Site-Specific or Random Chemistry — Who Will Win?

30 September 2025 by

Aboligo

Introduction

At AbOliGo, many customers ask about antibody–oligo conjugates made with random versus site-specific methods. To help, we’re publishing a series of short articles that distil key takeaways from the latest scientific papers.

Scientists want reliable Antibody oligo conjugates (AOCs) which maintain antibody affinity. This short guide compares core linking chemistries for AOCs, and signal removal methods for cyclic immunofluorescence (cyCIF), demonstrating how antibody-oligo conjugates (DNA barcoded antibodies) with Docking strands (DS)/Imaging strands (IS) behave in practice, and shares QC tips to maximise antibody binding affinity and antigenicity preservation.

AOCs and cyclic immunofluorescence

In an AOC cyclic immunofluorescence workflow, the antibody carries a short oligo Docking strand (DS). During imaging, a complementary oligo imaging strand (IS) bearing the fluorophore hybridises to the DS. Because the label is DNA-based, you can remove signal gently and repeat, enabling multiplexed proteomics imaging with cyclic immunofluorescence (cyCIF). This DS/IS modularity is the core reagent: you are able to separate antibody binding from the optical readout, which reduces harsh stripping steps and preserves epitopes. The chemistry you choose to attach the oligo to the antibody, and the way you remove IS after each round determines background, carryover, and data quality. Below, we compare conjugation chemistries then outline the pros/cons of the main signal removal methods.

Conjugation chemistries

Site specific conjugation: site click conjugation (Fc glycan engineering).

Site click conjugates azides on the Fc glycan, keeping the Fab untouched. Coupling your DS via Cu-free click chemistry (DBCO) at the Fc typically yields excellent antibody binding affinity retention and consistent labelling stoichiometry. Site click conjugation is useful for sensitive clones and low-abundance targets.

Site specific conjugation (cysteines) : Maleimide chemistry.

Targets reduced cysteines. Useful if interchain disulfide reduction can be tightly controlled, but partial reduction may compromise antibody structure. Good blocking, rapid quenching, and gentle handling are key to retaining antibody function.

Random conjugation : NHS ester chemistry (lysines).

Simple and accessible, but lysines are abundant and some are near the Fab binding domain. Over-labelling risks affinity loss and heterogeneous products. If you use NHS esters, control of pH, time, and molar ratios is key. It is important to consider lower DS excess and post-conjugation clean-up to limit nuclear background from free DS.

Random or site specific conjugation : DBCO (Cu-free click) coupling.

Fast, bioorthogonal attachment of azide-bearing partners without copper, ideal downstream of site click or other azide-installation strategies. Minimises protein damage relative to Cu-catalysed chemistry.

Conjugation Method	Antibody target site	Conjugation binding	AOC Impact	Ease/Scale	Notes
Site click + DBCO	Fc glycan	Site specific	High retention of affinity; good batch consistency	Medium	Best for sensitive clones/lowly expressed targets
Maleimide chemistry	Cysteines (reduced)	Site Specific	Good if reduction controlled	Medium	Watch structural integrity
NHS ester chemistry	Lysines	Random	Variable; risk to affinity if over-labelled	Easy	Tight control of pH/ratios essential
DBCO coupling (general)	Azide handles via NHS ester	Random	Gentle, clean, fast	Easy

Removal of the imaging strand (IS) between Immunofluorescence cycles while protecting tissue and epitopes.

During the cyclic immunofluorescence process there are several methods which may be adopted to remove the imaging strand (IS) without compromising tissue integrity. The authors decided to use only site-specific, site click AOC conjugates which installs azides on Fc glycans, keeping labels away from the antibody binding domain, theoretically yielding higher affinity binding.

Several approaches were investigated to identify which worked optimally to displace the IS from the DS:

Strand-mediated displacement (SMD): A “capture” oligo with a sequence similar to the DS out-competes the IS.

Pros: DNA-native, non-destructive.

Cons: Adds a third oligo and time; sequence design and kinetics matter.

Restriction enzyme (RE) cleavage: DS/IS pair encodes an RE site; enzyme cuts the fluorophore-bearing IS.

Pros: Specific mechanism.

Cons: Incomplete removal for some intracellular/nuclear targets; enzyme cost/time.

Thermal denaturation: Heat slightly above duplex Tm to melt DS/IS.

Pros: Simple, inexpensive.

Cons: Repeated heating can raise background and risk partial antibody

loss; shorter IS lowers Tm but can reduce on-target signal.

TCEP disulfide cleavage: IS carries a cleavable disulfide near the dye; TCEP releases the fluorophore.

Pros: Fast, non-destructive under mild conditions.

Cons: Extra chemistry steps can elevate background if timing/quenches aren’t tight.

Photocleavable linker (PCL): IS dye attached via PCL; UV exposure cleaves and washes off.

Pros: Rapid, clean removal with strong antigenicity preservation; supports full-length IS for brighter signal.

Cons: Requires UV control and PCL-modified IS.

Key point: For robust cyCIF across many cycles and targets, Photocleavable linker (PCL) IS often delivers the lowest carryover with excellent epitope protection.

Hints and tips

Before/after affinity checks. Benchmark each conjugate against the unconjugated antibody: Monitor antibody binding affinity retention across cycles.

Spectrophotometry caveat. Oligos dominate at 260 nm; 260/280 alone misleads. Cross-validate concentration/DoL and, where possible, use additional QC (e.g., SDS-PAGE with protein and DNA stains; optional mass photometry).

Blocking & purification. To minimise nuclear haze from free DS: block with sheared salmon sperm DNA, dextran sulfate, and saline sodium citrate (SSC).

IS length vs temperature. Shorter IS reduce thermal denaturation temperature but can weaken hybridisation; pick lengths that balance brightness with gentle removal.

Summary

The authors preferred choice for optimizing cyclic immunofluorescence was AOCs conjugated using the site-specific method. Preference does vary from assay to assay, so it appears one approach does not fit all, which will be discussed in future articles.

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Reference

Oligonucleotide conjugated antibody strategies for cyclic immunostaining

in Knowledge Hub