Journal: Signal Transduction and Targeted Therapy

Article Title: Peptide-substituted oligonucleotide synthesis and non-toxic, passive cell delivery

doi: 10.1038/sigtrans.2016.19

Figure Lengend Snippet: TP ODNs: sequences, molecular weight analysis and net charge at pH 7.4

Article Snippet: To 50 μl of a reduced serum, no antibiotic medium (2% FBS, 1× GlutaMAX, 1× non-essential amino acids and 1× sodium pyruvate) was added a stock solution of a TP ODN in water (Hyclone Cell Molecular biology Grade, Invitrogen) so as to obtain a final concentration of 0.1 μM TP ODN.

Techniques: Molecular Weight, Sequencing

Journal: Signal Transduction and Targeted Therapy

Article Title: Peptide-substituted oligonucleotide synthesis and non-toxic, passive cell delivery

doi: 10.1038/sigtrans.2016.19

Figure Lengend Snippet: ( a ) A stably transfected vector in HeLa cells expresses two luciferase genes. Firefly luciferase is expressed constitutively, whereas expression of the renilla luciferase gene is under the control of miR-15b as part of the RNA-induced silencing complex (RISC). In the presence of an anti-miR-15b oligonucleotide (TP ODN in this figure), the RISC dissociates from the renilla gene. This leads to an increase in renilla luciferase activity. Comparison of the activity of renilla luciferase to the unregulated firefly luciferase yields an indication of the activity of an oligonucleotide as an anti-miR-15b oligonucleotide. ( a ) Adapted from the Integrated DNA Technologies Inc. (Coralville, IA, USA) website. The stably transfected HeLa cell line and assay protocols were developed by miRagen Therapeutics, Inc. ( b ) Lipid transfection using DharmaFECT 1 (Dharmacon). ( c ) Passive transfection using TP ODNs. Positive and negative controls were purchased from Dharmacon: miRIDIAN Hairpin Inhibitor (IH-300587-07) and miRIDIAN Hairpin Inhibitor Negative Control #1 (IN-001005-01).

Article Snippet: To 50 μl of a reduced serum, no antibiotic medium (2% FBS, 1× GlutaMAX, 1× non-essential amino acids and 1× sodium pyruvate) was added a stock solution of a TP ODN in water (Hyclone Cell Molecular biology Grade, Invitrogen) so as to obtain a final concentration of 0.1 μM TP ODN.

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Luciferase, Expressing, Activity Assay, Negative Control

Journal: Signal Transduction and Targeted Therapy

Article Title: Peptide-substituted oligonucleotide synthesis and non-toxic, passive cell delivery

doi: 10.1038/sigtrans.2016.19

Figure Lengend Snippet: TP ODN melting temperatures

Article Snippet: To 50 μl of a reduced serum, no antibiotic medium (2% FBS, 1× GlutaMAX, 1× non-essential amino acids and 1× sodium pyruvate) was added a stock solution of a TP ODN in water (Hyclone Cell Molecular biology Grade, Invitrogen) so as to obtain a final concentration of 0.1 μM TP ODN.

Techniques:

Journal: Signal Transduction and Targeted Therapy

Article Title: Peptide-substituted oligonucleotide synthesis and non-toxic, passive cell delivery

doi: 10.1038/sigtrans.2016.19

Figure Lengend Snippet: Localization of a fluorescein-labeled, unsubstituted TP ODN (5′-FL-T u A u ACACGATACGCG u A u T-3′, unsubstituted TP linkages are designated by u) with mCherry-Gal-T. After labeling HeLa cells with Gal-T using TransIT-LT1 for 30 min, cells were incubated with unsubstituted TP ODN at 37 °C and monitored continuously for 15 h. Cells were stained with Hoechst 33258 to visualize the nuclei. ( a ) Before washing ODN. ( b ) After washing ODN and nucleus staining. Confocal microscopic image merging showed the unsubstituted TP ODN localizes near to the Golgi marker.

Article Snippet: To 50 μl of a reduced serum, no antibiotic medium (2% FBS, 1× GlutaMAX, 1× non-essential amino acids and 1× sodium pyruvate) was added a stock solution of a TP ODN in water (Hyclone Cell Molecular biology Grade, Invitrogen) so as to obtain a final concentration of 0.1 μM TP ODN.

Techniques: Labeling, Incubation, Staining, Marker

Journal: Signal Transduction and Targeted Therapy

Article Title: Peptide-substituted oligonucleotide synthesis and non-toxic, passive cell delivery

doi: 10.1038/sigtrans.2016.19

Figure Lengend Snippet: The effect of 1 μg ml −1 brefeldin A (BFA), 100 μM chloroquine (CQ) and 100 μM histidine (His) on endosomal release of a TP ODN (FL-4PA). Note that the suggested concentration of BFA is 10 μg ml −1 for endosomal release. In the research reported here, this concentration caused significant toxicity even after a short treatment. Thus, a lower and less toxic 1 μg ml −1 concentration was used for this experiment. Passive transfection was performed using 3 μM TP ODN FL-4PA for 24 h followed by washing. Cells were then incubated with BFA, CQ and His, respectively. Three-dimensional microscopy was taken at 0.5 μm per step for 30 steps. Fl-4PA: 5′-FL-T PepA GTAAAC PepA CATGA PepA TGTGCTGCT PepA A-3′. PepA, AcHN-Lys(N 3 )-Lys-Lys-Arg-Gly-NH 2 .

