A Platform for Site‐Specific DNA‐Antibody Bioconjugation by Using Benzoylacrylic‐Labelled Oligonucleotides

Abstract Many bioconjugation strategies for DNA oligonucleotides and antibodies suffer limitations, such as site‐specificity, stoichiometry and hydrolytic instability of the conjugates, which makes them unsuitable for biological applications. Here, we report a new platform for the preparation of DNA‐antibody bioconjugates with a simple benzoylacrylic acid pentafluorophenyl ester reagent. Benzoylacrylic‐labelled oligonucleotides prepared with this reagent can be site‐specifically conjugated to a range of proteins and antibodies through accessible cysteine residues. The homogeneity of the prepared DNA‐antibody bioconjugates was confirmed by a new LC‐MS protocol and the bioconjugate probes were used in fluorescence or super‐resolution microscopy cell imaging experiments. This work demonstrates the versatility and robustness of our bioconjugation protocol that gives site‐specific, well‐defined and plasma‐stable DNA‐antibody bioconjugates for biological applications.


Introduction
Cross-linking of various types of molecules leading to chimeric constructs with combined functionalities has been gaining attention across the fields of chemical biology, biotechnology,a nd medicine. [1] Among these,p roteins are one of the 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.I ti s mainly the highly specific and predictable base pairing that makes DNAorRNA oligonucleotides (ONs) unique tools for the design of biomolecular hybrids with new structures and improved functions.O Ns have been applied in the design of DNA-microarrays, [2] other nanostructures, [3] imaging [4] and as therapeutic agents. [5] By joining proteins and NAsinasingle biomolecular scaffold, NA-protein hybrids can combine the programmable sequence recognition properties of NAsa nd diverse functionalities of proteins.T wo major research areas that benefit from the dual functionality of NA-protein bioconjugate constructs are bioanalysis and nanofabrication. [6] In addition, conjugation of ONs to antibodies has been used for the antibody-mediated delivery of therapeutic ONs, [7] cytotoxic agents through intercalation [8] or hybridization of complementary DNAO Ns, [9] decoration of DNA nanostructures with multiple proteins [10] and fluorophores [11] and DNA-antibody bioconjugates have been used as probes for super-resolution imaging by means of the DNA-PAINT method. [12] However,for constructs like DNA-antibody 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] Thec hoice of conjugation method usually depends on the scaffold of the protein of interest, and the main challenges are scalability,c ontrol over efficacy, sitespecificity and stoichiometry of the NA bioconjugation. Strategies for the preparation of covalent DNA-antibody conjugates can be divided into two main categories:1 ) approaches based on proteins modified with functional groups suitable for biorthogonal reactions,s uch as strainpromoted azide-alkyne cycloaddition [14] or inverse electrondemand Diels-Alder reactions [14c, 15] (Figure 1a)o r2 )d irect conjugation of modified ONs to native proteins by using bifunctional cross-linkers (Figure 1b). [16] Whereas the former requires either genetic manipulation of the protein of interest or additional steps to introduce the functional groups for the biorthogonal ligation, the latter cross-links the reactive amino acid residues on the protein surface with modified ONs directly.H owever,p roteins usually contain multiple reactive residues,w hich often results in nonspecific labelling at sites important for protein activity and/or binding.T oa void formation of inactive conjugates,site-specific bioconjugation of NAst op roteins with control over modification site is required;often attempted by using bifunctional cross-linkers. Functionalized homo-or hetero-bifunctional linkers usually contain reactive activated esters for amine modification or Michael acceptor moieties for conjugation of thiol bearing molecules ( Figure 1c). N-hydroxysuccinimide (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 widely used for reactions with thiol-containing molecules but the selectivity of maleimide towards thiols highly depends on the reaction pH. At neutral pH, thiols react with maleimide 1000-times faster than amines but at higher pH reaction with amine is favored. [17] This non-specific labelling can lead to mixtures of heterogenous conjugates with various characteristics.F urthermore,a tm ore alkaline pH, hydrolysis of both the NHS ester and maleimide occurs,w hich 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 ar apid thiol-exchange reaction. [17,18] To avoid some of the downsides of these reagents,i mproved conjugation techniques that give site-specific,h omogenous and welldefined DNA-antibody conjugates are desired. In this work, we report as trategy that leads to chemically defined constructs by employing new benzoylacrylic acid pentafluorophenyl ester (BA-PFP) labelling reagent 2 (Figure 1d)a sa na lternative to the cross-linking of ONs and proteins by conventional maleimide-NHS ester-based reagents.I mportantly,w eu se the DNA-antibody conjugates generated as probes in fluorescence microscopy and super-resolution DNA-PAINT imaging experiments.

