Journal: Nucleic Acids Research

Article Title: A general strategy to enhance aptamer affinity by suppressing dissociation through symmetric assembly

doi: 10.1093/nar/gkag268

Figure Lengend Snippet: Symmetric aptamer trimerization enhances binding affinity. ( A ) Schematic illustration of aptamer assembly. The 3′ end of monomeric aptamer MSA52t was linked to the trident linker (trebler phosphoramidite) with a 15-thymidine spacer (T15) and assembled into dimeric (shDMSA52t) or trimeric (shTMSA52t) constructs. This design provides flexible orientation and optimal target accessibility for each aptamer unit. ( B ) Dot-blot binding assays showing the affinities of MSA52t, shDMSA52t, and shTMSA52t toward the monomeric wild-type S-protein (mS-protein). Apparent dissociation constants ( K d ) were determined by nonlinear curve fitting. Trimeric assembly resulted in a ∼344-fold affinity enhancement over the monomer.

Article Snippet: The wild-type SARS-CoV-2 spike protein subunit S1 (mS-protein, Cat. No. 40591-V08B1) and Troponin I (Cat. No. 501-TNNI0010) were purchased from Sino Biological Inc. VEGF 165 protein (his-tagged, Cat. No. VE5-H5248) was obtained from Acro Biosystems.

Techniques: Binding Assay, Construct, Dot Blot

Journal: Nucleic Acids Research

Article Title: A general strategy to enhance aptamer affinity by suppressing dissociation through symmetric assembly

doi: 10.1093/nar/gkag268

Figure Lengend Snippet: Trimeric assembly enhances affinity primarily by reducing dissociation rate. Binding kinetics of ( A ) MSA52t, ( B ) shDMSA52t, and ( C ) shTMSA52t interacting with the wild-type SARS-CoV-2 S1-protein (mS-protein) were measured by BLI. Concentration-dependent sensorgrams were globally fitted using 1:1 binding model to obtain the kinetic parameters k on , k off , and K d . The experimental curves are shown as solid lines and the fitted curves are shown as black dashed lines. Bar graphs comparing ( D ) k on , ( E ) k off , and ( F ) K d values for each aptamer. While only modest changes in k on were observed, trimerization led to a dramatic reduction in k off , resulting in a 348-fold improvement in K d for shTMSA52t compared to the monomer.

Article Snippet: The wild-type SARS-CoV-2 spike protein subunit S1 (mS-protein, Cat. No. 40591-V08B1) and Troponin I (Cat. No. 501-TNNI0010) were purchased from Sino Biological Inc. VEGF 165 protein (his-tagged, Cat. No. VE5-H5248) was obtained from Acro Biosystems.

Techniques: Binding Assay, Concentration Assay

Journal: Naunyn-Schmiedeberg's Archives of Pharmacology

Article Title: An endogenous human peptide derived from α 1 -antitrypsin as a novel pharmacological inhibitor of Bordetella pertussis toxin

doi: 10.1007/s00210-025-04744-1

Figure Lengend Snippet: Effect of α 1 AT-derived peptides 32–88 on the ADP-ribosylation status of Gαi in PT-treated CHO-K1 cells. a–f PT (10 ng/ml) and 20 µM α 1 AT peptides, 100 µM α 1 AT, or the respective amount of solvent (DMSO) were added directly to CHO-K1 cells in FCS-free medium and incubated for 4 h at 37 °C. For further control cells were left untreated. After the 4 h incubation, cell lysates were generated and Gαi, which had not been ADP-ribosylated during the initial intoxication of living cells with PT, was ADP-ribosylated and biotin-labeled via the subsequent incubation of the cell lysates with recombinant PTS1 and biotin-labeled NAD + . Next, the biotin-labeled Gαi was detected via Western blot, while Hsp90 served as a control for equal protein loading. The bar graph ( a ) shows the quantification of Western blot signals from four independent experiments, while ( b – f ) show the results of four representative experiments. The intensity values of the bar graph are given as x -fold of the untreated control (Con), normalized to Hsp90, mean ± SEM ( n = 3–14 values from four independent experiments). Significance was tested using one-way ANOVA followed by Dunnett’s multiple comparison test and refers to samples treated with PT only (* p < 0.1, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns not significant)

Article Snippet: For the detection of PT, the cells were incubated with the primary antibody for PTS1 (anti-PTS1, clone 63.1G9, sc-57639, Santa Cruz).

