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

Laura Teodori, Marjan Omer, Anders Märcher, Mads K Skaanning, Veronica L Andersen, Jesper S Nielsen, Emil Oldenburg, Yuchen Lin, Kurt V Gothelf, Jørgen Kjems*

*Corresponding author for this work

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

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Abstract

Camelid single-domain antibody fragments, also called nanobodies, constitute a class of binders that are small in size (~15 kDa) and possess antigen-binding properties similar to their antibody counterparts. Facile production of recombinant nanobodies in several microorganisms has made this class of binders attractive within the field of molecular imaging. Particularly, their use in super-resolution microscopy has improved the spatial resolution of molecular targets due to a smaller linkage error. In single-molecule localization microscopy techniques, the effective spatial resolution can be further enhanced by site-specific fluorescent labeling of nanobodies owing to a more homogeneous protein-to-fluorophore stoichiometry, reduced background staining and a known distance between dye and epitope. Here, we present a protocol for site-specific bioconjugation of DNA oligonucleotides to three distinct nanobodies expressed with an N- or C-terminal unnatural amino acid, 4-azido-L-phenylalanine (pAzF). Using copper-free click chemistry, the nanobody-oligonucleotide conjugation reactions were efficient and yielded highly pure bioconjugates. Target binding was retained in the bioconjugates, as demonstrated by bio-layer interferometry binding assays and the super-resolution microscopy technique, DNA points accumulation for imaging in nanoscale topography (PAINT). This method for site-specific protein-oligonucleotide conjugation can be further extended for applications within drug delivery and molecular targeting where site-specificity and stoichiometric control are required.

Original languageEnglish
Article numbere159
JournalJournal of Biological Methods
Volume9
Issue1
Number of pages14
ISSN2326-9901
DOIs
Publication statusPublished - Mar 2022

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