Journal: PLoS Genetics

Article Title: Single-molecule dynamics and genome-wide transcriptomics reveal that NF-kB (p65)-DNA binding times can be decoupled from transcriptional activation

doi: 10.1371/journal.pgen.1007891

Figure Lengend Snippet: (A) p65 forms a heterodimeric complex with p50 in the cytosol (cyt). The complex is bound by the cytosolic inhibitor IκBa that prevents its translocation into the nucleus. Following TNF-α stimulation and IκBα dissociation, p65/p50 translocate into the nucleus allowing subsequent DNA binding and gene activation. (B) We used a copy of the human p65 fused to a Halo tag as a basis for our mutational expression system. P65-Halo constructs were then expressed in HeLa cells and fluorescently labelled with a JF549 Halo ligand. Upon TNF-α stimulation, labelled p65-Halo translocates into the nucleus. Scale bar: 10 μm.

Article Snippet: Synthetic HPLC-purified sense and anti-sense oligo probes encoding the consensus binding sequence of p65 were purchased from Microsynth (Microsynth AG, Switzerland; Sense-p65_κB: 5’-AGTTGAG GGGACTTTCC CAGGC-3’; Anti-sense-p65_κB: 5’-GCCTG GGAAAGTCCC CTCAACT-3’).

Techniques: Translocation Assay, Binding Assay, Activation Assay, Expressing, Construct

Journal: PLoS Genetics

Article Title: Single-molecule dynamics and genome-wide transcriptomics reveal that NF-kB (p65)-DNA binding times can be decoupled from transcriptional activation

doi: 10.1371/journal.pgen.1007891

Figure Lengend Snippet: (A) Single particle tracking (SPT) was performed after TNF-α stimulation and p65-Halo translocation into the nucleus. All recorded trajectories were filtered based on a spatial threshold established using an immobile control (Histone subunit H2B, see ). After filtering out mobile molecules (i) and correction of photobleaching, the DNA binding time could be estimated from the length of each individual trajectory. ( B ) Schematic overview of p65 affinity mutants. (C) Normalized survival probability plots (1-CDF plot) of the DNA-bound fraction for p65-WT as well as the DNA affinity mutants KKAA and KKRR. The distributions were fitted using a bi-exponential function revealing the fast (t b fast) and slow (t b slow) DNA binding times. (D) Summary of the obtained fitting parameters together with the relative DNA dissociation constant K D for each construct. The pie chart shows the fraction of events associated to the fast (grey) or slow binding time. K on * was obtained from single step displacement histograms as described in Methods. While all constructs exhibit similar k on * as well as t b fast, the slow binding time t b slow correlates with the DNA affinity.

Article Snippet: Synthetic HPLC-purified sense and anti-sense oligo probes encoding the consensus binding sequence of p65 were purchased from Microsynth (Microsynth AG, Switzerland; Sense-p65_κB: 5’-AGTTGAG GGGACTTTCC CAGGC-3’; Anti-sense-p65_κB: 5’-GCCTG GGAAAGTCCC CTCAACT-3’).

Techniques: Single-particle Tracking, Translocation Assay, Control, Binding Assay, Construct

Journal: PLoS Genetics

Article Title: Single-molecule dynamics and genome-wide transcriptomics reveal that NF-kB (p65)-DNA binding times can be decoupled from transcriptional activation

doi: 10.1371/journal.pgen.1007891

Figure Lengend Snippet: (A) We assessed the level of gene activation using RNA sequencing (RNA-Seq). To this end, total mRNA was isolated and sequenced using next-generation sequencing. The total initial set of 1080 genes was cross-referenced using the Chip-Seq ENCODE database, providing a subset of 215 direct interacting genes. A second subset of 45 genes consist of known NFκB regulated genes. For each p65 mutant, the fold-change (FC) expression above the non-transfected (NT) control was calculated and for each gene compared with p65-WT using a log-log FC plot. As a general discrimination between up- and downregulated genes compared to p65-WT, we calculated the logFC ratio. Values with logFC ratio>1 are marked upregulated (red), those with logFC ratio<1 are marked downregulated (green). (B) FC values were standardized (per gene) and the different conditions clustered hierarchically to identify similarities. Interestingly, p65-WT co-clusters with p65-KKRR, which also shows the highest average z-score ( B , top plot) and was identified as the only gain-of-function mutant (C) . The two transactivation mutants as well as the low affinity mutant and p65-ΔDNA also co-cluster highlighting their functional similarity. (C) Classification of each p65 variant based on the logFC ratio estimator, showing that p65-KKRR (i.e. with higher DNA affinity) represents the only gain-of-function mutant. (D) RNA-Seq analysis comparing transcriptional activation of p65-KKAA and p65-KKRR with p65-WT. p65-KKAA shows very weak correlation with p65-WT as well as a strongly reduced gene activation (logFC ratio = -0.47) indicating a loss of gene specificity as well as activation potential. In contrast, p65-KKRR shows higher correlation as well as an increased gene activation (logFC ratio = 0.28).

