backbone oligonucleotides  (TaKaRa)

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: MicroRNA in cancer therapy: breakthroughs and challenges in early clinical applications

doi: 10.1186/s13046-025-03391-x

Figure Lengend Snippet: Most relevant recent clinical trial in the treatment of solid and hematological malignancies

Article Snippet: miR-155 , Cobomarsen , Antisense oligonucleotide (ASO) , PS backbone modification , Cutaneous T-cell lymphoma (Mycosis Fungoides Subtype), DLBCL , Phase I/ II , NCT02580552Completed NCT03713320Terminated , Intravenous , miRagen Therapeutics, Inc , .

Techniques: Modification

Journal: Molecular biology reports

Article Title: Protein kinase C iota (PKCι) and pVHL are both needed for lysosomal degradation of α5 integrin in renal carcinoma cells.

doi: 10.1007/s11033-025-10272-1

Figure Lengend Snippet: Fig. 2 Knockout of PKCι in pVHL-expressing 786-O cells disrupts tight junction forma- tion. 786-O cells without pVHL (1st row) and with pVHL rein- troduced (rows 2–4) were grown on coverslips to confluence and assayed by indirect immuno- fluorescence staining for ZO-1 as a tight junction marker. Left panels show ZO-1 localization, center panels show stain for DAPI (nuclei), and the right panel depicts a merged image of the ZO-1 and DAPI stains. Cell lines used and their sgRNA targets are indicated on the left of each row. The images were taken at 1000× magnification

Article Snippet: LentiCRISPRv2 plasmid was digested with BsmBI restriction enzyme and ligated with annealed sgRNA oligonucleotides as described in the Target Guide Sequence Cloning Protocol found at the Addgene website provided. sgRNA sequences targeting PKCι were TGT CTC GAA CCT CAT TGC AA (exon 2, antisense strand) and TCC AAG CCA AGC GTT TCA AC (exon 5, sense strand), hereafter named PKCι sgRNA 1 and 2, respectively. sgRNA sequence targeting PKCζ was CCA TCC ATC CCA TCG ATA AC (exon 9, antisense strand).

Techniques: Knock-Out, Expressing, Fluorescence, Staining, Marker

Journal: Molecular biology reports

Article Title: Protein kinase C iota (PKCι) and pVHL are both needed for lysosomal degradation of α5 integrin in renal carcinoma cells.

doi: 10.1007/s11033-025-10272-1

Figure Lengend Snippet: Fig. 4 Cycloheximide and bafilomycin assays reveal that pVHL-mediated down- regulation of α5 integrin levels requires lysosomal degrada- tion. (A) Parental 786-O cells (VHL-) and those with reintro- duced VHLp19 (VHL+) were incubated with cycloheximide (100 µg/ml for various time points and then a western blot for α5 integrin was performed, with an α-tubulin western blot as a loading control. Band intensities were divided by their corresponding α-tubulin band intensities (from the same lane) and presented as a percent, with the first lane set to 100%. (B) Similar analysis was performed as in (A) with 786-O cell lines with pVHL reintroduced (VHL+) containing sgRNA targeting LacZ (as a control) or PKCι (PKCι 1 and PKCι 2). (C) 786-O cell lines used in (A) and (B) were incubated with vehicle or bafilomycin (10 nM) overnight and a western blot for α5 integrin was performed, with an α-tubulin western blot performed as a loading control. Note that in (A), (B), and (C), the α-tubulin western blots were performed by reblotting the membranes in the top panel. (D) Cells with reintroduced VHLp19 (VHL+) were incu- bated with DMSO (vehicle) or bafilomycin (10 nM) overnight before ZO-1 immunostaining, as previously described. Images were taken at 1000× magnifica- tion

Article Snippet: LentiCRISPRv2 plasmid was digested with BsmBI restriction enzyme and ligated with annealed sgRNA oligonucleotides as described in the Target Guide Sequence Cloning Protocol found at the Addgene website provided. sgRNA sequences targeting PKCι were TGT CTC GAA CCT CAT TGC AA (exon 2, antisense strand) and TCC AAG CCA AGC GTT TCA AC (exon 5, sense strand), hereafter named PKCι sgRNA 1 and 2, respectively. sgRNA sequence targeting PKCζ was CCA TCC ATC CCA TCG ATA AC (exon 9, antisense strand).

Techniques: Incubation, Western Blot, Control, Immunostaining

Journal: Biomolecules

Article Title: Nucleoside Analogs in ADAR Guide Strands Enable Editing at 5′-G A Sites

doi: 10.3390/biom14101229

Figure Lengend Snippet: Dual luciferase assay measuring cellular editing with MECP2 R255X reporter plasmid and overexpressed ADARs in HEK293 cells. ( A ) Guide oligonucleotide design for directed editing of the MECP2 R255X in cellulo. Target A is highlighted in red. All nucleotides bear 2′-O-methyl modifications and contain phosphodiester linkages with the following exceptions: * indicates phosphorothioate modification, bold indicates 2′-deoxyribonucleotides. ( B ) Schematic of reporter plasmid construct encoding MECP2 R255X mutation sequence followed by sequence encoding nanoluciferase and an independent promoter for firefly luciferase expression. ( C ) Relative nLuc/fLuc luminescence signals observed for guide oligonucleotides with varying identity of nucleoside analogs at −1 position and a 3 nM, 10 nM, and 30 nM concentration of guide with overexpressed ADAR2. Cells were lysed and luminesced at 48 h. A two-tailed Welch’s t test was conducted, where ** p < 0.01, *** p < 0.001.

Article Snippet: Molecular weights for editing oligonucleotide (EON)-bearing phosphorothioate backbone modifications were obtained from Novatia Inc. (Newton, PA, USA) using ESI-MS. Sequences and mass spectrometry data for all oligonucleotides are found in .

Techniques: Luciferase, Plasmid Preparation, Modification, Construct, Mutagenesis, Sequencing, Expressing, Concentration Assay, Two Tailed Test