Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Computational design of an ultrapotent deltacoronavirus miniprotein inhibitor

doi: 10.1073/pnas.2533456123

Figure Lengend Snippet: Computational design of PDCoV miniprotein inhibitors. ( A ) Ribbon rendering of the predicted structures of the PDCoV IL121_2014 RBD (gold) in complex with the MBs selected for experimental testing (rainbow-colored from N- to C-terminus). The index next to each structure indicates the minibinder name. ( B ) BLI screen for binding of PDCoV IL121_2014 S 1 -Fc at a concentration of 500 nM to MBs immobilized at the surface of HIS1K biosensors. One batch of MBs and PDCoV S 1 -Fc were used for the screening experiment. ( C ) BLI analysis of binding kinetics of the PDCoV IL121_2014 RBD at multiple concentrations to the MB11 immobilized at the surface of streptavidin biosensors. The PDCoV RBD concentrations used are indicated with the color key and the fit to the data using a 1:1 binding model with global fit is shown as dashed black lines. K D : equilibrium dissociation constant. Independent batches of MB11 and PDCoV RBD were used for two biological replicates. ( D ) Neutralizing activity expressed as half-maximal inhibitory concentration (IC 50 ) of each MB determined using PDCoV IL121_2014 S VSV pseudoviruses and HEK293T target cells transiently expressing galline APN. Each data point represents a biological replicate obtained with distinct batches of minibinders and of pseudoviruses. The bar represents the geometric mean of 3 to 4 biological replicates.

Article Snippet: HEK293T cells (ATCC, Cat# CRL-11268) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Cytiva) and 1% penicillin-streptomycin (Thermo Fisher Scientific) at 37 °C and 5% CO 2 .

Techniques: Binding Assay, Concentration Assay, Activity Assay, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Computational design of an ultrapotent deltacoronavirus miniprotein inhibitor

doi: 10.1073/pnas.2533456123

Figure Lengend Snippet: MB11 mediates potent and broad DCoV neutralization. ( A ) Inferred phylogeny of the DCoV genus from RBD amino acid sequence, adapted from ( 39 ). The range of sequence identity to PDCoV IL121_2014 in each viral clade is indicated. Sequence identity was calculated using Sequence Manipulation Suite: Ident and Sim from bioinformatics.org. ( B ) Heat map showing the breadth of MB10 and MB11 binding to DCoV RBDs expressed on the surface of yeast. ( C ) BLI binding analysis of a panel of DCoV RBDs at a concentration of 50 nM to MB11 immobilized at the surface of streptavidin biosensors. ( D and E ) Neutralizing activity expressed as half-maximal inhibitory concentration (IC 50 ) of MB11 ( D ) and PD33 Fab ( E ) determined using PDCoV IL121_2014 , SparrowCoV ISU42824 , MuniaCoV HKU13 , and AvianCoV rub035cor1 S VSV pseudoviruses and HEK293T cells transiently expressing galline (PDCoV IL121_2014 ), sparrow (SparrowCoV ISU42824 ), or munia (MuniaCoV HKU13 and AvianCoV rub035cor1 ) APN orthologs. Dotted lines show the limits of detection of the assays. ( F ) PDCoV IL121_2014 RBD escape maps from MB binding obtained by deep mutational scanning. ( Left ) Line plots showing the sum of escape from MB binding due to mutations at each site in the PDCoV IL121_2014 RBD. Sites with strong escape are highlighted with pink bars. ( Right ) Logoplots showing individual mutations at each site in the PDCoV IL121_2014 RBD that escape with a 10-fold or greater reduction in MB binding. RBD mutations are colored by their orthogonally measured effects on gAPN receptor-binding avidity (increasing light yellow indicates increasingly deleterious and brown indicates neutral or advantageous effects on receptor binding). An alignment of DCoV S sequence isolates used in this manuscript at sites illustrated in the logoplots is shown below.

Article Snippet: HEK293T cells (ATCC, Cat# CRL-11268) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Cytiva) and 1% penicillin-streptomycin (Thermo Fisher Scientific) at 37 °C and 5% CO 2 .