Results and Discussion
Our approach towards the preparation of DNA-antibody conjugates was based on the method for cysteine protein modification using benzoylacrylic (BA) reagents recently developed in our laboratory. [19] With these reagents (sometimes in stoichiometric amounts) proteins with solventaccessible cysteine residues are irreversibly modified with reaction kinetics comparable to maleimide chemistry. [19a] This approach offers site-specific, stable constructs that overcome the disadvantages of maleimide bioconjugates,s uch as hydrolytic instability of reagents and problems with retro-Michael addition reaction. [19a] To introduce the BA moiety to DNAONs,weused bifunctional reagent 2,which is easily prepared by activation of commercially available trans-3-benzoylacrylic acid (1)( see Organic synthesis of BA reagents in the SI). PFP-activating ester group was chosen for its known higher hydrolytic stability over NHS-ester derivatives. [16] Bifunctional reagent 2 can be stored as asolid for months or in solution for weeks and used without any significant loss of the reactivity for the modification of various amino group-containing small molecules or biomolecules (see Organic synthesis of BA reagents in the SI).
As mall optimization of the DNA-labelling reaction was performed on the commercially available short single-stranded (ss) DNAO N5 '-NH2-ss11-mer that contains 11 nucleotides and a5 '-amino modifier (see 5'-NH 2 DNAO NB Alabelling reaction optimization in the SI). Conditions that used different amounts of reagent and dimethyl formamide (DMF) were screened. Theb est results were obtained with 100 equiv of 2 in 20 %D MF in phosphate (NaP i )b uffer at pH 8.0 and 37 8 8Co vernight (Table S1). Under these conditions,c omplete conversion of the starting ON into BAlabelled ON (determined by LC-MS) was obtained. Lower amounts of linker and shorter reaction times did not lead to complete conversions and more DMF or linker resulted in as mall fraction (< 10 %) of the second DNAm odification, probably on one of the nucleophilic sites on nucleobases, being detected. Thesecond DNAmodification was also noted after prolonged reaction times (> 48 h) and under more basic conditions (NaP i pH 10.0 buffer for 24 h). Labelling of aminomodified DNAO Ns by 2 can be efficiently performed on different reaction scales (20 mL-1 mL) and at ar ange of ON concentrations (25-100 mM). BA-labelled ONs can be easily purified by using size-exclusion spin columns for small-scale preparations or RP-HPLC for larger-scale labelling reactions.
Theoptimized labelling conditions were used to label four amino-modified ONs,5'-NH2-ss11-mer,5'-NH2-ss25-mer,5'-NH2-ss50-mer with different lengths,i ncluding doublestranded (ds) DNA-ON 5'-NH2-ds19-mer ( Figure 2). We chose examples of modified ONs based on their possible applications:s horter (ss11-mer) ONs are usually used in preparation of probes for super-resolution microscopy by DNA-PAINT, [12] whereas longer ones are in the range of sizes used for therapeutic [7] (ss25-mer or ds19-mer) or biomedical [3] (ss50-mer) applications.A ll BA-labelled ONs were successfully characterized by LC-MS ( Figure 2b) and in all cases, complete conversion of the starting material into expected product was observed. It is necessary to mention that for modification of 5'-NH2-ss50-mer more linker 2 (200 equiv instead of 100 equiv) was needed to ensure complete conversion of the starting ON ( Figure S22).