Article Snippet: Synthetic HPLC-purified sense and anti-sense oligo probes encoding the consensus binding sequence of p65 were purchased from Microsynth (Microsynth AG, Switzerland; Sense-p65_κB: 5’-AGTTGAG GGGACTTTCC CAGGC-3’; Anti-sense-p65_κB: 5’-GCCTG GGAAAGTCCC CTCAACT-3’).

Techniques: Activation Assay, RNA Sequencing, Isolation, Next-Generation Sequencing, ChIP-sequencing, Mutagenesis, Expressing, Transfection, Control, Functional Assay, Variant Assay

Journal: PLoS Genetics

Article Title: Single-molecule dynamics and genome-wide transcriptomics reveal that NF-kB (p65)-DNA binding times can be decoupled from transcriptional activation

doi: 10.1371/journal.pgen.1007891

Figure Lengend Snippet: ( A ) Schematic overview of p65 truncation mutants. ( B ) Normalized survival probability (1-CDF plot) plots of the DNA-bound fraction for p65-WT as well as the transactivation mutants ΔTA1 and ΔTAD as well as a mutant with removed DNA-binding domain (ΔDNA). The distributions were fitted using a bi-exponential function revealing the fast (t b fast) and slow (t b slow) DNA binding times. ( C ) As for the DNA affinity mutants, we found k on * to be in a similar range for all the tested constructs. ( D ) RNA-Seq analysis revealed very low residual transcriptional activation of p65-ΔDNA as evident by logFC ratio = -0.45. The two transactivation mutants showed good correlation with p65-WT (r ~ 0.8) but at strongly reduced transcript abundance resulting in logFC ratio around -0.25.

Article Snippet: Synthetic HPLC-purified sense and anti-sense oligo probes encoding the consensus binding sequence of p65 were purchased from Microsynth (Microsynth AG, Switzerland; Sense-p65_κB: 5’-AGTTGAG GGGACTTTCC CAGGC-3’; Anti-sense-p65_κB: 5’-GCCTG GGAAAGTCCC CTCAACT-3’).

Techniques: Mutagenesis, Binding Assay, Construct, RNA Sequencing, Activation Assay

Journal: PLoS Genetics

Article Title: Single-molecule dynamics and genome-wide transcriptomics reveal that NF-kB (p65)-DNA binding times can be decoupled from transcriptional activation

doi: 10.1371/journal.pgen.1007891

Figure Lengend Snippet: ( A ) The median log 2 FC ratio as retrieved from RNA-Seq data is plotted against t b slow . Note that ΔDNA affinity has been assigned to an arbitrarily low value. ( B ) Working model for p65 mediated transcriptional activation. (1) P65 can act as a pioneering TF, open the chromatin and bind its consensus DNA sequence. Transcriptional activation can then be initiated although the exact mechanism of RNA pol-II recruitment remains unclear. Following this model, the DNA binding time would correlate with the transcriptional output, while removal of TADs would not affect the complex stability. (2) An important extension of this model as suggested by our data is that TADs are required to efficiently translate p65 DNA binding into transcriptional output presumably through the recruitment of protein co-factors.

Article Snippet: Synthetic HPLC-purified sense and anti-sense oligo probes encoding the consensus binding sequence of p65 were purchased from Microsynth (Microsynth AG, Switzerland; Sense-p65_κB: 5’-AGTTGAG GGGACTTTCC CAGGC-3’; Anti-sense-p65_κB: 5’-GCCTG GGAAAGTCCC CTCAACT-3’).

