Journal: Nature Communications

Article Title: Dynamic and static control of the off-target interactions of antisense oligonucleotides using toehold chemistry

doi: 10.1038/s41467-023-43714-0

Figure Lengend Snippet: Sequence used in this study and melting temperature ( T m ) of ASO/RNA or ASO/PNA

Article Snippet: Fig. 6 Complementary PNAs suppress off-target hybridizations. a Schematic illustration of an in-cuvette FRET assay. b Hybridization kinetics were evaluated for the indicated ssASO and BROs using FRET ( n = 3 independent experiments). c The antisense effects of hApo1n/PNA(C9) and hApo1n/cGapmer on ApoB mRNA in Huh-7 cells using the CEM transfection method ( n = 6 biologically independent samples for control, n = 3 for BRO series; one-way ANOVA followed by two-sided Dunnett’s multiple comparison tests comparing to control, p values from left side; <0.0001, 0.0006, 0.6547). d The antisense effects of hApo1n and hApo1n/PNA(C5-C13) BROs in Huh-7 cells using the CEM transfection method ( n = 3 biologically independent samples; one-way ANOVA followed by two-sided Dunnett’s multiple comparison tests comparing to hApo1n, p values from left side; <0.0001, 0.9854, 0.99398, 0.4175, 0.0388, 0.0011, <0.0001, <0.0001, <0.0001, <0.0001). e The van’t Hoff analysis for the indicated ssASO and BROs ( n = 3 independent experiments). f Knockdown activities of on-target ApoB gene and three single-mismatch genes were compared for indicated hApo1n and BROs (hApo1n/PNA(C8), hApo1n/PNA(C9)) in Huh-7 cells exposed to 1 µM after 24 h of CEM transfection ( n = 3 biologically independent samples; one-way ANOVA followed by two-sided Dunnett’s multiple comparison tests comparing to control, p values from left side; ApoB: <0.0001, <0.0001, <0.0001; Copg: 0.0299, 0.1307, 0.1071; Mast2: 0.0012, 0.1826, 0.8253; Hltf: 0.0262, 0.9132, 0.4780). g Hybridization kinetics for on-target mRNA ( ApoB ) were evaluated for the indicated ssASO and BROs using FRET ( n = 3 independent experiments). h FRET assay of TAMRA-labeled hApo1nF and BHQ2-labeled mRNA mimics (Copg, Hltf). i FRET assay of TAMRA-labeled hApo1nF/PNA(C9) BRO and BHQ2-labeled mRNA mimics (Copg, Hltf).

Techniques: Sequencing

Journal: Nature Communications

Article Title: Dynamic and static control of the off-target interactions of antisense oligonucleotides using toehold chemistry

doi: 10.1038/s41467-023-43714-0

Figure Lengend Snippet: a Procedure for evaluation of cytotoxicity of mPCS1n ssASO and mPCS1n/PNA(C8) BRO in original and galactose-modified media. b PI staining of Huh-7 cells exposed to 2 µM mPCS1n ssASO and mPCS1n/PNA(C8) BRO after 48 h of CEM transfection ( n = 3 biologically independent samples). c Drug concentration-dependent assessment of cell viability ( n = 5 biologically independent samples) d Activity of caspases 3/7, 8, and 9 ( n = 4 biologically independent samples; one-way ANOVA followed by two-sided Dunnett’s multiple comparison tests comparing to control, p values from left side; glucose conditions: 0.0674, 0.5360 for caspase 8; 0.0077, 0.9104 for caspase 9; 0.032, 0.9571 for caspase 3/7; galactose conditions: <0.0001, 0.8379 for caspase 8; <0.0001, 0.7712 for caspase 9; <0.0001, 0.7933 for caspase 3/7). e the depolarization of the mitochondrial membrane potential (ΔΨm) with JC−1 dye were compared and evaluated for mPCS1n or mPCS1n/PNA(C8) BRO through transfection into Huh-7 cells using the CEM method ( n = 1) ( e ). ** p < 0.01, * p < 0.05. “ns” indicates not significant ( p > 0.05). Data in Fig. 4c and d are presented as mean values ± SD. Source data are provided as a Source Data file.

