monophosphate  (InvivoGen)

Journal: Nucleic Acids Research

Article Title: Generation of precise and accurate engineered circRNAs using enzymatic ligation

doi: 10.1093/nar/gkag405

Figure Lengend Snippet: Evaluation of circularization efficiency and accuracy by different ligases. ( A ) Schematic depicting requirements and features of DNA ligase, RNA ligase 1, and RNA ligase 2. ( B ) Workflow of circRNA generation using enzymatic ligation and RNase R based purification which can be improved by addition of poly(A) tails to linear RNAs. ( C ) 3% urea–PAGE showed that all ligases were able to circularize 5′-monophosphate RNAs. Boxed bands depict the circRNAs that run slower than their linear counterparts. Contaminating RNAs of lower and higher size than circular or linear RNA were also observed suggesting that poly(A) tailing and RNase R treatments were insufficient to degrade them. Efficiency of ligation was calculated as percentage yields of RNAs remaining after all treatments divided by input RNA for each ligation reaction. CircRNAs derived from modified transcription templates (mod) had higher efficiencies particularly for DNA ligase and RNA ligase 2 than those derived from unmodified templates (unmod). RNA ligase 2 had the highest circularization efficiency, especially with circRNAs derived from mod templates in presence of an RNA splint. Representative data are from a mean of n = 3 technical replicates with SEM; (*) P ≤.05 (unpaired t -test). ( D ) Sanger sequencing of ligation junctions showed accurate sequences with circRNAs derived from modified transcription templates using all ligases. CircRNAs made with RNA Ligase 1 had errors with the linear RNAs from unmodified templates which were corrected with the use of modified templates.

Article Snippet: One approach involved dephosphorylation of 1 pmol of 5′-triphosphate transcripts using 5 units of quick calf intestinal phosphatase (NEB #M0525L) at 37°C for 15 min, followed by purification and addition of monophosphate to transcripts using 10 units of T4 polynucleotide kinase (NEB #M0201L) at 37°C for 30 min.

Techniques: Ligation, Purification, Derivative Assay, Modification, Sequencing

Journal: Nucleic Acids Research

Article Title: Generation of precise and accurate engineered circRNAs using enzymatic ligation

doi: 10.1093/nar/gkag405

Figure Lengend Snippet: Purification of circRNAs and extending the RNA ligase 2 (RL2)-dependent circularization method to other RNAs. ( A ) CircRNAs synthesized with RNA ligase 2 using DNA splint were purified using three different approaches: from 3% urea–PAGE using crush and soak method, or from EX E-gels either using the crush and soak method or using column-based kit. As a control, linear RNAs were also extracted using the same methods. CircRNAs extracted from 3% urea–PAGE or EX E-gel using a crush and soak method had more intact circRNAs with less nicking compared to those extracted from EX E-gel using column-based kits. Linear RNAs on the other hand remained intact with each of the approaches. ( B ) Schematic of RNase-H based circularity confirmation assay that uses a short ssDNA probe which cleaves intact circRNAs into a single linear band, while nicked circRNAs or linear RNAs are cut into two shorter bands. ( C ) RNase-H based assay confirmed circularity of EGFP-IRES circRNAs. Linear RNAs were cleaved into two shorter bands of expected sizes while circRNAs derived from modified DNA templates were linearized to the size of full-length linear precursor. ( D, E ) 5′-monophosphate linear precursors of human immunodeficiency virus (HIV) and mCherry were ligated using RNA ligase 2 and respective DNA splints. For circHIV, urea–PAGE purification of circRNAs derived from modified templates had the highest yields with the least contaminating RNAs. Yields of mCherry circRNAs were much higher with polyA + RNase R approach on RNAs from modified template ligated using RNA ligase 2, however urea–PAGE showed higher and lower sized undesired RNAs. Representative data are from a mean of n = 3 technical replicates with SEM. ( F ) Sanger sequencing confirmed accuracy of circRNAs. Clean chromatograms were observed for ligation junctions of both HlV and mCherry circRNAs derived from modified DNA templates purified either through poly(A) tailing and RNase R treatment or from urea–PAGE purification.

