Dataset Information

Biological Applications of Synthetic Binders Isolated from a Conceptually New Adhiron Library.

ABSTRACT:

Background

Adhirons are small (10 kDa) synthetic ligands that might represent an alternative to antibody fragments and to alternative scaffolds such as DARPins or affibodies.

Methods

We prepared a conceptionally new adhiron phage display library that allows the presence of cysteines in the hypervariable loops and successfully panned it against antigens possessing different characteristics.

Results

We recovered binders specific for membrane epitopes of plant cells by panning the library directly against pea protoplasts and against soluble C-Reactive Protein and SpyCatcher, a small protein domain for which we failed to isolate binders using pre-immune nanobody libraries. The best binders had a binding constant in the low nM range, were produced easily in bacteria (average yields of 15 mg/L of culture) in combination with different tags, were stable, and had minimal aggregation propensity, independent of the presence or absence of cysteine residues in their loops.

Discussion

The isolated adhirons were significantly stronger than those isolated previously from other libraries and as good as nanobodies recovered from a naïve library of comparable theoretical diversity. Moreover, they proved to be suitable reagents for ELISA, flow cytometry, the western blot, and also as capture elements in electrochemical biosensors.

SUBMITTER: D'Ercole C

PROVIDER: S-EPMC10605594 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature

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Publications

Biological Applications of Synthetic Binders Isolated from a Conceptually New Adhiron Library.

D'Ercole Claudia C De March Matteo M Veggiani Gianluca G Oloketuyi Sandra S Svigelj Rossella R de Marco Ario A

Biomolecules 20231017 10

<h4>Background</h4>Adhirons are small (10 kDa) synthetic ligands that might represent an alternative to antibody fragments and to alternative scaffolds such as DARPins or affibodies.<h4>Methods</h4>We prepared a conceptionally new adhiron phage display library that allows the presence of cysteines in the hypervariable loops and successfully panned it against antigens possessing different characteristics.<h4>Results</h4>We recovered binders specific for membrane epitopes of plant cells by panning ...[more]

PMID: 37892215

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Project description:Bisbenzimides, or Hoechst 33258 (H258), and its derivative Hoechst 33342 (H342) are archetypal molecules for designing minor groove binders, and widely used as tools for staining DNA and analyzing side population cells. They are supravital DNA minor groove binders with AT selectivity. H342 and H258 share similar biological effects based on the similarity of their chemical structures, but also have their unique biological effects. For example, H342, but not H258, is a potent apoptotic inducer and both H342 and H258 can induce transgene overexpression in in vitro studies. However, the molecular mechanisms by which Hoechst dyes induce apoptosis and enhance transgene overexpression are unclear.To determine the molecular mechanisms underlying different biological effects between H342 and H258, microarray technique coupled with bioinformatics analyses and multiple other techniques has been utilized to detect differential global gene expression profiles, Hoechst dye-specific gene expression signatures, and changes in cell morphology and levels of apoptosis-associated proteins in malignant mesothelioma cells. H342-induced apoptosis occurs in a dose-dependent fashion and is associated with morphological changes, caspase-3 activation, cytochrome c mitochondrial translocation, and cleavage of apoptosis-associated proteins. The antagonistic effect of H258 on H342-induced apoptosis indicates a pharmacokinetic basis for the two dyes' different biological effects. Differential global gene expression profiles induced by H258 and H342 are accompanied by unique gene expression signatures determined by DNA microarray and bioinformatics software, indicating a genetic basis for their different biological effects.A unique gene expression signature associated with H342-induced apoptosis provides a new avenue to predict and classify the therapeutic class of minor groove binders in the drug development process. Further analysis of H258-upregulated genes of transcription regulation may identify the genes that enhance transgene overexpression in gene therapy and promote recombinant protein products in biopharmaceutical companies.The microarray data reported in this article have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no.GSE28616).

Dataset Information

Biological Applications of Synthetic Binders Isolated from a Conceptually New Adhiron Library.

Background

Methods

Results

Discussion

Publications

Biological Applications of Synthetic Binders Isolated from a Conceptually New Adhiron Library.

Similar Datasets

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