Article Snippet: Fig. 6 Complementary PNAs suppress off-target hybridizations. a Schematic illustration of an in-cuvette FRET assay. b Hybridization kinetics were evaluated for the indicated ssASO and BROs using FRET ( n = 3 independent experiments). c The antisense effects of hApo1n/PNA(C9) and hApo1n/cGapmer on ApoB mRNA in Huh-7 cells using the CEM transfection method ( n = 6 biologically independent samples for control, n = 3 for BRO series; one-way ANOVA followed by two-sided Dunnett’s multiple comparison tests comparing to control, p values from left side; <0.0001, 0.0006, 0.6547). d The antisense effects of hApo1n and hApo1n/PNA(C5-C13) BROs in Huh-7 cells using the CEM transfection method ( n = 3 biologically independent samples; one-way ANOVA followed by two-sided Dunnett’s multiple comparison tests comparing to hApo1n, p values from left side; <0.0001, 0.9854, 0.99398, 0.4175, 0.0388, 0.0011, <0.0001, <0.0001, <0.0001, <0.0001). e The van’t Hoff analysis for the indicated ssASO and BROs ( n = 3 independent experiments). f Knockdown activities of on-target ApoB gene and three single-mismatch genes were compared for indicated hApo1n and BROs (hApo1n/PNA(C8), hApo1n/PNA(C9)) in Huh-7 cells exposed to 1 µM after 24 h of CEM transfection ( n = 3 biologically independent samples; one-way ANOVA followed by two-sided Dunnett’s multiple comparison tests comparing to control, p values from left side; ApoB: <0.0001, <0.0001, <0.0001; Copg: 0.0299, 0.1307, 0.1071; Mast2: 0.0012, 0.1826, 0.8253; Hltf: 0.0262, 0.9132, 0.4780). g Hybridization kinetics for on-target mRNA ( ApoB ) were evaluated for the indicated ssASO and BROs using FRET ( n = 3 independent experiments). h FRET assay of TAMRA-labeled hApo1nF and BHQ2-labeled mRNA mimics (Copg, Hltf). i FRET assay of TAMRA-labeled hApo1nF/PNA(C9) BRO and BHQ2-labeled mRNA mimics (Copg, Hltf).

Techniques: Modification, Staining, Transfection, Concentration Assay, Activity Assay, Comparison, Membrane

Journal: Nature Communications

Article Title: Dynamic and static control of the off-target interactions of antisense oligonucleotides using toehold chemistry

doi: 10.1038/s41467-023-43714-0

Figure Lengend Snippet: a Schematic illustration of an in-cuvette FRET assay. b Hybridization kinetics were evaluated for the indicated ssASO and BROs using FRET ( n = 3 independent experiments). c The antisense effects of hApo1n/PNA(C9) and hApo1n/cGapmer on ApoB mRNA in Huh-7 cells using the CEM transfection method ( n = 6 biologically independent samples for control, n = 3 for BRO series; one-way ANOVA followed by two-sided Dunnett’s multiple comparison tests comparing to control, p values from left side; <0.0001, 0.0006, 0.6547). d The antisense effects of hApo1n and hApo1n/PNA(C5-C13) BROs in Huh-7 cells using the CEM transfection method ( n = 3 biologically independent samples; one-way ANOVA followed by two-sided Dunnett’s multiple comparison tests comparing to hApo1n, p values from left side; <0.0001, 0.9854, 0.99398, 0.4175, 0.0388, 0.0011, <0.0001, <0.0001, <0.0001, <0.0001). e The van’t Hoff analysis for the indicated ssASO and BROs ( n = 3 independent experiments). f Knockdown activities of on-target ApoB gene and three single-mismatch genes were compared for indicated hApo1n and BROs (hApo1n/PNA(C8), hApo1n/PNA(C9)) in Huh-7 cells exposed to 1 µM after 24 h of CEM transfection ( n = 3 biologically independent samples; one-way ANOVA followed by two-sided Dunnett’s multiple comparison tests comparing to control, p values from left side; ApoB: <0.0001, <0.0001, <0.0001; Copg: 0.0299, 0.1307, 0.1071; Mast2: 0.0012, 0.1826, 0.8253; Hltf: 0.0262, 0.9132, 0.4780). g Hybridization kinetics for on-target mRNA ( ApoB ) were evaluated for the indicated ssASO and BROs using FRET ( n = 3 independent experiments). h FRET assay of TAMRA-labeled hApo1nF and BHQ2-labeled mRNA mimics (Copg, Hltf). i FRET assay of TAMRA-labeled hApo1nF/PNA(C9) BRO and BHQ2-labeled mRNA mimics (Copg, Hltf). Data in Fig. 6b–d and g–i are presented as mean values ± SD. ** p < 0.01, * p < 0.05. “ns” indicates not significant ( p > 0.05). Source data are provided as a Source Data file.