Techniques: Neutralization, Sequencing, Binding Assay, Concentration Assay, Activity Assay, Expressing

Journal: bioRxiv

Article Title: Unveiling DNA Origami Interaction Dynamics on Living Cell Surfaces by Single Particle Tracking

doi: 10.1101/2024.12.23.628980

Figure Lengend Snippet: Immunostaining of the EGFR on Hek 293T cells (left) and the MDA MD 468 cells (right). Images were taken via confocal laser scanning microscopy.

Article Snippet: The MDA-MB-468 (ATCC cat. HTB-132) and Hek 293T (ATCC cat. CRL-3519) cell lines were cultured in Thermo ScientificTM NuncTM Cell Culture Treated Flasks with Filter Caps.

Techniques: Immunostaining, Confocal Laser Scanning Microscopy

Journal: bioRxiv

Article Title: Unveiling DNA Origami Interaction Dynamics on Living Cell Surfaces by Single Particle Tracking

doi: 10.1101/2024.12.23.628980

Figure Lengend Snippet: a) Schematic representation of the experimental findings on NR selectivity: the differential binding profile between the targeted cell line (MDA MD 468) and the non-targeted cell line (Hek 293T) with the targeted NRs (NRs_18Ab and NRs_18Apt). For the MDA MD 468 cells, binding events with the targeted NRs are characterized by very long trajectories, indicating specific binding, while the binding trajectories with the Hek 293T are typically much shorter, indicating non-specific binding. b) Representative image of NRs_18Ab trajectories at 60 minutes after their incubation with MDA MD 468 and Hek 293T cells. Cell contours are indicated by the dotted line. Color bar indicates the diffusion coefficients (ranging from 0 to 4 µm 2 /s). c) Scatter plot of all the binding events for non-functionalized NRs, NR_18Ab and NR_18Apt (i.e. all the trajectories with D ≤ 1), plotted against their respective trajectory length (y-axis). d) Bar plot displaying the differential specific binding percentage between MDA MD 468 and Hek 293T for the different NR designs (NR_18Ab and NR_18Apt) at 3 distinct time points (10 min, 30 min and 60 min). Results are shown as the mean +-standard error of the mean. n = 3 biological replicates (per biological replicate, the trajectories of 5 different movies were combined, i.e. 5 technical replicates) *: significant difference between groups with p ≤ 0.05, ** : significant difference between groups with p ≤ 0.01, *** : significant difference between groups with p ≤ 0.001.

Article Snippet: The MDA-MB-468 (ATCC cat. HTB-132) and Hek 293T (ATCC cat. CRL-3519) cell lines were cultured in Thermo ScientificTM NuncTM Cell Culture Treated Flasks with Filter Caps.

Techniques: Binding Assay, Incubation, Diffusion-based Assay

Journal: bioRxiv

Article Title: Unveiling DNA Origami Interaction Dynamics on Living Cell Surfaces by Single Particle Tracking

doi: 10.1101/2024.12.23.628980

Figure Lengend Snippet: a) Scheme of the binding kinetics between targeted NRs (NR_18Ab and NR_18Apt) and cells (left panel). The corresponding formula of the binding kinetics is displayed in the right panel. b) Comparison of the total number of specific binding events between NRs with 8 or 18 binding ligands (antibody or aptamer) in both MDA MD 468 and Hek 293T cells. The amount of binding events can be directly related to k on . c) Example of the exponential decay fitting for the binding time of NR functionalized with 18 antibodies in MDA MD 468 cells, where dotted line represents the fitted courve and the corresponding equation is displayed. d) τ B values obtained via an exponential decay fitting of all the binding events for each NR design and cell type. This value is inversely proportional to k off . A comparison was made between NRs with 8 or 18 binding ligands (antibody or aptamer) in both MDA MD 468 or Hek 293T. Results are shown as mean fitted value, where error bars represent the standard error of the fitting for each NR design in the different cell lines.

Article Snippet: The MDA-MB-468 (ATCC cat. HTB-132) and Hek 293T (ATCC cat. CRL-3519) cell lines were cultured in Thermo ScientificTM NuncTM Cell Culture Treated Flasks with Filter Caps.

Techniques: Binding Assay, Comparison