxy-propyl)uridine, * = 2-methylthio-N6- isopentenyladenosine, S = 5-methylaminomethyl-2-thiouridine, and 6 = N6-threonylcarbamoyl-adenosine). The falloff in 5′ coverage of synthetic tRNA Lys (B, lower panel) and tRNA Phe (C, lower panel) is likely due to incomplete 5′ phosphorylation of these substrates.

Article Snippet: The standards included 5-methyluridine (TCI America), B -pseudouridine (TRC Canada), uridine (Sigma), 4-thiouridine (MP Biomedicals), 2′- O -methylcytidine (Alfa Aesar), and 7-methylguanosine (Sigma).

Techniques: Purification, Generated, Modification, Sequencing

Journal: Molecules

Article Title: Synthesis and Antiviral Activity of a Series of 2′- C -Methyl-4′-thionucleoside Monophosphate Prodrugs

doi: 10.3390/molecules25215165

Figure Lengend Snippet: Examples of early modified nucleoside drugs ( a – c ) and the aryloxy phosphoramidate monophosphate prodrug sofosbuvir ( d ).

Article Snippet: In 2014 Idenix filed a patent claiming the triphosphates of 4′-thioguanosine, 4′-thiouridine, and 2′-β-methyl-4′-thiouridine, as well as monophosphate prodrugs of the latter two, for the treatment of HCV [ ]; they were acquired by Merck that same year, purportedly for their anti-HCV portfolio.

Techniques: Modification