Dataset Information

Side-by-side comparison of G-quadruplex (G4) capture efficiency of the antibody BG4 versus the small-molecule ligands TASQs.

ABSTRACT: The search for G-quadruplex (G4)-forming sequences across the genome is motivated by their involvement in key cellular processes and their putative roles in dysregulations underlying human genetic diseases. Sequencing-based methods have been developed to assess the prevalence of DNA G4s genome wide, including G4-seq to detect G4s in purified DNA (in vitro) using the G4 stabilizer PDS, and G4 chromatin immunoprecipitation sequencing (G4 ChIP-seq) to detect G4s in in situ fixed chromatin (in vivo) using the G4-specific antibody BG4. We recently reported on G4-RNA precipitation and sequencing (G4RP-seq) to assess the in vivo prevalence of RNA G4 landscapes transcriptome wide using the small molecule BioTASQ. Here, we apply this technique for mapping DNA G4s in plants (rice) and compare the efficiency of this new technique, G4-DNA precipitation and sequencing, G4DP-seq, to that of BG4-DNA-IP-seq that we developed for mapping of DNA G4s in rice using BG4. By doing so, we compare the G4 capture ability of small-sized ligands (BioTASQ and BioCyTASQ) versus the antibody BG4.

SUBMITTER: Feng Y

PROVIDER: S-EPMC10212998 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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Side-by-side comparison of G-quadruplex (G4) capture efficiency of the antibody BG4 versus the small-molecule ligands TASQs.

Feng Yilong Y He Zexue Z Luo Zhenyu Z Sperti Francesco Rota FR Valverde Ibai E IE Zhang Wenli W Monchaud David D

iScience 20230506 6

The search for G-quadruplex (G4)-forming sequences across the genome is motivated by their involvement in key cellular processes and their putative roles in dysregulations underlying human genetic diseases. Sequencing-based methods have been developed to assess the prevalence of DNA G4s genome wide, including G4-seq to detect G4s in purified DNA (<i>in vitro</i>) using the G4 stabilizer PDS, and G4 chromatin immunoprecipitation sequencing (G4 ChIP-seq) to detect G4s in <i>in situ</i> fixed chrom ...[more]

PMID: 37250775

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Project description:Fc γ receptors (FcγRs) are one of the structures that can initiate effector function for monoclonal antibodies. FcγRIa has the highest affinity toward IgG1-type monoclonal antibodies among all FcγRs. In this study, a comprehensive characterization was performed for FcγRIa as a potential affinity ligand for IgG1-type monoclonal antibody binding. The binding interactions were assessed with the SPR technique using different immobilization techniques such as EDC-NHS coupling, streptavidin-biotin interaction, and His-tagged FcγRIa capture. The His-tagged FcγRIa capture was the most convenient method based on assay repeatability. Next, a crude IgG1 sample and its fractions with different monomer contents obtained from protein A affinity chromatography were used to evaluate FcγRIa protein in terms of monoclonal antibody binding capacity. The samples were also compared with a protein A-immobilized chip (a frequently used affinity ligand) for IgG1 binding responses. The antibody binding capacity of the protein A-immobilized chip surface was significantly better than that of the FcγRIa-immobilized chip surface due to its 5 Ig binding domains. The antibody binding responses changed similarly with protein A depending on the monomer content of the sample. Finally, a different configuration was used to assess the binding affinity of free FcγRs (FcγRIa, FcγRIIa, and FcγRIIIa) to three different immobilized IgGs by immobilizing protein L to the chip surface. Unlike previous immobilization techniques tested where the FcγRIa was utilized as a ligand, nonimmobilized or free FcγRIa resulted in a significantly higher antibody binding response than free protein A. In this configuration, kinetics data of FcγRI revealed that the association rate (ka 50-80 × 105 M-1 s-1) increased in comparison to His capture method (1.9-2.4 × 105 M-1 s-1). In addition, the dissociation rate (kd 10-5 s-1) seemed slower over the His capture method (10-4 s-1) and provided stability on the chip surface during the dissociation phase. The KD values for FcγRIa were found in the picomolar range (2.1-10.33 pM from steady-state affinity analysis and 37.5-46.2 pM from kinetic analysis) for IgG1-type antibodies. FcγRIa possesses comparable ligand potential as well as protein A. Even though the protein A-immobilized surface bound more antibodies than the FcγRIa-captured surface, FcγRIa presented a significant antibody binding capacity in protein L configuration. The results suggest FcγRIa protein as a potential ligand for site-oriented immobilization of IgG1-type monoclonal antibodies, and it needs further performance investigation on different surfaces and interfaces for applications such as sensing and antibody purification.

Dataset Information

Side-by-side comparison of G-quadruplex (G4) capture efficiency of the antibody BG4 versus the small-molecule ligands TASQs.

Publications

Side-by-side comparison of G-quadruplex (G4) capture efficiency of the antibody BG4 versus the small-molecule ligands TASQs.

Similar Datasets

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