Techniques: Derivative Assay, Solvent, Incubation, Control, Generated, Labeling, Recombinant, Western Blot, Comparison

Journal: Naunyn-Schmiedeberg's Archives of Pharmacology

Article Title: An endogenous human peptide derived from α 1 -antitrypsin as a novel pharmacological inhibitor of Bordetella pertussis toxin

doi: 10.1007/s00210-025-04744-1

Figure Lengend Snippet: Effect of α 1 AT-derived peptides 32–88 on enzyme activity of PTS1 in vitro. a–c PTS1 (84 nM) and 20 µM α 1 AT peptides, 20 µM α 1 AT, or the respective amount of solvent (DMSO) (Con) were preincubated for 15 min at room temperature in buffer and afterwards mixed with Gαi (825 nM) and biotin-NAD + and incubated for a further 40 min at room temperature. During the incubation with recombinant PTS1 and biotin-labeled NAD + , Gαi was biotin-ADP-ribosylated. The biotin-labeled Gαi was subsequently detected via Western blot. The bar graph ( a ) shows the quantifications of Western blot signals from five independent experiments, while ( b , c ) shows the results of two representative experiments. The intensity values of the bar graph are given as x -fold of the untreated control (Con), mean ± SEM ( n = 3–12 values from five independent experiments). Significance was tested using one-way ANOVA followed by Dunnett’s multiple comparison test and refers to the untreated control (Con) (* p < 0.1, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns not significant)

Article Snippet: For the detection of PT, the cells were incubated with the primary antibody for PTS1 (anti-PTS1, clone 63.1G9, sc-57639, Santa Cruz).

Techniques: Derivative Assay, Activity Assay, In Vitro, Solvent, TNKS1 Histone Ribosylation Assay, Incubation, Recombinant, Labeling, Western Blot, Control, Comparison

Journal: Naunyn-Schmiedeberg's Archives of Pharmacology

Article Title: An endogenous human peptide derived from α 1 -antitrypsin as a novel pharmacological inhibitor of Bordetella pertussis toxin

doi: 10.1007/s00210-025-04744-1

Figure Lengend Snippet: Effect of the endogenous α 1 AT peptide from human hemofiltrate on the ADP-ribosylation status of Gαi in PT-treated CHO-K1 cells and enzyme activity of PTS1 in vitro. a–b PT (10 ng/ml) and 100 µM α 1 AT HF peptide, 100 µM α 1 AT, or the respective amount of solvent (H 2 O) were added directly to CHO-K1 cells in FCS-free medium and incubated for 4 h at 37 °C. For further control cells were left untreated. After the 4 h incubation, cell lysates were generated, and Gαi, which had not been ADP-ribosylated during the initial intoxication of living cells with PT, was ADP-ribosylated and biotin-labeled via the subsequent incubation of the cell lysates with recombinant PTS1 and biotin-labeled NAD + . Next, the biotin-labeled Gαi was detected via western blot, while Hsp90 served as a control for equal protein loading. The bar graph ( a ) shows the quantification of Western blot signals from four independent experiments, while ( b ) shows the results of a representative experiment. The intensity values of the bar graph are given as x -fold of the untreated control (Con), normalized to Hsp90, mean ± SEM ( n = 7–15 values from four independent experiments). c–d PTS1 (125.6 nM) and different concentrations of α 1 AT HF peptide, 50 µM α 1 AT, or the respective amount of solvent (H 2 O) (Con) were mixed with Gαi (825 nM) and biotin-NAD + in buffer and incubated for 40 min at room temperature. As further control Gαi was incubated for 40 min at room temperature with solvent control and biotin-NAD + . During the incubation with recombinant PTS1 and biotin-labeled NAD + , Gαi was ADP-ribosylated and subsequently detected via Western blot. The bar graph ( c ) shows the quantifications of Western blot signals from four independent experiments, while ( d ) shows the results of a representative experiment. The intensity values of the bar graph are given as x -fold of the untreated control (Con), mean ± SEM ( n = 4–17 values from four independent experiments). Significance was tested using one-way ANOVA followed by Dunnett’s multiple comparison test and refers to samples treated with PT only ( a , b ) or untreated control (Con) ( c , d ) (* p < 0.1, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns not significant)