With awell-working protocol for the labelling of amino-modified ONs,w ew anted to proceed with the bioconjugation of BA-labelled ONs to proteins and antibodies.B ecause we were interested in developing ap rotocol for the generation of site-specific and stoichiometric (1:1) DNA-protein conjugates, we investigated an analytical method for the precise characterization of these constructs. Bioconjugation reactions between ONs and proteins are typically analyzed by gel electrophoresis under reducing or non-reducing conditions. [7a,20] Although this analytical method is useful to confirm successful bioconjugation reaction has taken place,i ts accuracy and resolution is inferior to results obtained by mass spectrometry (MS), which is nowadays the prevailing analytical tool for structural characterization of bioconjugates.Accuracy of characterization is needed for applications that require use of well-defined and homogenous conjugates,f or example development of improved therapies [21] or single-molecule techniques in biophysics. [22] One of the earliest examples of DNA-protein hybrids characterization by MS involved bioconjugates that were enzymatically pre-digested and then analyzed by LC-MS/MS. [23] Besides this,o nly ah andful of reports on the characterization of DNA-protein bioconjugates by MS methods have been published. These include use of matrix-assisted laser desorption/ionization MS that usually requires specific sample and matrix preparation. [24] Electrospray ionization (ESI) MS was used to characterize NA-protein conjugates but gave low mass accuracy and errors in mass ranges of tens of Da. [7b,25] Since MS provides more precise results than gel electrophoresis,w eb elieved that al iquid chromatographymass spectrometry (LC-MS) method for the accurate characterization of DNA-protein bioconjugates could be developed. Analysis by LC-MS is well-established for the accurate characterization of bioconjugates with small molecules,however,its use for the analysis of DNA-protein conjugates is not that straightforward. To the best of our knowledge as imple, accurate,r eproducible and reliable LC-MS method for routine characterization of intact DNA-protein hybrids has not been reported.
Bioconjugate 594Nup98 G85C-ss11-mer was chosen as am odel for the optimization of the LC-MS method. The conjugate was prepared by reaction of as hort BA-labelled ON 5'-BA-ss11-mer with the single-domain antibody against Xenopus nucleoporin complex 594Nup98 G85C [26] bearing as ingle available cysteine residue (Figure 3a). Complete conversion of the starting protein was obtained under mild reaction conditions (pH 8.0, 25 8 8C, 2equiv of the BA-labelled ON;F igure S30). At first, this bioconjugation reaction was analyzed by standard LC-MS conditions suitable for proteins with formic acid as the LC-MS additive (Figure 3b). Under these conditions,i on series with multiple ion species was obtained, which was deconvoluted by the MaxEnt1 algorithm and led to apeak with amolecular weight that corresponded to the expected product (ca. 17.5 kDa). This was observed together with other ions with higher masses (Figure 3b), which were assigned to ion species with different levels of saturation with counter cations,s uch as Na + and K + on the phosphate backbone of the ON part of the bioconjugate.W e assumed that this was due to the phosphate buffer used in the labelling reaction or the mobile phase used for the LC-MS analysis.S o, to eliminate these species,w et ested whether ad ifferent LC-MS mobile phase additive would provide better results.W ith ammonium acetate [27] at 10 mM concentration, much cleaner extracted ion series were obtained relative to experiments that used formic acid as the LC-MS additive (Figure 3b)a nd deconvolution led to as ingle peak with the mass corresponding to the calculated mass of the desired conjugate (17 516 Da). This was probably due to the saturation of the ON phosphate backbone by volatile ammonium counter cations and overall stabilization of the charge of the DNA-protein