Stoichiometrically Defined Antibody-DNA Conjugates for Quantitative Super-Resolution Imaging

Area of Science:

• Biophysics
• Molecular Imaging
• Bioconjugation Chemistry

Background:

• DNA-PAINT super-resolution microscopy allows precise protein copy number determination via qPAINT.
• Current methods using amine-reactive labeling create heterogeneous antibody-DNA conjugates, limiting quantitative accuracy.
• Stoichiometric control of DNA docking strands on antibodies is crucial for reliable qPAINT analysis.

Purpose of the Study:

• To develop a site-selective conjugation method for creating stoichiometrically defined antibody-DNA conjugates.
• To evaluate the impact of controlled DNA loading on qPAINT accuracy.
• To enable reliable quantitative super-resolution imaging in complex biological samples.

Main Methods:

• Site-selective disulfide rebridging using pyridazinedione chemistry for antibody conjugation.
• Generation of antibody-DNA conjugates with defined stoichiometry: one (OAR1) or four (OAR4) DNA strands per antibody.
• Application of OAR1 and OAR4 probes to nuclear pore complexes for qPAINT analysis.

Main Results:

• OAR1 and OAR4 probes produced narrow qPAINT index distributions, indicating stoichiometric control.
• Quantitative DNA-PAINT analysis with defined conjugates showed accurate protein copy number dependence.
• Site-selective conjugation enabled discrimination between singly and doubly labeled Nup96 dimers.
• Conventional lysine-based conjugation resulted in broadened distributions and overestimation of copy numbers.

Conclusions:

• Site-selective antibody-DNA conjugation is essential for accurate quantitative DNA-PAINT.
• Pyridazinedione chemistry provides a robust method for generating stoichiometrically defined antibody-DNA conjugates.
• This approach enhances the reliability of super-resolution microscopy for quantitative biological studies.