Antibody Oligonucleotide Conjugation

AOCs (Antibody−oligonucleotide conjugates) are a new class of chimeric biomolecules synthesized by coupling antibodies with oligonucleotides, combining the specific target recognition ability of antibodies with the functionality of oligonucleotides in the areas of detection, therapeutics, imaging, and materials science, and have become powerful tools in the areas of detection, imaging and drug development. BOC Sciences can easily chimerize antibodies with oligonucleotides through their amino acid residues, making them suitable for most in vitro applications.

Antibody Oligonucleotide Conjugates Development

With years of experience in oligonucleotide conjugation, BOC Sciences offers a full range of AOCs development strategies to help customers develop customized AOCs in a time-saving and cost-effective manner. Strategies currently available include binding to amines, sulfhydryl suffixes and binding to carbohydrates. The choice of coupling includes both non-covalent and covalent approaches, each of which can be selected for different purposes.

Binding to amines

Antibodies contain several amines (NH₂), which can be distributed throughout the antibody as lysine side chain ε-amines and N-terminal α-amines. These residues are often targets for affixation because their spatial accessibility can be easily modified. In addition to amines, glutamate and aspartate residues and carboxylic acids can also be suffixed.

Suffixation with sulfhydryl groups

Antibody-ON affixation can also be achieved with a reactive thiol (thihydrogen group) moiety. Antibodies contain oxidized sulfhydryl groups (-SH) in the form of disulfide bond (SS) bridges, which must first be reduced by a reducing agent to expose their reactive moieties

Binding to carbohydrates

Another method of site-specific affixation can occur on carbohydrate residues, which occur primarily in the Fc region because they are far from the antigen-binding site and are less susceptible to spatial site-blocking. In order to perform the affixation by this complex method, the carbohydrate moiety must be oxidized to an aldehyde (-CHO) using periodate.

Synthesis of Antibody Oligonucleotide Conjugates

Synthesis and modification of AOCs are produced under a strict quality control process, with HPLC and MS analyses performed for each development cycle.

Preparation process:

• The oligomer is modified with NHS ester maleimide-activated amine.
• Excess cross-linker is removed by gel filtration.
• The antibody is reduced, and then conjugated to the maleimide-activated oligonucleotide.

Analysis of Antibody Oligonucleotide Conjugates

In many cases, simple dialysis removes unreacted reagents from the reaction solution. Additional purification, tangential flow filtration (TFF) or gel filtration chromatography can also be used to remove excess reagents or to isolate and characterize cross-linking products. Reagents that are similar or larger in size to antibodies (primarily proteins and other biomolecules) may require additional purification techniques such as affinity chromatography, ion exchange chromatography, and hydrophobic interaction chromatography.

After product purification, many different types of studies may be required, including spectroscopy (MALDI-TOF, ESI, LC-MS fluorescence), electrophoresis, immunochemical biochemistry, and/or enzyme analysis.

Antibody Oligonucleotide Conjugates Applications

Antibody Oligonucleotide Conjugates (AOCs) have been developed for both detection and therapeutic applications. The detection methodologies encompass immuno-PCR (iPCR), proximity ligation assay (PLA), proximity extension assay (PEA), electrochemical proximity assay (ECPA), DNA-PAINT imaging and protein arrays. AOCs facilitate the translation of the detection signals from the proteins to the DNA level. This enables the application of highly sensitive DNA amplification methods to amplify the readout signal. While AOCs initially emerged as a diagnostic tool, they have recently evolved into targeted therapeutic approach for many diseases.

Immuno-PCR

Site-specific AOCs could be used for antigen detection with improved sensitivity and lower nonspecific background when employed in immuno-PCR for antigen detection, compared to conventional methods that rely on lysine conjugation. Historically, this first application for high-sensitivity detection of antigens based on antibody−oligonucleotide chimeras was proposed by Sano et al. in 1992. Streptavidin-protein A fusion protein was first prepared and served as a scaffold for the noncovalent linking of a biotinylated DNA label with an IgG antibody targeting a specific antigen. This linkage utilized the interactions between biotin−streptavidin and antibody−protein A. By amplifying the DNA label of this construct using PCR, followed by gel electrophoresis and staining with ethidium bromide, detection sensitivity reached as low as 580 antigen molecules within a sample. Importantly, compared to the classically used amplification systems based on enzyme-linked immunosorbent assays, the technique demonstrated a remarkable five-order-of-magnitude enhancement in the limit of detection.

Proximity Ligation Assay (PLA)

Proximity ligation assay (PLA) involves conjugating single-stranded oligonucleotides to protein affinity binders, followed by signal amplification through DNA polymerization and hybridization of complementary oligonucleotides labeled with fluorogenic or chromogenic readout. This assay utilizes a pair of AOCs with an affinity against different epitopes on the same protein for detection purposes or targeting different proteins to study protein-protein interactions.

Proximity Extension Assay (PEA)

Proximity extension assay (PEA) is a technology based on a pair of AOC probes. Once the antibodies are labeled with complementary DNA oligonucleotide tags, the oligonucleotides come into close proximity, facilitating hybridization and subsequent extension through standard PCR.

DNA-PAINT imaging

DNA-PAINT (DNA point accumulation in nanoscale topology) is a super-resolution method predicated on the dynamic binding and dissociation of DNA imager strands. It achieves stochastic switching of fluorescence signals between the ON- and OFF-states through the transient binding of short fluorescently labeled oligonucleotides ("imager" strands) to complementary "docking" strands anchored to specific targets. Upon the binding of an imager strand, its fluorescence emission is detected and subsequently localized with nanometer precision. AOCs are used to translate DNA-based multiplexing to other super-resolution techniques, such as STORM (Stochastic Optical Reconstruction Microscopy), STED (Stimulated Emission Depletion) microscopy, and SIM (Structured Illumination Microscopy).

Therapeutics

AOCs serve as therapeutic agents, encapsulating payloads like anti-sense oligonucleotides (ASO) or siRNA, and have been utilized both as pretargeting modules for radionuclide therapy and imaging. By selectively binding to specific cell surface antigens, AOCs facilitate the delivery of oligonucleotides to target cells, resulting in gene regulation, gene silencing, enzyme suppression, etc. AOCs have been recently used in systemic delivery of oligonucleotide-based drugs. AOC 1001, a leading AOC drug candidate designed for treating patients with DM1, has shown positive results from the phase I/II MARINA ® trial. This formulation combines a monoclonal antibody targeting transferrin receptor 1 (TfR1) with an siRNA that modulates DMPK messenger RNA. In preclinical studies, AOC 1001 successfully delivered siRNAs to muscle cells, resulting in durable, dose-dependent reductions of DMPK RNA across a broad range of muscles types, including skeletal, cardiac, and smooth muscles. The U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) have granted Orphan Designation for AOC 1001 and the FDA has granted AOC 1001 Fast Track Designation.

Therapeutic Applications of AOCs (Dovgan, I., et al. 2019)

These days more AOC drug candidates have entered the clinical field such as AOC1020, AOC 1044 and DYNE-101. In the past few years, AOC therapeutic research has developed rapidly for the treatment of rare diseases.

BOC Sciences follows quality control and quality assurance procedures to provide you with a double guarantee of the highest quality of each coupling delivered. In addition, our dedicated Technical Account Manager will guide your project through every step of the process and keep you constantly updated on the progress of your project.

Our Advantages

• Performed by trained professionals
• Evidence of successful conjugation provided
• Conjugate at required antibody–oligo ratio and scale
• Fast turnaround time
• Full technical support
• High levels of antibody and oligo recovery so you save precious reagents

Project Workflow