conjugate sample.Anion series of an individual species could be then extracted and deconvoluted into am ass spectrum that contained as ingle peak for the expected product. Concentration of LC-MS additive had little effect on the signal of the extracted ion series or deconvoluted mass spectra at higher (10 mM) or lower (5 mM, 2mMo r1 mM) ammonium acetate concentrations (Figures S32-S35). However,t he use of higher concentrations was noticeable in the UV trace of the LC whereby background noise was detected. To examine the impact of the counter anion of the LC-MS additive on the analysis,1 0mMa mmonium formate was also tested. We observed ac lear effect on the ionization of the sample,b ut the deconvolution and reconstruction of the total mass ion gave as imilar result as with 10 mM ammonium acetate (Figure 3b). At 1mMc oncentration, extracted ion series clearly show differences in the presence and intensity of ion species,w hich we attributed to the less efficient ionization by ammonium formate at lower concentration ( Figure S36). Based on all these observations,w ec hose 1-10 mM ammonium acetate as the LC-MS additive for the accurate analysis and characterization of our DNA-antibody conjugates.W ethen tested the applicability and accuracyo fo ur LC-MS method for the characterization of 11-mer conjugates with other variants of Xenopus nucleoporin nanobodies. [26] Constructs 576Nup98 A75C-ss11-mer,4 27Nup93 C4-ss11-mer and 443Nup98 C4-ss11-mer were prepared similarly to 594Nup98 G85C-ss11-mer conjugate ( Figures S38-S40). In all the cases,our optimized LC-MS method gave accurate results for the deconvoluted mass spectra of prepared conjugates with observed masses in the range of AE 5Daf rom calculated values (Figure 3c). The advantages of site-specificity and reproducibility of the conjugation reaction combined with the accuracy of our LC-MS analysis could allow application for the construction of well-defined, functionally active DNA-protein conjugates.
Having optimized the conditions for the LC-MS analysis and characterization of the DNA-antibody conjugates,w e wanted to explore the applicability of our bioconjugation protocol to proteins of various sizes with cysteine residues with different accessibility and reactivity.W em anaged to successfully conjugate 5'-BA-ss11-mer to four different proteins including Ubiquitin K63C (with an engineered cysteine residue), [28] C2A domain of Synaptotagmin-I C2Am-C95 (derivatives of which are used as probes for in vivo imaging of apoptosis), [29] Annexin V-C315 [30] and an recombinant human serum albumin-Recombumin (Albumedix Ltd) HSA-C34 [31] (which often serves as aversatile carrier for therapeutic and diagnostic agents). In all cases,b ioconjugation conditions were optimized to obtain complete conversion of the starting protein into desired bioconjugate with no more than 10 equiv of BA-labelled ON used (Figure 4a). All reactions were performed in 50 mM NaP i buffer at pH 8.0, 25 8 8C, 30 min or 1hto obtain UbK63C-ss11-mer,C2Am-C95-ss11-mer and HSA-C34-ss11-mer conjugates (Figure 4b). Annexin V-315-ss11-mer conjugate required ar eaction tem- perature of 37 8 8Ca nd ap rolonged reaction time because of the more buried and consequently less reactive Cys-315 residue ( Figure S44). Moreover,byconjugating longer ssONs 5'-BA-ss25-mer,5'-BA-ss50-mer and even dsON 5'-BA-ds19mer to C2Am-C95 (Figure 4c)wehave shown that our DNAprotein bioconjugation protocol is versatile and suitable for conjugation of different types of BA-labelled ONs.A s ac ontrol experiment, we mixed Ubiquitin K63C with an unmodified 5'-NH2-ss11-mer ( Figure S42). Formation of conjugate was not observed under these conditions,w hich demonstrates that unspecific bioconjugation reactions do not occur in the absence of the BA moiety.A ll the prepared DNA-protein bioconjugates were successfully characterized by our optimized LC-MS method thus proving its reliability and accuracy in the analysis of DNA-protein constructs for aw ide range of masses and types of bioconjugates.