Techniques: RNA Sequencing, Activation Assay, Sequencing, Binding Assay

Journal: Rsc Applied Interfaces

Article Title: New buffer systems for photopainting of single biomolecules †

doi: 10.1039/d3lf00125c

Figure Lengend Snippet: Schematic representation of the 2D acrylate scaffold functionalization via LAPAP at a single molecule level (smLAPAP). (a) A 2D acrylate scaffold (4 : 1 weight ratio Ormocomp : PEGDA, 1 wt% Irgacure 819) was prepared via MPL on glass substrate. (b) Passivation of the surrounding glass substrate with BSA. (c) Incubation with Cy3 conjugated to NP77- (Cy3-NP77) model oligonucleotide and photobleaching of the fluorophores in the focal spot of the laser (515 nm) (LAPAP). (d) After washing, only bleached fluorophores bound to the polymer-substrate remain on the scaffold. (e) Incubation with ATTO 647N labelled cNP77 (ATTO647N-cNP77) complementary oligonucleotide. (f) Immobilized oligonucleotide helix on the polymer surface. (g) Oligonucleotide helix composed of NP77 and cNP77. Sketches are not to drawn scale.

Article Snippet: For visualization of the fluorophore–biomolecule conjugates bound via LAPAP, ATTO 647N conjugated to the complementary oligonucleotide cNP77 (sequence: ATA CCT GAC GCG ATC TGT TCG TAC CGA TCA AGG CAG TCT GCC AAT TGT ACT AGT CTA GCG, 5′ modification: C6-spacer amino modification – ATTO 647N) (ATTO647N-cNP77, Microsynth AG, Switzerland) was used for Cy-LAPAP and ATTO 655 conjugated to streptavidin (ATTO655-SA, ATTO-TEC GmbH, Germany) was used for Cou-LAPAP and Rho-LAPAP.

Techniques: Incubation, Polymer

Journal: Rsc Applied Interfaces

Article Title: New buffer systems for photopainting of single biomolecules †

doi: 10.1039/d3lf00125c

Figure Lengend Snippet: LAPAP on 2D scaffolds. 3 buffer systems (buffer I: neat water for Cy-LAPAP and PBS for Cou-LAPAP and Rho-LAPAP; buffer II: buffer I + Mn 2+ ; buffer III: buffer I + Mn 2+ + RuPo) with different fluorophore families were tested for LAPAP. Immobilized molecules were confirmed with ATTO647N-cNP77 and ATTO655-SA, respectively. (a) Cy-LAPAP cyanine dye: Cy3-NP77. With buffer I, 1.01 ± 0.35 BM at LAPAP-efficiency of 21% was observed. With buffer II, BM increases to 1.35 ± 0.49 with a LAPAP-efficiency of 24%. Buffer III yielded 1.87 ± 0.64 BM, and an increase of LAPAP-efficiency by three-fold (64%). (b) Cou-LAPAP coumarin dye: ATTO390-biotin. With buffer I, 1.00 ± 0.23 BM at LAPAP-efficiency of 64% was estimated. With buffer II, LAPAP-efficiency increased to 86% and 0.93 ± 0.16 BM. Buffer III yields 1.20 ± 0.23 BM with a LAPAP-efficiency of 90%. (c) Rho-LAPAP rhodamine dye: ATTO514-biotin. With buffer I, 0.91 ± 0.12 BM at LAPAP-efficiency of 48% was determined. With buffer II, LAPAP-efficiency increased to 78% and 0.98 ± 0.20 BM. Buffer III yielded similar results as buffer II. Scale bar for all fluorescence images 2 μm.

Article Snippet: For visualization of the fluorophore–biomolecule conjugates bound via LAPAP, ATTO 647N conjugated to the complementary oligonucleotide cNP77 (sequence: ATA CCT GAC GCG ATC TGT TCG TAC CGA TCA AGG CAG TCT GCC AAT TGT ACT AGT CTA GCG, 5′ modification: C6-spacer amino modification – ATTO 647N) (ATTO647N-cNP77, Microsynth AG, Switzerland) was used for Cy-LAPAP and ATTO 655 conjugated to streptavidin (ATTO655-SA, ATTO-TEC GmbH, Germany) was used for Cou-LAPAP and Rho-LAPAP.

Techniques: Fluorescence

Journal: Rsc Applied Interfaces

Article Title: New buffer systems for photopainting of single biomolecules †

doi: 10.1039/d3lf00125c

Figure Lengend Snippet: LAPAP on a 3D scaffold (a) model sketch and SEM image of 3D MPL acrylate scaffold. The laser was focused on the bottom of the hanging horizontal bars (green lines). (b) Fluorescence microscopy image of fluorescent spots on 3D MPL acrylate scaffold. Cy-LAPAP with buffer III was used. An average BM of 1.45 ± 0.36 was calculated. (c) Localized positions of emitters in yellow insert in (b), in the red insert two bound ATTO647N-cNP77 separated by ∼200 nm (average position accuracy PA: 24 ± 5 nm) within one diffraction limited spot are resolved. Gray value scaled to maximum.

