Teodori Laura, Omer Marjan, Märcher Anders, Skaanning Mads K, Andersen Veronica L, Nielsen Jesper S, Oldenburg Emil, Lin Yuchen, Gothelf Kurt V, Kjems Jørgen
Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Gustav Wieds Vej 14, 8000 Aarhus C, Denmark.
Center for Cellular Signal Patterns (CellPAT), Aarhus University, Gustav Wieds Vej 14, 8000 Aarhus C, Denmark.
J Biol Methods. 2022 Mar 1;9(1):e159. doi: 10.14440/jbm.2022.381. eCollection 2022.
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--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.
骆驼科单域抗体片段，也称为纳米抗体，是一类大小约为15 kDa的结合物，具有与抗体类似的抗原结合特性。在多种微生物中可简便地生产重组纳米抗体，这使得这类结合物在分子成像领域颇具吸引力。特别是，它们在超分辨率显微镜中的应用，由于连接误差较小，提高了分子靶点的空间分辨率。在单分子定位显微镜技术中，通过纳米抗体的位点特异性荧光标记，由于蛋白质与荧光团的化学计量比更均匀、背景染色减少以及染料与表位之间的已知距离，有效空间分辨率可进一步提高。在此，我们展示了一种将DNA寡核苷酸位点特异性生物偶联到三种用N端或C端非天然氨基酸4-叠氮基-L-苯丙氨酸（pAzF）表达的不同纳米抗体上的方案。使用无铜点击化学，纳米抗体-寡核苷酸偶联反应高效，并产生了高纯度的生物偶联物。生物层干涉术结合分析和超分辨率显微镜技术DNA纳米级形貌成像中的点积累（PAINT）表明，生物偶联物中保留了靶点结合能力。这种位点特异性蛋白质-寡核苷酸偶联方法可进一步扩展到药物递送和分子靶向等需要位点特异性和化学计量控制的应用中。