Next, we extended our DNAbioconjugation method to antibodies.S ite-specific and stoichiometric conjugation of DNAo rR NA ONs to monoclonal antibodies (mAbs) or antibody fragments is desired for applications like structural DNAn anotechnology and imaging, [32] preparation of well-defined DNA-antibody nanostructures [33] or delivery of therapeutic ONs,s uch as antisense ONs (AOs) [7a] or small-interfering RNAs (siRNAs). [7b] As only al imited number of bioconjugation methods for such constructs are available,w ew anted to test the reliability of our method in the preparation of ON-antibody conjugates. With our bioconjugation platform, light (LC) and heavy chain (HC) subunits of immunoglobulin G( IgG) mAbs can be easily modified with BA-labelled ONs via engineered cysteine residues.T o demonstrate this we used Thiomab LC-V205C, [34] am Ab targeting the human epidermal growth factor receptor 2 (HER2) that is over-expressed on the cell surface of certain types of breast cancer, and Gemtuzumab mAb variants, which recognize the myeloid differentiation antigen CD33 on acute myeloid leukemia (AML) cells. [35] By using our bioconjugation protocol, cysteine residues on LCs of Thiomab LC-V205C and Gemtuzumab LC-V205C and cysteine residues on HCs of Gemtuzumab i239C and Gemtuzumab HC-S442C were the only amino acid residues modified with 5'-BA-ss11-mer ON (Figure 4d). Forthe bioconjugation reaction conditions we used 5equiv of the BA-labelled ON per cysteine residue in 50 mM NaP i buffer at pH 8.0 and 37 8 8Cfor 1h,which led in all cases to complete conversion of starting antibody materials into desired bioconjugates.T or ule out any chemoselectivity issues,5 '-BA-ss11mer was also reacted with Tr astuzumab,aHER2 targeting mAb containing no additional accessible cysteine residues. LC-MS analysis revealed no modification in the light or heavy chain after 1hat 37 8 8C(identical results were obtained when leaving the reaction for 48 h; Figure S53), demonstrating the site-specificity of the BA-labelled ON reagents towards cysteine residues.I ti sw orth noting that LC-MS analysis on ON-modified HC antibody fragments was challenging.A s seen in Figure 4d (see also Figures S51 and S52), the signal of the main peak in the deconvoluted mass spectra of HCs of the ss11-mer-modified Gemtuzumab i239C and Gemtuzumab HC-S442C variants is of lower intensity and accompanied by other peaks.A lthough with as lightly larger error,o bserved peaks are in the range of expected masses for bioconjugate products,w hich suggests the DNA-antibody conjugation reaction was successful. Theinaccuracyinthe LC-MS analysis for this type of conjugate could be attributed to their inefficient ionization caused by glycosylation of mAb with various glycans attached to HCs. [36] In addition to full-length IgG mAbs,wehave applied our DNA-protein bioconjugation protocol to single domain anti-HER2 antibody 2Rb17c-C138 [37] with all four prepared BAlabelled ONs (Figure 4e). Here,c omplete conversion was observed and all products were characterized by LC-MS with masses as expected. Therange of BA-labelled ONs of various lengths successfully conjugated to an array of various proteins and antibodies clearly demonstrates the versatility of this simple and powerful, bioconjugation method to give sitespecific and stoichiometric DNA-protein conjugates.2Rb17c-C138 was also reacted with RMss11 (1:9 5'-maleimide-ss11mer:5'-maleamic acid-ss11-mer) under the same conditions as those used for 5'-BA-ss11-mer.O nly ca. 64 %c onversion of the starting material to the desired conjugate was observed ( Figure S55). As ac ontrol, 2Rb17c-C138 was also reacted with 5'-maleamic acid-ss11-mer and as expected, formation of conjugate did not occur ( Figure S28). Thel ow conversion of 2Rb17c-138 to 2Rb17cc-C138-ss11-mer using RMss11 can be attributed to the high percentage of 5'-maleamic acid-ss11mer present in the reaction mixture.Altogether,the results of both antibody bioconjugation and ON labelling experiments confirm the superior performance of the BA functionality over maleimides in DNA-antibody bioconjugation reactions.