Article Snippet: For the detection of PT, the cells were incubated with the primary antibody for PTS1 (anti-PTS1, clone 63.1G9, sc-57639, Santa Cruz).

Techniques: Activity Assay, In Vitro, Solvent, Incubation, Control, Generated, Labeling, Recombinant, Western Blot, TNKS1 Histone Ribosylation Assay, Comparison

Journal: Naunyn-Schmiedeberg's Archives of Pharmacology

Article Title: An endogenous human peptide derived from α 1 -antitrypsin as a novel pharmacological inhibitor of Bordetella pertussis toxin

doi: 10.1007/s00210-025-04744-1

Figure Lengend Snippet: Effect of the endogenous α 1 AT peptide from human hemofiltrate on PT binding to CHO-K1 cells. PT (1 µg/ml), α 1 AT HF, α 1 AT, or the respective amount of solvent (H 2 O) were added to CHO-K1 cells and incubated for 40 min at 4 °C. This enabled PT binding but not the internalization of PT into cells. As further control cells were left untreated. Next, washing, fixation, permeabilization, and quenching were performed. After blocking, the cells were incubated with primary and secondary antibodies to detect PTS1 (green). Nuclei were stained with Hoechst (blue). Representative images are shown from three independent experiments ( n = 3)

Article Snippet: For the detection of PT, the cells were incubated with the primary antibody for PTS1 (anti-PTS1, clone 63.1G9, sc-57639, Santa Cruz).

Techniques: Binding Assay, Solvent, Incubation, Control, Blocking Assay, Staining

Journal: Naunyn-Schmiedeberg's Archives of Pharmacology

Article Title: An endogenous human peptide derived from α 1 -antitrypsin as a novel pharmacological inhibitor of Bordetella pertussis toxin

doi: 10.1007/s00210-025-04744-1

Figure Lengend Snippet: Effect of the substitution of amino acids with alanine of endogenous α 1 AT peptide from human hemofiltrate on ADP-ribosylation of Gαi in PT-treated CHO-K1 cells. a–c PT (10 ng/ml) and 100 µM α 1 AT HF peptide, 200 µM α 1 AT HF peptide P1-11, 100 µM α 1 AT, or the respective amount of solvent (H 2 O) were added directly to CHO-K1 cells in FCS-free medium and incubated for 4 h at 37 °C. For further control cells were left untreated. After the 4-h incubation, cell lysates were generated, and Gαi, which had not been ADP-ribosylated during the initial intoxication of living cells with PT, was ADP-ribosylated and biotin-labeled via the subsequent incubation of the cell lysates with recombinant PTS1 and biotin-labeled NAD + . Next, the biotin-labeled Gαi was detected via western blot, while Hsp90 served as a control for equal protein loading. The bar graph ( a ) shows the quantification of Western blot signals from three independent experiments, while ( b , c ) show the results of a representative experiment. The intensity values of the bar graph are given as x -fold of the untreated control (Con), normalized to Hsp90, mean ± SEM ( n = 4–24 values from six independent experiments). Significance was tested using one-way ANOVA followed by Dunnett’s multiple comparison test and refers to samples treated with PT only (* p < 0.1, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns not significant)

Article Snippet: For the detection of PT, the cells were incubated with the primary antibody for PTS1 (anti-PTS1, clone 63.1G9, sc-57639, Santa Cruz).

Techniques: Solvent, Incubation, Control, Generated, Labeling, Recombinant, Western Blot, Comparison