Antibody–oligonucleotide conjugates have emerged as powerful tools for highly multiplexed and amplified protein detection in imaging and sequencing workflows. Yet when single‑stranded DNA (ssDNA) is conjugated to antibodies for use in assays such as SABER, or immuno‑PCR, non‑specific background, especially nuclear staining, remains a persistent obstacle. A recent study (Wang and colleagues) systematically dissects this issue and presents an optimized protocol that dramatically improves specificity.
The Problem: Why Non‑Specific Binding Occurs
- ssDNA hybridization: Conjugated DNA can hybridize to intracellular nucleic acids or DNA‑binding proteins.
- Electrostatic interactions: Negatively charged ssDNA binds positively charged cellular proteins (like histones).
The team observed massive nuclear background when using antibody-oligo conjugated primary antibodies (e.g., anti‑β‑tubulin E7 with HCR initiator DNA), even when amplified by conventional fluorophore‑secondary antibodies.
The Approach: Optimizing the Staining Buffer
Based on existing literature and their own investigations, the authors developed a protocol to eliminate the non-specific binding:
- Pre‑hybridizing the antibody’s ssDNA to a short complementary DNA to form double‑stranded DNA, blocking hybridization.
- Incorporating dextran sulfate (0.02–0.1%) as a polyanion to out compete conjugated DNA for electrostatic binding.
- Raising ionic strength with 150 mM NaCl to shield electrostatic forces.
- Optional additives: low‑level salmon‑sperm DNA or poly‑TTG sequences, plus standard Triton X‑100, BSA, and normal IgG.
Method.
To optimize the protocol with the aim to minimize background staining, the team employed protein imaging using antibody-oligo conjugates with extended hybridization chain reactions (HCR):
For protein imaging, primary antibodies are tagged with unique DNA oligo sequences to label multiple targets simultaneously (diagram above, figure A). Fluorophore-labeled HCR hairpins (figure B) through opening of the DNA hairpins binds to the complimentary antibody-oligo conjugate (figure C). The hairpins stack (figure C) together resulting in signal amplification and are visualized using fluorescence microscopy.
Results: Cleaner Signal Without Compromising Binding
Comparing protocols across multiple antibodies, including anti‑α and β‑tubulin, the new buffer (with complementary DNA + dextran sulfate) consistently:
- Abolished nuclear background, while
- Preserving strong specific staining of microtubules and other cellular targets.
Key findings included:
- Complementary DNA works much better than salmon‑sperm DNA alone.
- Dextran sulfate remains effective even at low concentrations; higher amounts can reduce antibody affinity.
- Higher salt helps background suppression.
- The improved protocol worked successfully across cultured cells and FFPE tissue.
Protocol Summary
Blocking buffer:
1–3% BSA + 0.1 mg/mL normal IgG + 0.1% Triton X‑100 (in PBS). Optionally 1 μM poly(TTG) or 0.2 mg/mL salmon sperm DNA.
Antibody incubation buffer:
– pre‑hybridized complementary DNA,
– 0.02–0.1% dextran sulfate,
– 150 mM NaCl,
– 5 mM EDTA.
This protocol addresses a general problem: non‑specific interactions by conjugated ssDNA. While the study focuses on HCR‑based imaging, the principles apply to any Ab‑Oligo assays, for instance SABER. Additionally, the authors optimized oligo labeling to ~1–3 DNA molecules per antibody to balance signal and minimize disruption, avoiding over-labeling which potentially impairs binding.
Take‑Home Points
- Convert ssDNA → dsDNA by hybridizing complementary strands.
- Mask electrostatic binding using low‑percent dextran sulfate.
- Boost ionic shielding with moderate salt.
- Fine‑tune optional salmon sperm DNA and oligo‑to‑antibody ratios.
By integrating these simple yet effective steps into Ab‑Oligo workflows, researchers can significantly reduce non‑specific background—yielding cleaner, more reliable imaging and detection without sacrificing multiplexing or amplification capacity.
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