As we were keen to develop ab ioconjugation method which provides DNA-antibody conjugates suitable for in vivo studies,weassessed the plasma stability of 2Rb17c-C138-ss11mer. Theconjugate was added to 10 %human plasma in PBS buffer pH 7.4 at 37 8 8C, and samples were taken at four different time points (0 h, 24 h, 48 hand 72 h) and analyzed by LC-MS (Figure 5a). No decomposition of 2Rb17c-C138-ss11mer was seen, which strongly suggests that it is stable in the presence of human plasma and opens up opportunities for this method to be used for the preparation of hybrid biomolecular constructs for in vivo applications,such as improved targeted cancer therapy by antibody-mediated delivery of siRNA, antisense ONs or other therapeutic ONs. [38] Ultimately,T hiomab LC-V205C-ss11-mer and 2Rb17c-C138-ss11-mer conjugates were used as probes in superresolution microscopy experiments with the DNA-PA INT method. [12,14b,24c] Thes equence of the conjugated ON, [39] called "docking strand", is designed to be complementary to another short 10-mer fluorophore-labelled DNAO N, referred to as "imager strand". Thel ow melting temperature of the corresponding duplex causes the "imager strand" to repeatedly bind and dissociate from the target "docking strand" and this transient binding enables to locate "docking strand"-labelled antibody.T he detected "blinking" is then used to reconstruct the super-resolution image.B oth Thiomab LC-V205C full-length IgG and 2Rb17c-C138 single-domain antibody selectively bind to HER2 receptors on breast cancer cells, so we investigated whether HER2 receptor binding of both bioconjugate probes was retained even after DNAO Nc onjugation. These experiments were performed on paraformaldehyde-fixed SKBR3 breast cancer cells (see DNA-PAINT imaging ex- c) Epifluorescence microscopyimaging of the HER2 receptor-mediated internalization of the 2Rb17c-C138-Cy3-3'-ss25-mer conjugate on SKBR3 breast cancer cells. Obtained images of SKBR3 cells incubatedf or 2h: without any probe, with 2Rb17c-C138 antibody,5 '-NH2-Cy3-3'-ss25-mer ON, 2Rb17c-C138-Cy3-3'-ss25-mer or with 2Rb17c-C138-Cy3-3'-ss25-mer after 1hincubation with Trastuzumab. Images were processed using ImageJ software, scale bars represent 100 mm. Experiments were performed two independent times. Representative data from one experiment is shown. Forfull experimental details see SI.
periments in the SI) which are known for their high HER2 receptor expression levels. [40] High localization numbers were detected in both cases and super-resolution images of HER2 receptor networks on the surface of SKBR3 cells were acquired (Figure 5b). Results obtained from these experiments confirm that both prepared DNA-antibody conjugates retained their target receptor binding activity.