Article Snippet: One approach involved dephosphorylation of 1 pmol of 5′-triphosphate transcripts using 5 units of quick calf intestinal phosphatase (NEB #M0525L) at 37°C for 15 min, followed by purification and addition of monophosphate to transcripts using 10 units of T4 polynucleotide kinase (NEB #M0201L) at 37°C for 30 min.

Techniques: Purification, Synthesized, Control, Rnase H Assay, Derivative Assay, Modification, Virus, Sequencing, Ligation

Journal: bioRxiv

Article Title: Metabolic glues as a means of purine sensing and chemotherapeutic response

doi: 10.64898/2026.05.05.723063

Figure Lengend Snippet: a. Example cryo-EM density of the PPAT-NUDT5 6-meTIMP molecular glue interface with model fit. b,c. PPAT activity assay measuring inhibitory effects of 6-meTIMP in the presence and absence of wildtype NUDT5 and indicated mutants and c. compared to AMP only. d. Left – Representative Western blot of immunoprecipitations from endogenous NUDT5 3xFLAG HEK293T cells treated with indicated drugs for 16 hours: methotrexate (MTX; 2 µM), lometrexol (LMX; 10 µM), 6-mercaptopurine (6-MP; 50 µM), MLN4924 (1 µM), brequinar (2 µM), and rapamycin (1 µM). Right – quantification of PPAT immunoprecipitation relative to NUDT5 3xFLAG bait and normalized to a DMSO-treated control condition. Data are individual values from n=3 biological replicates from independent experiments and error bars are SEM. e. Western blot of immunoprecipitations from endogenous NUDT5 3xFLAG HEK293T cells treated with MTX (2 µM) for the indicated amounts of time. Similar results were obtained in two independent experiments. f. Western blot of endogenous PPAT 3xFLAG immunoprecipitations following 16-hour treatment with MTX (2 µM), 6-MP (50 µM), and MTX + 6-MP g. Time-resolved microscopy (incucyte) growth assays of wildtype and mutant HEK293T cells treated with the indicated drugs. Data are the mean and error bars are SEM of n=6 biological replicates. h. Levels of intracellular 6-TIMP and 6-meTIMP metabolites following 16-hour treatment with 6-MP (20 µM). Data are individual values and error bars are SEM from n=3 biological replicates. i. PPAT activity assay measuring inhibitory effects of 6-meTGMP in the presence and absence of wildtype NUDT5 and indicated mutants. Activity data shown in panels b, c and i are the mean and error bars are SEM of n=3 independent experiments.

Article Snippet: The following drugs and chemicals were used in this study at amounts specified in figures and legends: Pevonedistat; MLN4924 (MedChemExpress, HY-70062), methotrexate; MTX (MedChemExpress, HY-14519), lometrexol; LMX (MedChemExpress, HY-14521), brequinar (MedChemExpress, HY-108325) , rapamycin (Adooq Biosciences, A10782), 5-Phospho-D-ribose 1-diphosphate; PRPP (Sigma-Aldrich, P8296) , L-glutamine (Sigma-Aldrich, G8540); adenosine-5’-monophosphate; AMP (Sigma-Aldrich 01930), inosine-5’-monophosphate; IMP (MedChemExpress, HY-W010759), guanosine-5’-monophosphate; GMP (Sigma-Aldrich, G8377), AICA-ribonucleotide (Cayman Chemicals 33907), adenine (Thermo Scientific, A17622.14), hypoxanthine (MedChemExpress, HY-N0091), 6-thioguanine; 6-TG (Thermo Scientific, B21280.03), 6-mercaptopurine; 6-MP (Adooq Biosciences, A15898), 6-thioinosine-5’-monophosphate; 6-TIMP (Jena Biosciences, NU-1148), 6-methylthioinosine-5’-monophosphate; 6-meTIMP (Jena Biosciences, NU-1226), 6-methylthioguanosine-5’-monophosphate; 6-meTGMP (Jena Biosciences, NU-1128), 6-benzylthioinosine-5’-monophosphate; 6-benzylTIMP (WuXi, custom synthesis), 6-ethylthioinosine-5’-monophosphate 6-etTIMP (WuXi, custom synthesis), 6-ethylmercaptopurine riboside; 6-EMPR (WuXi, custom synthesis).

