Site-Specific or Random Chemistry — Who Will Win?
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 distill 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 maximize 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 hybridizes 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 labeling 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-labeling 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. Minimizes protein damage relative to Cu-catalyzed 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-labeled | 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 minimize 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 hybridization; 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.