To provide further insight into intracellular trafficking and localization of generated bioconjugates,a nother experiment which provides qualitative evidence on the receptor-mediated internalization of the DNA-antibody bioconjugate was carried out by using epifluorescence microscopy.I nternalizing anti-HER2 single-domain antibody 2Rb17c-C138, which upon binding to its target cell surface HER2 receptors is taken up by cells via receptor-mediated endocytosis,w as chosen as delivery vehicle.T his feature together with the smaller format of this antibody relative to full-length anti-HER2 IgG Thiomab LC-V205C,makes this antibody an ideal candidate for cell-specific (in this case HER2 +), antibodymediated delivery of drugs and therapeutic ONs.2 Rb17c-C138-Cy3-3'-ss25-mer DNA-antibody bioconjugate probe with aC y3 fluorophore attached to the 3'-end of as s25-mer ON was prepared ( Figure S59). SKBR3 cells were incubated with this bioconjugate or controls,f ixed and imaged (see epifluorescence microscopy imaging experiments in the SI). Obtained images demonstrate that Cy3-labelled DNAO N was localized inside SKBR3 cells only when formulated and delivered as an ON-antibody conjugate (Figure 5c)a nd not detected when cells were incubated with "naked" Cy3-ON without the antibody vehicle (Figure 5c). Ac ontrol experiment in which HER2 receptors were blocked with full-length anti-HER2 IgG antibody Tr astuzumab resulted in 75 % decrease of the Cy3-ON signal ( Figure S64) which was localized mainly on the cell surface presumably as ar esult of competition for HER2 receptor binding between 2Rb17c-C138-Cy3-3'-ss25-mer and Tr astuzumab.Additional evidence was obtained from an imaging experiment in which the same nanobody directly modified with afluorophore dye was used. Conjugate 2Rb17c-C138-C5-AF488 was prepared by modifying the cysteiner esidue of the nanobody with AF-488-C5maleimide under standard conditions ( Figure S65). Asimilar intracellular localization pattern to 2Rb17c-C138-Cy3-3'-ss25-mer was observed with 2Rb17c-C138-C5-AF488 conjugate ( Figure S66). Ther esults provided by super-resolution and fluorescence microscopy imaging experiments support the potential of the developed DNA-antibody bioconjugation platform to be applied for targeted antibody-mediated delivery of therapeutic ONs.

Conclusion
We have developed an improved protocol for the bioconjugation of DNAONs to proteins and antibodies,a nd an optimized method for the LC-MS characterization of prepared bioconjugates.B yu sing easy-to-prepare BA-PFP reagent 2,v arious types of ss-or ds-amino-modified DNA ONs can be labelled with aB Am oiety.P repared BAmodified ONs are then conjugated to proteins and antibodies through cysteine selective thiol-Michael addition reactions. By conjugating different types of DNAONs to aw ide range of proteins and antibodies,wedemonstrate the generality and versatility of our DNA-protein bioconjugation platform that gives site-specific and homogenous DNA-protein conjugates. Importantly,b ioconjugates prepared by this method are stable in the presence of human plasma, which makes them useful for various in vitro applications,i ncluding imaging, nanofabrication, biosensing or immunological detection methods [6a] and suitable for in vivo bioassays and potentially for therapeutic applications. [13] All these features (confirmed by side-by-side comparative experiments) make our BAbased DNA-antibody bioconjugation platform superior to the most popular approaches utilizing maleimides.A sa ne xample,wehave shown that DNA-antibody probes 2Rb17c-C138-ss11-mer and Thiomab LC-V205C-ss11-mer prepared by our method can be used to image HER2 receptors on the surface of HER2 + SKBR3 breast cancer cells by means of DNA-PA INT super-resolution microscopy. [12,39] Such studies could open up the door for development of improved diagnostic methods for the quantification of expression levels of HER2 or other receptors in breast and other types of cancer by adapting the method for the quantitative point accumulation in nanoscale topography (qPAINT). [41] In addition, fluorescent DNA-antibody bioconjugate 2Rb17c-C138-Cy3-3'-ss25mer was used as ap robe in epifluorescence microscopy imaging experiments to investigate HER2 receptor-mediated internalization of this DNA-antibody construct in SKBR3 cancer cells.Images obtained from these experiments clearly showed intracellular localization of 2Rb17c-C138-Cy3-3'-ss25-mer. Ther esults of this work support the potential of the developed bioconjugation platform for therapeutic applications by using antibodies as delivery vehicles for therapeutic ONs.Further studies and developments towards strategies for more efficient cancer therapies by constructing tumortargeting nanostructures or various types of NA-antibody bioconjugates for targeted delivery of therapeutic ONs are underway in our laboratories.