Oligonucleotide-Antibody Conjugation Service

Introduction

Our Oligonucleotide-Antibody Conjugation service accelerates drug discovery with high-quality bioconjugates via advanced chemical modification and purification. Combining antibodies' precise targeting with oligonucleotides' diverse functions, we offer solutions for specific drug delivery, enhanced diagnostic sensitivity, and robust research tools.

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Oligonucleotide-antibody Conjugation

OAC is a type of biopharmaceutical formed by chemically conjugating oligonucleotides (such as siRNA, miRNA, ASO, or aptamer) with antibodies or their fragments. It combines the targeting specificity of antibodies with the gene regulatory function of oligonucleotides and represents an important advancement in the field of precision medicine.

Fig.1 Antibody fragments in different forms and their derivatives.¹

Classification

According to the types and mechanisms of action of oligonucleotides, OACs are mainly classified as:

• siRNA/ miRNA-antibody conjugate: Guide RNA induces the silencing complex (RISC) to cleave the target mRNA;
• Antisense oligonucleotides (ASO)-antibody conjugate: Inhibits mRNA translation or regulates splicing
• Aptamer-antibody conjugate: Blocks protein activity;
• Immune-stimulating oligonucleotide-antibody conjugate: Activate innate immune signaling pathways.

Characteristics

• Dual targeting: Antibody-mediated cell-specific delivery and gene-level regulation of oligonucleotides.
• Enhance stability: Antibodies protect oligonucleotides from degradation by nucleases and extend their half-life.
• Reduce off-target effects: Antibody-oriented reduction of non-target tissue exposure enhances safety.
• Multi-functional collaboration: It can be designed to simultaneously block protein function (antibodies) and gene expression (oligonucleotides).
• Precise and controllable release: Local activation of the lesion is achieved through cleavable linkers (such as pH-sensitive and enzyme-sensitive linkers).

Mechanism

• Receptor-mediated endocytosis: Antibodies bind to the surface antigens of target cells and enter the cells through endocytosis, releasing oligonucleotides.
• Lysosome escape: The conjugate needs to escape from endosomes/lysosomes to avoid degradation and enter the cytoplasm (such as through membrane fusion or perforated peptide assistance).
• The bystander effect: Some oligonucleotides can diffuse from the initial cell to adjacent cells, expanding their range of action.

Workflow

1. Synthesis

   Oligonucleotide-antibody conjugate synthesis involves precise chemical linking, with attention to conjugation chemistry, linker design, and reaction conditions to ensure high yield, homogeneity, and preserved biological activity of both components. Key methods include:

   • Ionic Interactions: Uses oligonucleotide's negative charge with antibody-fused/conjugated positive moieties. Simple, flexible but reversible (risk of in vivo instability); polycationic complexes may aid lysosomal escape.
   • Avidin-Based Conjugation: Relies on strong biotin-avidin binding, conjugating biotinylated oligonucleotides to avidin-modified antibodies. High stability but requires chemical modification of both components, less general than direct methods.
   • Direct Conjugation: Versatile, preferred method (similar to ADC synthesis). Links chemically modified oligonucleotides to reactive antibody sites.
     • Click Chemistry: Reacts azide-modified oligonucleotides with DBCO-modified antibodies (or vice versa). Efficient, biocompatible, widely used.
     • Maleimide-Thiol Conjugation: Thiol-modified oligonucleotides react with maleimide-activated antibodies, enabling precise oligonucleotide-to-antibody ratio (OAR) control.
     • Enzymatic Conjugation: Uses enzymes (e.g., transglutaminase) for site-specific linking, yielding highly homogeneous conjugates.
     • Linker Design: Critical choice of cleavable/non-cleavable linkers impacts oligonucleotide release, in vivo activity, and stability.
   • DNA Origami or Hybridization Conjugation: This technique conjugates a single-strand oligonucleotide to an antibody, then hybridizes with a complementary strand. It builds complex nanostructures for carrying multiple therapeutics or as multiplexed diagnostic probes.

2. Purification

   Rigorous purification is critical for high-quality AOCs, given conjugation heterogeneity and unreacted materials. Oligonucleotides' distinct properties (larger size, high negative charge) vs. ADC small molecules require specialized purification strategies.

   • Ion-Exchange Chromatography (IEX)
   • Dialysis and Ultrafiltration
   • Size-Exclusion Chromatography (SEC)

3. Quality Control

   • OAR Determination:
     • Mass Spectrometry (MS)
     • UV-Vis Spectroscopy
     • SDS-PAGE and IEX
     • PCR-based methods
   • Purity Assessment:
     • SDS-PAGE (reducing and non-reducing)
     • IEX and SEC
   • Functional Characterization:
     • Binding Affinity (ELISA or binding assays)
     • Cellular Internalization and Trafficking (confocal microscopy)
     • Gene Silencing Activity (qPCR, Western blot, luciferase reporter assays)
     • Cytotoxicity (in vitro cytotoxicity assays)
   • Stability Studies:
     • Serum Stability
     • Storage Stability

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References

1. Jin, Shijie, et al. "Emerging new therapeutic antibody derivatives for cancer treatment." Signal transduction and targeted therapy 7.1 (2022): 39. DOI: 10.1038/s41392-021-00868-x. Distributed under Open Access license CC BY 4.0, without modification.
2. Wiener, Julius, et al. "Preparation of single-and double-oligonucleotide antibody conjugates and their application for protein analytics." Scientific reports 10.1 (2020): 1457. DOI: 10.1038/s41598-020-58238-6. Distributed under Open Access license CC BY 4.0, the figures were cropped.