Article Snippet: Fig. 6 Complementary PNAs suppress off-target hybridizations. a Schematic illustration of an in-cuvette FRET assay. b Hybridization kinetics were evaluated for the indicated ssASO and BROs using FRET ( n = 3 independent experiments). c The antisense effects of hApo1n/PNA(C9) and hApo1n/cGapmer on ApoB mRNA in Huh-7 cells using the CEM transfection method ( n = 6 biologically independent samples for control, n = 3 for BRO series; one-way ANOVA followed by two-sided Dunnett’s multiple comparison tests comparing to control, p values from left side; <0.0001, 0.0006, 0.6547). d The antisense effects of hApo1n and hApo1n/PNA(C5-C13) BROs in Huh-7 cells using the CEM transfection method ( n = 3 biologically independent samples; one-way ANOVA followed by two-sided Dunnett’s multiple comparison tests comparing to hApo1n, p values from left side; <0.0001, 0.9854, 0.99398, 0.4175, 0.0388, 0.0011, <0.0001, <0.0001, <0.0001, <0.0001). e The van’t Hoff analysis for the indicated ssASO and BROs ( n = 3 independent experiments). f Knockdown activities of on-target ApoB gene and three single-mismatch genes were compared for indicated hApo1n and BROs (hApo1n/PNA(C8), hApo1n/PNA(C9)) in Huh-7 cells exposed to 1 µM after 24 h of CEM transfection ( n = 3 biologically independent samples; one-way ANOVA followed by two-sided Dunnett’s multiple comparison tests comparing to control, p values from left side; ApoB: <0.0001, <0.0001, <0.0001; Copg: 0.0299, 0.1307, 0.1071; Mast2: 0.0012, 0.1826, 0.8253; Hltf: 0.0262, 0.9132, 0.4780). g Hybridization kinetics for on-target mRNA ( ApoB ) were evaluated for the indicated ssASO and BROs using FRET ( n = 3 independent experiments). h FRET assay of TAMRA-labeled hApo1nF and BHQ2-labeled mRNA mimics (Copg, Hltf). i FRET assay of TAMRA-labeled hApo1nF/PNA(C9) BRO and BHQ2-labeled mRNA mimics (Copg, Hltf).

Techniques: Hybridization, Transfection, Comparison, Labeling

Journal: Nature Communications

Article Title: dsRNAi-mediated silencing of PIAS2beta specifically kills anaplastic carcinomas by mitotic catastrophe

