Brown, D. is usually a simple and practical reagent that enables preparation of a range of diverse sitespecific, homogenous DNAprotein/antibody bioconjugates under mild conditions. The generated constructs can be conveniently characterized by an optimized LCMS method and utilized as probes for cell imaging using fluorescence or superresolution DNAPAINT microscopy. == Introduction == Crosslinking of various types of molecules leading to chimeric constructs with combined functionalities has been gaining attention across the fields of chemical biology, biotechnology, and medicine.[1]Among these, proteins are one of the Nalfurafine hydrochloride most important and valuable class of biomolecules owing to the wide range of functions they fulfil in living organisms. Numerous studies have been undertaken to extend the diversity of protein functionalities by introducing nonnatural labels or tags in the emerging field of chemical protein modification.[1a]Besides proteins, nucleic acids (NAs) are also an extensively studied class of biomolecules. It is mainly the highly specific and predictable base pairing that makes DNA or RNA oligonucleotides (ONs) unique tools for the design of biomolecular hybrids with new structures and improved functions. ONs have been applied in the design of DNAmicroarrays,[2]other nanostructures,[3]imaging[4]and as therapeutic agents.[5]By joining proteins and NAs in a single biomolecular scaffold, NAprotein hybrids can combine the programmable sequence recognition properties of NAs and diverse functionalities of proteins. Two major research areas that benefit from the dual functionality of NAprotein bioconjugate constructs are bioanalysis and nanofabrication.[6]In addition, conjugation of ONs to antibodies has been used for the antibodymediated delivery of therapeutic ONs,[7]cytotoxic agents through JAG1 intercalation[8]or hybridization of complementary DNA ONs,[9]decoration of DNA nanostructures with multiple proteins[10]and fluorophores[11]and DNAantibody bioconjugates have been used as probes for superresolution imaging by means of the DNAPAINT method.[12] However, for constructs like DNAantibody conjugates to become viable for bioanalytical and biological applications, efficient methods for their production are needed. Many different chemical approaches are available to prepare these bioconjugates.[13]The choice of conjugation method usually depends on the scaffold of the protein of interest, and the main challenges are scalability, control over efficacy, sitespecificity and stoichiometry of the NA bioconjugation. Strategies for the preparation of covalent Nalfurafine hydrochloride DNAantibody conjugates can be divided into two main categories: 1) approaches based on proteins modified with functional groups suitable for biorthogonal reactions, such as strainpromoted azidealkyne cycloaddition[14]or inverse electrondemand DielsAlder reactions[14c,15] (Physique1 a) or 2) direct conjugation of altered ONs to native proteins by using bifunctional crosslinkers (Physique1 b).[16]Whereas the former requires either genetic manipulation of the protein of interest or additional actions to introduce the functional groups for the biorthogonal ligation, the latter crosslinks the reactive amino acid residues around the protein surface with modified ONs directly. However, proteins usually contain multiple reactive residues, which often results in nonspecific labelling at sites important for protein activity and/or binding. To avoid formation of inactive conjugates, sitespecific bioconjugation of NAs to proteins with control over modification site is required; often attempted by using bifunctional crosslinkers. Functionalized homo or heterobifunctional linkers usually contain reactive activated esters for amine modification or Michael acceptor moieties for conjugation of thiol bearing molecules (Physique1 Nalfurafine hydrochloride c).Nhydroxysuccinimide (NHS) ester and maleimide moieties are some of the most frequently used scaffolds but various other crosslinkers are available.[16]Although popular, bifunctional linkers based on NHS and maleimide functionalities have some drawbacks. Maleimide is usually widely used for reactions with thiolcontaining molecules but the selectivity of maleimide towards thiols highly depends on the reaction pH. At neutral pH, thiols react with maleimide 1000times faster than amines but at higher pH reaction with amine is usually favored.[17]This nonspecific labelling can lead to mixtures of heterogenous conjugates with various characteristics. Furthermore, at more alkaline pH, hydrolysis of both the NHS ester and maleimide occurs, which results in an unreactive carboxylic acid and maleamic acid, respectively.[16]Another disadvantage of the maleimide reagents is the limited stability of the formed thioether bond, which can undergo a rapid thiolexchange reaction.[17,18] To avoid some of the downsides of these reagents, improved conjugation techniques that give sitespecific, homogenous and welldefined DNAantibody conjugates are desired. In this work, we report a strategy that leads to chemically defined constructs by Nalfurafine hydrochloride employing new benzoylacrylic acid pentafluorophenyl ester (BAPFP) labelling reagent2(Physique1 d) as an alternative to the crosslinking of ONs and proteins by conventional maleimideNHS esterbased reagents. Importantly, we use the DNAantibody conjugates generated as probes in fluorescence microscopy and superresolution DNAPAINT imaging.