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