doi: 10.1038/s41467-024-47751-1

Figure Lengend Snippet: PSMC5 and TUBB3 PIAS2b-dependent SUMOylation at mitosis, using non-denaturing extracts with SUMO-protease inhibitors ( a ), or 10xHis-SUMO1 transfected denaturing extracts ( b ). From top-to bottom re-stripped westerns presented. a GFP-Trap pull-downs of EGFP or EGFP-PSMC5 in asynchronous (As), DT/R-0 h, DT/R-6 h (Left); or treated with ns-dsRNAi or PIAS2b -dsRNAi at DT/R-5 h (Right). EGFP-PSMC5 and endogenous PSMC5 enhance SUMO1ylation at mitosis, lost with PIAS2b silencing. Endogenous PSMC5 associates to exogenous EGFP-PSMC5. PIAS2b is associated to- and SUMO1ylated in the GFP pull-downs. b 10xHis-SUMO1 Ni-NTA pull-downs in denatured extracts reveal covalent SUMO1ylated – or poly-SUMOylated, PSMC5, TUBB3 and PIAS2b at DT/R-6 h, that are lost with PIAS2b silencing. mSUMO1 and His-Tag patterns show enhanced SUMO1ylation at DT/R-6h, and reduced SUMO1ylation in PIAS2b absence. Unspecific bands (-u) in Ni-NTA pull-downs of double consecutively transfected, 10xHis-Tag-SUMO1+dsRNAi, required longer gel running (right). (Controls in Supplementary Fig. ). Western blots (anti-SUMO1 and anti-His-Tag) in Eluted lanes show similar patterns due to detection of identical SUMOylated proteins, while some variation in Input lanes reflects both SUMO1ylation and overexpressed 10xHis-tagged SUMO1ylation. However, PIAS2b depletion accumulates free mono-His-SUMO1. c Imaris quantification using 3D super-resolution microscopy of EGFP-PSMC5 or rPSMC5, mPIAS2 or rPIAS2b, TUBB-647 or SUMO1-647 combinations presented as violin plots of double (green, orange) and triple (blue, pink) colocalization. d Surface rendering of progressive mitotic phases with increasing association at the spindle (planes in Supplementary Fig. ). e – g Nuclear accumulation of PSMC5 is promoted by PIAS2b -dsRNAi silencing at DT/R-5 h, with earlier abnormal proteasome activity increase and reduced pSer10-HH3. h Mitotic perturbations at the kinetochore and centrosome at DT/R-5 h in PIAS2b -dsRNAi cells (pictures in Supplementary Fig. d–f): gTubulin + CREST colocalizing nuclear spots increase to 23, coincident with kinetochores, while the few in ns-dsRNAI are side-to-side with CREST; increased nuclear gTubulin colocalized with BUB3 + CREST (kinetochore); duplicated PLK1 (mitosis onset) and PCNT do not colocalize; gTubulin and pericentrin (PCNT) colocalization reveals qualitative and quantitative centrosome alterations. a , b n = 3 independent experiments. c , d Violin plots with cells per condition indicated, from n = 5 independent experiments; two-sided one-way ANOVA with Holm–Sidak’s multiple comparison test. f n = 2 independent experiments. g n = 6 independent experiments, two-sided unpaired T-test. h (upper part) at least 100 cells analyzed per condition, and h (bottom) PLK1 + PCNT n = 4, Centrosomes analysis n = 8 independent experiments; two-sided Mann–Whitney. Bar indicate means ± SEM; exact p value is indicated in the Figure. Source Data are provided.

Article Snippet: However, PIAS2b depletion accumulates free mono-His-SUMO1. c Imaris quantification using 3D super-resolution microscopy of EGFP-PSMC5 or rPSMC5, mPIAS2 or rPIAS2b, TUBB-647 or SUMO1-647 combinations presented as violin plots of double (green, orange) and triple (blue, pink) colocalization. d Surface rendering of progressive mitotic phases with increasing association at the spindle (planes in Supplementary Fig. ). e – g Nuclear accumulation of PSMC5 is promoted by PIAS2b -dsRNAi silencing at DT/R-5 h, with earlier abnormal proteasome activity increase and reduced pSer10-HH3. h Mitotic perturbations at the kinetochore and centrosome at DT/R-5 h in PIAS2b -dsRNAi cells (pictures in Supplementary Fig. d–f): gTubulin + CREST colocalizing nuclear spots increase to 23, coincident with kinetochores, while the few in ns-dsRNAI are side-to-side with CREST; increased nuclear gTubulin colocalized with BUB3 + CREST (kinetochore); duplicated PLK1 (mitosis onset) and PCNT do not colocalize; gTubulin and pericentrin (PCNT) colocalization reveals qualitative and quantitative centrosome alterations. a , b n = 3 independent experiments. c , d Violin plots with cells per condition indicated, from n = 5 independent experiments; two-sided one-way ANOVA with Holm–Sidak’s multiple comparison test. f n = 2 independent experiments. g n = 6 independent experiments, two-sided unpaired T-test. h (upper part) at least 100 cells analyzed per condition, and h (bottom) PLK1 + PCNT n = 4, Centrosomes analysis n = 8 independent experiments; two-sided Mann–Whitney.

