Site-specific nanobody-oligonucleotide conjugation for super-resolution imaging

Area of Science:

• Bioconjugation Chemistry
• Molecular Imaging
• Nanotechnology

Background:

• Camelid single-domain antibody fragments (nanobodies) are small, potent binders useful in molecular imaging.
• Nanobodies enhance super-resolution microscopy resolution due to their small size and reduced linkage error.
• Site-specific labeling of nanobodies can further improve spatial resolution, stoichiometry, and reduce background staining.

Purpose of the Study:

• To present a protocol for site-specific bioconjugation of DNA oligonucleotides to nanobodies.
• To demonstrate the efficiency and purity of the resulting nanobody-oligonucleotide bioconjugates.
• To validate the retention of target binding and utility in super-resolution microscopy.

Main Methods:

• Expression of three distinct nanobodies with an N- or C-terminal unnatural amino acid, 4-azido-L-phenylalanine (pAzF).
• Copper-free click chemistry for efficient site-specific conjugation of DNA oligonucleotides to nanobodies.
• Bio-layer interferometry and DNA points accumulation for imaging in nanoscale topography (PAINT) for validation.

Main Results:

• Efficient and high-purity nanobody-oligonucleotide bioconjugates were successfully synthesized.
• Site-specific conjugation via unnatural amino acids and click chemistry was achieved.
• Target binding affinity was retained, and super-resolution microscopy demonstrated enhanced imaging capabilities.

Conclusions:

• A robust protocol for site-specific protein-oligonucleotide conjugation using nanobodies was established.
• This method offers improved stoichiometric control and specificity for molecular imaging.
• The conjugation strategy holds potential for applications in drug delivery and molecular targeting.