Article Snippet: For visualization of the fluorophore–biomolecule conjugates bound via LAPAP, ATTO 647N conjugated to the complementary oligonucleotide cNP77 (sequence: ATA CCT GAC GCG ATC TGT TCG TAC CGA TCA AGG CAG TCT GCC AAT TGT ACT AGT CTA GCG, 5′ modification: C6-spacer amino modification – ATTO 647N) (ATTO647N-cNP77, Microsynth AG, Switzerland) was used for Cy-LAPAP and ATTO 655 conjugated to streptavidin (ATTO655-SA, ATTO-TEC GmbH, Germany) was used for Cou-LAPAP and Rho-LAPAP.

Techniques: Fluorescence, Microscopy

Journal: Molecular Therapy. Nucleic Acids

Article Title: Targeting EGFR/HER2/HER3 with a Three-in-One Aptamer-siRNA Chimera Confers Superior Activity against HER2 + Breast Cancer

doi: 10.1016/j.omtn.2017.12.015

Figure Lengend Snippet: Schematic Illustration of HER2 Aptamer-EGFR siRNA-HER3 Aptamer, H2EH3, and Characterization of H2EH3 (A) Structure of H2EH3. HER2 aptamer was conjugated with HER3 aptamer through 21 bases of EGFR siRNA and 2–4 unpaired base linkers. (B) Western blot detection of the expression levels of HER2, HER3, and EGFR in a panel of breast cancer cell lines. (C) Evaluation of the cytotoxicity of H2EH3 by CCK-8 assay. Breast cancer cell lines, including BT474, SKBR3, MCF7, MDA-MB-231, and Hs587T cells, were treated with varying concentrations of H2EH3 or controls for 72 hr, and cell viability was detected with the CCK-8 agent. Data are the mean ± SD from three independent experiments. (D) Evaluation of EGFR-silencing capability of H2EH3 using western blot. (E) Evaluation of H2EH3-binding capability compared with HER2 aptamer and HER3 aptamer.

Article Snippet: 2′-Fluoro-2′-deoxycytidine-5′-triphosphate, 2′-fluoro-2′-deoxyuridine-5′-triphosphate, and Cy5-labeled 2′-fluoro-labeled HER2 aptamer were purchased from TriLink Biotechnologies (San Diego, CA).

Techniques: Western Blot, Expressing, CCK-8 Assay, Binding Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Targeting EGFR/HER2/HER3 with a Three-in-One Aptamer-siRNA Chimera Confers Superior Activity against HER2 + Breast Cancer

doi: 10.1016/j.omtn.2017.12.015

Figure Lengend Snippet: Analysis of Cell Cycle and Apoptosis (A) Effects of H2EH3 on cell cycle progression of HER2 + HER3 + cells. SKBR3 and BT474 cells were treated with H2EH3 (1 μM, 2 μM) for 24 and 48 hr. The cell cycle was then analyzed by flow cytometry. (B) Quantitative analysis of apoptosis after H2EH3 treatment by flow cytometry. BT474 and SKBR3 cells were treated with the different concentrations of H2EH3 for 72 hr, and cells were stained with Alexa Fluor 488 annexin-V-propidium iodide and analyzed by flow cytometry.

Article Snippet: 2′-Fluoro-2′-deoxycytidine-5′-triphosphate, 2′-fluoro-2′-deoxyuridine-5′-triphosphate, and Cy5-labeled 2′-fluoro-labeled HER2 aptamer were purchased from TriLink Biotechnologies (San Diego, CA).