Techniques: Cryo-EM Sample Prep, Activity Assay, Western Blot, Immunoprecipitation, Control, Microscopy, Mutagenesis

Journal: Bioactive Materials

Article Title: Screening of a quinonoid compounds library identifies decylubiquinone as an antioxidant and anti-apoptotic agent against glucocorticoid-induced osteoporosis via CD39/CD73/adenosine axis

doi: 10.1016/j.bioactmat.2026.03.062

Figure Lengend Snippet: Roles of A 2b R in ADO-mediated activation of the cAMP/PKA/CREB pathway in primary BMSCs. ( A ) Principal component analysis (PCA) of RNA-seq data from primary BMSCs treated with Dex or Dex + ADO. ( B ) The volcano plot presented the differentially expressed genes (DEGs) as determined by RNA-Seq in primary BMSCs treated with Dex or Dex + ADO. ( C ) Gene Ontology (GO) enrichment analysis in the biological process category for DEGs as determined by RNA-Seq in primary BMSCs treated with Dex, or Dex + ADO. ( D ) The molecular docking of ADO with mus musculus A 1 R, A 2a R, A 2b R, and A 3 R proteins. ADO is displayed in Cyan. The surrounding residues in the binding pocket are shown in green (forming a non-hydrogen bond with ADO) or magenta (forming a hydrogen bond with ADO). The hydrogen bond is labeled as yellow dashed lines. The backbone of the receptor is depicted as gray. ( E ) RT-qPCR analysis of the mRNA levels of Adora1 , Adora2a , Adora2b , and Adora3 in primary BMSCs treated with vehicle, Dex, or Dex + ADO. ( F ) RT-qPCR analysis for the expression of Runx2 in primary BMSCs of different groups. (G) Gene Set Enrichment Analysis (GSEA) plot showing the differentially expressed pathway (cAMP) between the Dex group and the Dex + ADO group as indicated by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. ( H ) Western blot validation for the knockdown deficiency of A 2b R after transfection with si Adora2b . ( I ) ELISA analysis for the relative intracellular cAMP levels in BMSCs of different groups. ( J ) Western blot and quantification for the expression of PKA, p-PKA, CREB, and p-CREB in primary BMSCs. ( K ) Representative images and quantitative analysis of Alizarin Red S staining for mineralization deposit in primary BMSCs of different groups under osteogenic conditions. n = 4 independent repeats by using different biological samples in each group for in vitro experiments. Data were means ± s.e.m. ns p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 by one-way ANOVA. Scale bar: 200 μm (K).

Article Snippet: The intracellular cAMP level was examined by using a cAMP ELISA Kit (E-EL-0056, Elabscience, Wuhan, China) according to the manufacturer's instructions.

Techniques: Activation Assay, RNA Sequencing, Binding Assay, Labeling, Quantitative RT-PCR, Expressing, Western Blot, Biomarker Discovery, Knockdown, Transfection, Enzyme-linked Immunosorbent Assay, Staining, In Vitro

Journal: Bioactive Materials

Article Title: Screening of a quinonoid compounds library identifies decylubiquinone as an antioxidant and anti-apoptotic agent against glucocorticoid-induced osteoporosis via CD39/CD73/adenosine axis

