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.
Oligonucleotides are increasingly being used as a programmable connection material to assemble molecules and proteins in well‐defined structures. For the application of such assemblies for in vivo diagnostics or therapeutics it is crucial that the oligonucleotides form highly stable, non‐toxic, and non‐immunogenic structures. Only few oligonucleotide derivatives fulfil all of these requirements. Here we report on the application of acyclic l‐threoninol nucleic acid (aTNA) to form a four‐way junction (4WJ) that is highly stable and enables facile assembly of components for in vivo treatment and imaging. The aTNA 4WJ is serum‐stable, shows no non‐targeted uptake or cytotoxicity, and invokes no innate immune response. As a proof of concept, we modify the 4WJ with a cancer‐targeting and a serum half‐life extension moiety and show the effect of these functionalized 4WJs in vitro and in vivo, respectively.
The question of how nucleosides might have formed as essential precursor molecules on the early Earth is one of the many challenges associated with the origin of life. In this context, the prebiotic synthesis of pyrimidine nucleosides is controversially discussed. For the pyrimidines, two at first glance contradictory prebiotically plausible reaction pathways have been proposed, based on either oxazole or isoxazole chemistry. This study shows that these two reaction sequences can be merged under prebiotically reasonable conditions, suggesting that both pathways could have co-existed and possibly interacted. The key precursor 3-aminoisoxazole was found to react with the key intermediate of the oxazole route (ribo-2-(methylthio)oxazoline), to form a ribo-isoxazole-oxazoline hybrid structure, which collapses upon reductive NÀ O bond cleavage to give the nucleoside cytidine. The data suggest that different, interacting prebiotically plausible chemical pathways may have created the key molecules of life on the early Earth.
Oligonucleotides are increasingly being used as a programmable connection material to assemble molecules and proteins in well‐defined structures. For the application of such assemblies for in vivo diagnostics or therapeutics it is crucial that the oligonucleotides form highly stable, non‐toxic, and non‐immunogenic structures. Only few oligonucleotide derivatives fulfil all of these requirements. Here we report on the application of acyclic l‐threoninol nucleic acid (aTNA) to form a four‐way junction (4WJ) that is highly stable and enables facile assembly of components for in vivo treatment and imaging. The aTNA 4WJ is serum‐stable, shows no non‐targeted uptake or cytotoxicity, and invokes no innate immune response. As a proof of concept, we modify the 4WJ with a cancer‐targeting and a serum half‐life extension moiety and show the effect of these functionalized 4WJs in vitro and in vivo, respectively.
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