Techniques: Flow Cytometry, Staining

Journal: Molecular Therapy. Nucleic Acids

Article Title: Targeting EGFR/HER2/HER3 with a Three-in-One Aptamer-siRNA Chimera Confers Superior Activity against HER2 + Breast Cancer

doi: 10.1016/j.omtn.2017.12.015

Figure Lengend Snippet: Evaluation of Binding Specificity and In Vivo Bio-distribution of H2EH3 (A) Western blot analysis of HER2 and/or HER3 gene knockdown. (B) Flow cytometry to quantitatively measure cell binding of H2EH3 upon HER2 and/or HER3 knockdown in BT474 cells. Untreated: solid gray; mock transfected cells: light blue; normal cells stained with H2EH3-Cy5: red line; control siRNA transfected cells stained with H2EH3: green line, HER2-silenced cells stained with H2EH3-Cy5: purple line; HER3-silenced cells stained with H2EH3-Cy5: black line; both HER2- and HER3-silenced cells stained with H2EH3: blue line. (C) Time course whole-body imaging to show the binding profile of H2EH3. Tumor-bearing mice were intravenously injected with Cy5-H2EH3 or non-targeting control aptamer. Cy5 fluorescence of whole body was captured at the time points of 2 and 6 hr with the Xenogen IVIS100 imaging system. (D) Ex vivo organ imaging. Following whole-body imaging, major organs were removed and detected with the Xenogen IVIS100 imaging system. The result is the representative of two independent experiments.

Article Snippet: 2′-Fluoro-2′-deoxycytidine-5′-triphosphate, 2′-fluoro-2′-deoxyuridine-5′-triphosphate, and Cy5-labeled 2′-fluoro-labeled HER2 aptamer were purchased from TriLink Biotechnologies (San Diego, CA).

Techniques: Binding Assay, In Vivo, Western Blot, Flow Cytometry, Transfection, Staining, Imaging, Injection, Fluorescence, Ex Vivo

Journal: Molecular Therapy. Nucleic Acids

Article Title: Targeting EGFR/HER2/HER3 with a Three-in-One Aptamer-siRNA Chimera Confers Superior Activity against HER2 + Breast Cancer

doi: 10.1016/j.omtn.2017.12.015

Figure Lengend Snippet: H2EH3 Significantly Suppresses Tumor Growth in Breast Cancer Xenografts (A) Inhibition of tumor growth by H2EH3 through intratumoral injection. Mice with subcutaneous tumors were intratumorally injected with H2EH3, a mixture of HER2 aptamer, HER3 aptamer, and EGFR siRNA, or PBS every other day for 5 weeks (n = 5 per group). (B) Dissected tumors after treatment though intratumoral injection (n = 5). (C) Quantitation of dissected tumor sizes from (B) (n = 5). (D) Body weight of mice treated with H2EH3 through intratumoral injection (n = 5). (E) Inhibition of tumor growth by H2EH3 through intravenous injection. Mice with orthotopic tumors were intratumorally injected with H2EH3, a mixture of HER2 aptamer, HER3 aptamer, and EGFR siRNA, or PBS every 3 days for 4 weeks (n = 4). (F) Dissected tumors after treatment through intravenous injection (n = 4). (G) Quantitation of dissected tumor sizes from (F) (n = 4). (H) Body weight of mice treated with H2EH3 through intravenous injection (n = 4). *p < 0.05; **p < 0.005. Data represent the mean ± SEM; n = 4–5 for each group.

Article Snippet: 2′-Fluoro-2′-deoxycytidine-5′-triphosphate, 2′-fluoro-2′-deoxyuridine-5′-triphosphate, and Cy5-labeled 2′-fluoro-labeled HER2 aptamer were purchased from TriLink Biotechnologies (San Diego, CA).

Techniques: Inhibition, Injection, Quantitation Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Targeting EGFR/HER2/HER3 with a Three-in-One Aptamer-siRNA Chimera Confers Superior Activity against HER2 + Breast Cancer

doi: 10.1016/j.omtn.2017.12.015

Figure Lengend Snippet: Evaluation of Gene Expression and Apoptosis after H2EH3 Treatment In Vivo Formalin-fixed paraffin-embedded sections of xenograft tumors were stained with antibodies targeting EGFR, HER2, HER3, P21, and cleaved caspase-3. (A) IHC analysis of subcutaneous tumors after intratumoral injection of H2EH3. (B) IHC analysis of orthotopic tumors after intravenous injection of H2EH3. Scale bar, 50 μm.

Article Snippet: 2′-Fluoro-2′-deoxycytidine-5′-triphosphate, 2′-fluoro-2′-deoxyuridine-5′-triphosphate, and Cy5-labeled 2′-fluoro-labeled HER2 aptamer were purchased from TriLink Biotechnologies (San Diego, CA).

Techniques: Expressing, In Vivo, Formalin-fixed Paraffin-Embedded, Staining, Injection