doi: 10.1016/j.bioactmat.2026.03.062

Figure Lengend Snippet: Screening of the quinonoid compounds for the treatment of GIOP. (A) Flowchart depicting the screening process of the quinonoid compounds library. The schematic diagram was created by using BioRender.com. (B) Volcano diagram showing the effects of the 153 quinonoid compounds on Runx2 expression in BMSCs. Red and blue dots indicate the specific compounds that up- and down-regulate Runx2 expression in BMSCs, respectively. (C) Heat map showing the effect of the compounds on ALP activity in primary BMSCs. Color from blue to red indicates the ALP activity in primary BMSCs from low to high. (D) Measurement of intracellular ROS level in primary BMSCs treated with three potential compounds by using the fluorescent dye DCFDA. (E) Chemical structure of DUB, the final candidate among the screened drugs. (F) MTT assay for the proliferation of BMSCs treated with different doses of DUB for 2 and 10 days, under osteogenic induction conditions with or without 10 μM Dex. (G) Representative images and quantitative analysis of mineralized nodule formation via Alizarin Red S (ARS) staining in primary BMSCs treated with DUB at a series of concentrations, under osteogenic induction conditions with or without 10 μM Dex. (H) Western blot and quantification for the expression of osteogenesis-related proteins in primary BMSCs under different treatments. (I) Oil Red O staining and quantifications for lipid droplets in primary BMSCs of different groups. n = 4 independent repeats by using different biological samples in each group for in vitro experiments. Data were means ± s.e.m. ∗∗∗ p < 0.001 by one-way ANOVA. Scale bars: 200 μm (G), and 50 μm (I).

Article Snippet: The intracellular cAMP level was examined by using a cAMP ELISA Kit (E-EL-0056, Elabscience, Wuhan, China) according to the manufacturer's instructions.

Techniques: Expressing, Activity Assay, MTT Assay, Staining, Western Blot, In Vitro

Journal: Bioactive Materials

Article Title: Screening of a quinonoid compounds library identifies decylubiquinone as an antioxidant and anti-apoptotic agent against glucocorticoid-induced osteoporosis via CD39/CD73/adenosine axis

doi: 10.1016/j.bioactmat.2026.03.062

Figure Lengend Snippet: Roles of A 2b R in ADO-mediated activation of the cAMP/PKA/CREB pathway in primary BMSCs. ( A ) Principal component analysis (PCA) of RNA-seq data from primary BMSCs treated with Dex or Dex + ADO. ( B ) The volcano plot presented the differentially expressed genes (DEGs) as determined by RNA-Seq in primary BMSCs treated with Dex or Dex + ADO. ( C ) Gene Ontology (GO) enrichment analysis in the biological process category for DEGs as determined by RNA-Seq in primary BMSCs treated with Dex, or Dex + ADO. ( D ) The molecular docking of ADO with mus musculus A 1 R, A 2a R, A 2b R, and A 3 R proteins. ADO is displayed in Cyan. The surrounding residues in the binding pocket are shown in green (forming a non-hydrogen bond with ADO) or magenta (forming a hydrogen bond with ADO). The hydrogen bond is labeled as yellow dashed lines. The backbone of the receptor is depicted as gray. ( E ) RT-qPCR analysis of the mRNA levels of Adora1 , Adora2a , Adora2b , and Adora3 in primary BMSCs treated with vehicle, Dex, or Dex + ADO. ( F ) RT-qPCR analysis for the expression of Runx2 in primary BMSCs of different groups. (G) Gene Set Enrichment Analysis (GSEA) plot showing the differentially expressed pathway (cAMP) between the Dex group and the Dex + ADO group as indicated by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. ( H ) Western blot validation for the knockdown deficiency of A 2b R after transfection with si Adora2b . ( I ) ELISA analysis for the relative intracellular cAMP levels in BMSCs of different groups. ( J ) Western blot and quantification for the expression of PKA, p-PKA, CREB, and p-CREB in primary BMSCs. ( K ) Representative images and quantitative analysis of Alizarin Red S staining for mineralization deposit in primary BMSCs of different groups under osteogenic conditions. n = 4 independent repeats by using different biological samples in each group for in vitro experiments. Data were means ± s.e.m. ns p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 by one-way ANOVA. Scale bar: 200 μm (K).

Article Snippet: The intracellular cAMP level was examined by using a cAMP ELISA Kit (E-EL-0056, Elabscience, Wuhan, China) according to the manufacturer's instructions.

Techniques: Activation Assay, RNA Sequencing, Binding Assay, Labeling, Quantitative RT-PCR, Expressing, Western Blot, Biomarker Discovery, Knockdown, Transfection, Enzyme-linked Immunosorbent Assay, Staining, In Vitro