Bacterially Derived Antibody Binders as Small Adapters for DNA-PAINT Microscopy

Area of Science:

• Biophysics
• Cell Biology
• Microscopy

Background:

• Super-resolution microscopy achieves nanometer-scale resolution.
• Large antibody sizes limit resolution for cellular targets.
• Nanobodies and aptamers offer smaller alternatives but lack availability.

Purpose of the Study:

• To develop a labeling protocol for bacterial protein A and G conjugated to DNA for DNA-PAINT microscopy.
• To assess the intracellular performance of these bacterial binders for super-resolution imaging.
• To quantify improvements in obtainable resolution using these novel binders.

Main Methods:

• Utilized bacterial-derived proteins A and G, commonly used in antibody purification.
• Developed a DNA conjugation protocol for these protein binders.
• Employed DNA-based point accumulation for imaging in nanoscale topography (DNA-PAINT) microscopy.
• Targeted intracellular proteins: tubulin, TOM20, and epidermal growth factor receptor (EGFR).

Main Results:

• Successfully conjugated bacterial proteins A and G with DNA for DNA-PAINT.
• Demonstrated intracellular labeling of tubulin, TOM20, and EGFR.
• Quantified increased resolution achievable with these smaller binders compared to traditional secondary antibodies.

Conclusions:

• Bacterial proteins A and G, when conjugated with DNA, serve as effective and small labeling reagents for DNA-PAINT.
• This approach overcomes limitations posed by large antibody sizes in super-resolution microscopy.
• Offers a widely available and efficient alternative for nanoscale imaging of cellular targets.