Techniques: Transfection, Western Blot, Super-Resolution Microscopy, Activity Assay, Comparison, MANN-WHITNEY

Journal: Stem Cell Reports

Article Title: Pathogenic Pathways in Early-Onset Autosomal Recessive Parkinson's Disease Discovered Using Isogenic Human Dopaminergic Neurons

doi: 10.1016/j.stemcr.2019.12.005

Figure Lengend Snippet: Quantification of Cell Death Events in TH + DNs (A) Dissociated spheres were analyzed via flow cytometry, TH quantification in isogenic HUES1 lines at d35 showing decreased numbers of TH + cells in the PARKIN −/− line. Statistical significance p value derived from one-way ANOVA followed by Bonferroni-Holm multiple comparison test (n = 6, 6, 3, and 3 independent differentiation experiments). (B) TH quantification in WT and PARKIN −/− isogenic lines in a time course experiment in HUES1 line (n = 1 except d35 n = 6). (C) TH quantification in WT and PARKIN −/− isogenic lines in a time course experiment in WA01 line (d0–d21, WT, n = 3 differentiation replicates; PARKIN −/− , n = 3 independent clones; d26–d38, WT, n = 4 differentiation replicates; PARKIN −/− , n = 5 or 3 independent clones, 2 differentiation replicates; d38, ∗ p < 0.05, unpaired two-sided t test). (D–F) Nikon Biostation CT live cell fluorescence imaging of TH:TdTomato expression during differentiation of WT and PARKIN −/− lines after dissociation at d15. Image acquisition starts 24 h post plating. Images were acquired every 6 h, time points indicated. Squares depict areas of interest that are shown in higher magnification. (D) WT cell line. (E) PARKIN −/− cell line. (F) Representative images of cell death events in the PARKIN −/− cell line. Zoomed images with red arrows indicating cells of interest before and after cell death. (G) Cell death quantification of burst events in WT and PARKIN −/− cell lines at the indicated time points, shown as log 2 fold change between PARKIN −/− and WT cell lines (n = 3 wells per cell line). ∗ p < 0.05, unpaired two-sided t test. (H) Bar chart showing normalized relative abundance of SNCA protein across all samples, WT samples set to 1. (I) Bar chart showing normalized relative abundance of CALB1 protein across all samples. WT samples set to 1. Statistical significance was analyzed by one-way ANOVA followed by post hoc test (1% FDR) Bonferroni-Holm for multiple comparisons.

Article Snippet: Statistical significance p value derived from one-way ANOVA followed by Bonferroni-Holm multiple comparison test (n = 6, 6, 3, and 3 independent differentiation experiments). (B) TH quantification in WT and PARKIN −/− isogenic lines in a time course experiment in HUES1 line (n = 1 except d35 n = 6). (C) TH quantification in WT and PARKIN −/− isogenic lines in a time course experiment in WA01 line (d0–d21, WT, n = 3 differentiation replicates; PARKIN −/− , n = 3 independent clones; d26–d38, WT, n = 4 differentiation replicates; PARKIN −/− , n = 5 or 3 independent clones, 2 differentiation replicates; d38, ∗ p < 0.05, unpaired two-sided t test). (D–F) Nikon Biostation CT live cell fluorescence imaging of TH:TdTomato expression during differentiation of WT and PARKIN −/− lines after dissociation at d15.

Techniques: Flow Cytometry, Derivative Assay, Comparison, Clone Assay, Fluorescence, Imaging, Expressing