Suppressing Nonspecific Binding in Biolayer Interferometry Experiments for Weak Ligand–Analyte Interactions

Dubrow, Alyssa; Zuniga, Bryan; Topo, Elias; Cho, Jae-Hyun

doi:10.1021/acsomega.1c05659

Cited by 12 publications

(10 citation statements)

References 34 publications

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“…After immobilizing CA121 HEX : 6HIS to the HIS‐affinity probes, we report the biochemical constants for cyFG‐3 are K D =0.963±0.06 μM, k on =8.90±0.53×10 3 M −1 s −1 , k off =8.58±0.136×10 −3 s −1 (Figure 6E). We confirmed our result with a cyclic peptide by carrying out a BLI assay with a positive control, a small molecule antiviral Lenacapvir (LEN) that was recently approved for use in highly treatment‐experienced individuals who are HIV‐1–positive and on a failing antiretroviral therapy (ART) regimen [34,38–42] . The biochemical constants for LEN to CA HEX : 6HIS are K D =28.6±0.2 nM, k on =1.040±0.007×10 4 M −1 s −1 , k off =2.978±0.015×10 −4 s −1 (Supplementary Figure S12).…”

Section: Resultssupporting

confidence: 73%

A Tag‐Free Platform for Synthesis and Screening of Cyclic Peptide Libraries

Bruce,

Adebomi,

Czabala

et al. 2024

Angewandte Chemie

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In the realm of high‐throughput screening (HTS), macrocyclic peptide libraries traditionally necessitate decoding tags, essential for both library synthesis and identifying hit peptide sequences post‐screening. Our innovation introduces a tag‐free technology platform for synthesizing cyclic peptide libraries in solution and facilitates screening against biological targets to identify peptide binders through unconventional intramolecular CyClick and DeClick Chemistries (CCDC) discovered within our research. This combination allows for the synthesis of diverse cyclic peptide libraries, the incorporation of various amino acids, and facile linearization and decoding of cyclic peptide binder sequences. Our sensitivity‐enhancing derivatization method, utilized in tandem with nano LC‐MS/MS, enables the sequencing of peptides even at exceedingly low picomolar concentrations. Employing our technology platform, we've successfully unearthed novel cyclic peptide binders against a monoclonal antibody and the first cyclic peptide binder of HIV CAPSID protein responsible for viral infections as validated by microscale thermalshift assays (TSA), biolayer Interferometry (BLI) and functional assays.

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Section: Resultssupporting

confidence: 73%

A Tag‐Free Platform for Synthesis and Screening of Cyclic Peptide Libraries

Bruce,

Adebomi,

Czabala

et al. 2024

Angewandte Chemie

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“…We show, in particular, that the combination of BSA and trehalose exhibits good performance as a blocking agent. Although the significant bioactivity of nonspecifically bound BMP2 was an issue in our case, this study demonstrated the potential of biomaterials with bare glass in effectively retaining and delivering biologically active BMP2, as previously investigated [38] .…”

Section: Discussionsupporting

confidence: 58%

“…Trehalose, recognized for preventing protein aggregation, has been studied in particular for treating neurodegenerative diseases, such as Alzheimer’s and Huntington’s 37 . It also showed some interesting properties in BioLayer Interferometry studies as an additive for reducing non-specific binding 38 . Interestingly, a patent described using 5% trehalose to block functionalized surfaces, reducing serum protein adsorption 39 .…”

Section: Discussionmentioning

confidence: 99%

BMP2 binds non-specifically to PEG-passivated biomaterials and induces substantial signaling

Le Pennec,

Guibert,

Vivès

et al. 2024

Preprint

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Biomaterials are widely employed across diverse biomedical applications and represent an attractive strategy to explore physiologically how extracellular matrix components influence the cellular response. In this study, we aimed to use previously developed biomimetic streptavidin platforms to investigate the role of glycosaminoglycans (GAGs) in bone morphogenetic protein 2 (BMP2) signaling. However, we observed that the interpretation of our findings was skewed due to the GAG-unrelated, non-specific adsorption of BMP2 on components of our biomaterials. Non-specific adsorption of proteins is a recurrent and challenging issue for biomaterial studies. Despite the initial incorporation of anti-fouling poly(ethylene glycol) (PEG) chains within our biomaterials, the residual non-specific BMP2 adsorption still triggered BMP2 signaling within the same range as our conditions of interest. To tackle this issue, we explored various options to prevent BMP2 non-specific adsorption. Specifically, we tested alternative constructions of our biomaterials on gold or glass substrate using distinct PEG-based linkers. We identified the aggregation of BMP2 at neutral pH as a potential cause of non-specific adsorption and thus determined specific buffer conditions to prevent it. We also investigated the induced BMP2 signaling over different culture periods. Nevertheless, none of these options resulted in a viable suitable solution to reduce the non-specific BMP2 signaling. Next, we studied the effect of various blocking strategies. We identified a blocking condition involving a combination of bovine serum albumin and trehalose that successfully reduced the unspecific attachment of BMP2 and the non-specific signaling. Furthermore, the effect of this blocking step was improved when using gold platforms instead of glass, particularly with Chinese hamster ovary (CHO) cells that seemed less responsive to non-specifically bound BMP2 than C2C12 cells.

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“…The N-terminal His 6 and SUMO-tagged NS1 proteins were used for immobilization. The buffer was 20 mM sodium phosphate (pH 7), 150 mM NaCl, 1% bovine serum albumin, and 0.6 M sucrose 56 . All reported values are the average and standard deviation of three repeated measurements.…”

Section: Methodsmentioning

confidence: 99%

Energy landscape reshaped by strain-specific mutations underlies epistasis in NS1 evolution of influenza A virus

et al. 2022

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Elucidating how individual mutations affect the protein energy landscape is crucial for understanding how proteins evolve. However, predicting mutational effects remains challenging because of epistasis—the nonadditive interactions between mutations. Here, we investigate the biophysical mechanism of strain-specific epistasis in the nonstructural protein 1 (NS1) of influenza A viruses (IAVs). We integrate structural, kinetic, thermodynamic, and conformational dynamics analyses of four NS1s of influenza strains that emerged between 1918 and 2004. Although functionally near-neutral, strain-specific NS1 mutations exhibit long-range epistatic interactions with residues at the p85β-binding interface. We reveal that strain-specific mutations reshaped the NS1 energy landscape during evolution. Using NMR spin dynamics, we find that the strain-specific mutations altered the conformational dynamics of the hidden network of tightly packed residues, underlying the evolution of long-range epistasis. This work shows how near-neutral mutations silently alter the biophysical energy landscapes, resulting in diverse background effects during molecular evolution.

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Suppressing Nonspecific Binding in Biolayer Interferometry Experiments for Weak Ligand–Analyte Interactions

Cited by 12 publications

References 34 publications

A Tag‐Free Platform for Synthesis and Screening of Cyclic Peptide Libraries

A Tag‐Free Platform for Synthesis and Screening of Cyclic Peptide Libraries

BMP2 binds non-specifically to PEG-passivated biomaterials and induces substantial signaling

Energy landscape reshaped by strain-specific mutations underlies epistasis in NS1 evolution of influenza A virus

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