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. 2024 Aug 9;25(16):8681.
doi: 10.3390/ijms25168681.

Virtual Screening and Validation of Affinity DNA Functional Ligands for IgG Fc Segment

Qianyu Yang  1 , Zhiwei Liu  1 , Xinrui Xu  1 , Jiang Wang  1 , Bin Du  1 , Pengjie Zhang  1 , Bing Liu  1 , Xihui Mu  1 , Zhaoyang Tong  1
Affiliations

Virtual Screening and Validation of Affinity DNA Functional Ligands for IgG Fc Segment

Qianyu Yang et al. Int J Mol Sci. .

Abstract

The effective attachment of antibodies to the immune sensing interface is a crucial factor that determines the detection performance of immunosensors. Therefore, this study aims to investigate a novel antibody immobilization material with low molecular weight, high stability, and excellent directional immobilization effect. In this study, we employed molecular docking technology based on the ZDOCK algorithm to virtually screen DNA functional ligands (DNAFL) for the Fc segment of antibodies. Through a comprehensive analysis of the key binding sites and contact propensities at the interface between DNAFL and IgG antibody, we have gained valuable insights into the affinity relationship, as well as the principles governing amino acid and nucleotide interactions at this interface. Furthermore, molecular affinity experiments and competitive binding experiments were conducted to validate both the binding ability of DNAFL to IgG antibody and its actual binding site. Through affinity experiments using multi-base sequences, we identified bases that significantly influence antibody-DNAFL binding and successfully obtained DNAFL with an enhanced affinity towards the IgG Fc segment. These findings provide a theoretical foundation for the targeted design of higher-affinity DNAFLs while also presenting a new technical approach for immunosensor preparation with potential applications in biodetection.

Keywords: DNA functional ligand; Fc segment; affinity; molecular docking; virtual screening.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(A) 3D structure of mouse IgG (PDB code: 1IGT) with the light chain A and C colored in pink and orange while the heavy chain B and D is represented in blue and green (left), and the cavity in the middle of the Fc segment, as well as the oligosaccharide chain E and F within it can be observed by rotating the left figure 90° outward from the right (right); (B) three binding modes between DNAFL and IgG and their possible binding at the biosensing interface while DNAFLs were linked to the sensor chip using an biotin-avidin system.
Figure 2
Figure 2
Process and outcomes of molecular docking for (A) distribution map of 2000 possible poses (left) and pose1 with the highest score (right) while the intensity of the red dot corresponds to a higher pose score, while the blue color indicates a lower score; (B) frontal view (left) and right-side view (right) of DNAFL binding sites in the Fc segment about single-stranded nucleotide from protein complex whose PDB code is 3HXQ.
Figure 3
Figure 3
(A) Binding frequencies of amino acid sites in the Fc segment (the Fc segment of Chain B and D spans approximately from amino acids 230 to 444(Lys242-Arg474)); (B) binding frequency of monosaccharide sites in the Fc segment (Chain E and F both include 9 monosaccharides with the first eight components being identical while the 9th of E and F is furanose and fructose, respectively); (C) distribution of high-frequency amino acid sites in Fc segment (left) and position of Glu423 on chain B and D, respectively (right).
Figure 4
Figure 4
Distribution of binding sites before and after the mutation.
Figure 5
Figure 5
Contact propensity of (A) 20 amino acids and (B) 6 monosaccharides to bases, respectively, and the dashed line represents the scenario where the Pij = 1.
Figure 6
Figure 6
Association−dissociation curve of (A) DNAFL (derived from an oligonucleotide strand in a protein compound whose PDB code is 6GN7 as an example) binding to mouse IgG, and (B) signal response of DNAFL to SPA in competitive binding assay.
Figure 7
Figure 7
Association-dissociation curve of poly-A20, poly-C20, poly-G20 and poly-T20.
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