Article Snippet: To 50 μl of a reduced serum, no antibiotic medium (2% FBS, 1× GlutaMAX, 1× non-essential amino acids and 1× sodium pyruvate) was added a stock solution of a TP ODN in water (Hyclone Cell Molecular biology Grade, Invitrogen) so as to obtain a final concentration of 0.1 μM TP ODN.

Techniques: Concentration Assay, Transfection, Incubation, Microscopy

Journal: Signal Transduction and Targeted Therapy

Article Title: Peptide-substituted oligonucleotide synthesis and non-toxic, passive cell delivery

doi: 10.1038/sigtrans.2016.19

Figure Lengend Snippet: TP ODNs: sequences, molecular weight analysis and net charge at pH 7.4

Article Snippet: The concentration of a 5′-fluorescein-labeled TP ODN in HyPure Molecular Biology Grade Water (Thermo Scientific, Chino, CA, USA) was measured by ultraviolet spectroscopy.

Techniques: Molecular Weight, Sequencing

Journal: Signal Transduction and Targeted Therapy

Article Title: Peptide-substituted oligonucleotide synthesis and non-toxic, passive cell delivery

doi: 10.1038/sigtrans.2016.19

Figure Lengend Snippet: ( a ) A stably transfected vector in HeLa cells expresses two luciferase genes. Firefly luciferase is expressed constitutively, whereas expression of the renilla luciferase gene is under the control of miR-15b as part of the RNA-induced silencing complex (RISC). In the presence of an anti-miR-15b oligonucleotide (TP ODN in this figure), the RISC dissociates from the renilla gene. This leads to an increase in renilla luciferase activity. Comparison of the activity of renilla luciferase to the unregulated firefly luciferase yields an indication of the activity of an oligonucleotide as an anti-miR-15b oligonucleotide. ( a ) Adapted from the Integrated DNA Technologies Inc. (Coralville, IA, USA) website. The stably transfected HeLa cell line and assay protocols were developed by miRagen Therapeutics, Inc. ( b ) Lipid transfection using DharmaFECT 1 (Dharmacon). ( c ) Passive transfection using TP ODNs. Positive and negative controls were purchased from Dharmacon: miRIDIAN Hairpin Inhibitor (IH-300587-07) and miRIDIAN Hairpin Inhibitor Negative Control #1 (IN-001005-01).

Article Snippet: The concentration of a 5′-fluorescein-labeled TP ODN in HyPure Molecular Biology Grade Water (Thermo Scientific, Chino, CA, USA) was measured by ultraviolet spectroscopy.

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Luciferase, Expressing, Activity Assay, Negative Control

Journal: Signal Transduction and Targeted Therapy

Article Title: Peptide-substituted oligonucleotide synthesis and non-toxic, passive cell delivery

doi: 10.1038/sigtrans.2016.19

Figure Lengend Snippet: TP ODN melting temperatures

Article Snippet: The concentration of a 5′-fluorescein-labeled TP ODN in HyPure Molecular Biology Grade Water (Thermo Scientific, Chino, CA, USA) was measured by ultraviolet spectroscopy.

Techniques:

Journal: Signal Transduction and Targeted Therapy

Article Title: Peptide-substituted oligonucleotide synthesis and non-toxic, passive cell delivery

doi: 10.1038/sigtrans.2016.19

Figure Lengend Snippet: Localization of a fluorescein-labeled, unsubstituted TP ODN (5′-FL-T u A u ACACGATACGCG u A u T-3′, unsubstituted TP linkages are designated by u) with mCherry-Gal-T. After labeling HeLa cells with Gal-T using TransIT-LT1 for 30 min, cells were incubated with unsubstituted TP ODN at 37 °C and monitored continuously for 15 h. Cells were stained with Hoechst 33258 to visualize the nuclei. ( a ) Before washing ODN. ( b ) After washing ODN and nucleus staining. Confocal microscopic image merging showed the unsubstituted TP ODN localizes near to the Golgi marker.

Article Snippet: The concentration of a 5′-fluorescein-labeled TP ODN in HyPure Molecular Biology Grade Water (Thermo Scientific, Chino, CA, USA) was measured by ultraviolet spectroscopy.

Techniques: Labeling, Incubation, Staining, Marker

Journal: Signal Transduction and Targeted Therapy

Article Title: Peptide-substituted oligonucleotide synthesis and non-toxic, passive cell delivery

doi: 10.1038/sigtrans.2016.19

Figure Lengend Snippet: The effect of 1 μg ml −1 brefeldin A (BFA), 100 μM chloroquine (CQ) and 100 μM histidine (His) on endosomal release of a TP ODN (FL-4PA). Note that the suggested concentration of BFA is 10 μg ml −1 for endosomal release. In the research reported here, this concentration caused significant toxicity even after a short treatment. Thus, a lower and less toxic 1 μg ml −1 concentration was used for this experiment. Passive transfection was performed using 3 μM TP ODN FL-4PA for 24 h followed by washing. Cells were then incubated with BFA, CQ and His, respectively. Three-dimensional microscopy was taken at 0.5 μm per step for 30 steps. Fl-4PA: 5′-FL-T PepA GTAAAC PepA CATGA PepA TGTGCTGCT PepA A-3′. PepA, AcHN-Lys(N 3 )-Lys-Lys-Arg-Gly-NH 2 .

Article Snippet: The concentration of a 5′-fluorescein-labeled TP ODN in HyPure Molecular Biology Grade Water (Thermo Scientific, Chino, CA, USA) was measured by ultraviolet spectroscopy.

Techniques: Concentration Assay, Transfection, Incubation, Microscopy