Template-directed covalent conjugation of DNA to native antibodies, transferrin and other metal-binding proteins

Abstract

DNA-protein conjugates are important in bioanalytical chemistry, molecular diagnostics and bionanotechnology, as the DNA provides a unique handle to identify, functionalize or otherwise manipulate proteins. To maintain protein activity, conjugation of a single DNA handle to a specific location on the protein is often needed. However, preparing such high-quality site-specific conjugates often requires genetically engineered proteins, which is a laborious and technically challenging approach. Here we demonstrate a simpler method to create site-selective DNA-protein conjugates. Using a guiding DNA strand modified with a metal-binding functionality, we directed a second DNA strand to the vicinity of a metal-binding site of His6-tagged or wild-type metal-binding proteins, such as serotransferrin, where it subsequently reacted with lysine residues at that site. This method, DNA-templated protein conjugation, facilitates the production of site-selective protein conjugates, and also conjugation to IgG1 antibodies via a histidine cluster in the constant domain.

Selective Metal-Site-Guided Arylation of Proteins

“…It should be noted that detailed analysis (see SI) of the structure MGS shows that D100, D102, T139 contribute to part of the metal/ion-binding site; remaining backbone interactions and protein shell may explain retained residual directing activity of even triple DDT→AAA mutants. As poly-His sites have been used to guide other protein chemistries, [18][19][20] we tested endogenous vs such unnatural sites. The out-competing, directing effect of the identified metal/ion-binding D100-D102-T139 motif over artificial (i.e.…”

“…9,10 Metal-mediated examples include azidealkyne 'cycloadditions', 11,12 cross-couplings, 13 and olefin crossmetathesis. 14 [18][19][20] or -cysteine 21 motifs. However, covalent protein modifications based on naturally occurring motifs to achieve site-selectivity are rare.…”

Assessing histidine tags for recruiting deoxyribozymes to catalyze peptide and protein modification reactions

“…However, where tested, DNA-catalyzed protein modification using the recruiting strategy was not observed. Unlike nonenzymatic protein modification reactions such as those studied by Rosen et al, 8 where simple proximity is responsible for the enhanced reactivity (Fig. 1A), achieving DNA-catalyzed reactions of proteins faces additional challenges, even with the benefit of histidine tag recruiting.…”

“…(A) The reported use of hexahistidine (His 6 ) tags and divalent metal ions (M 2+ ) for directing nonenzymatic, DNA-templated reaction of protein side chains. 8 In one case, the authors instead used a natively metal-binding protein. NTA = nitrilotriacetic acid.…”

Using modified aptamers for site specific protein–aptamer conjugations

“…15 Very recently, Gothelf and coworkers applied DNA-templated synthesis to accomplish site-selective labeling of antibodies, transferrin and metal-binding proteins. 16 As our probe molecules of choice, several DNA aptamers that recognize membrane proteins on cancer cells have been developed. 17-18 Identification of these membrane proteins is important not only because they are potential biomarkers, 19-21 but also because they hold the keys to understanding biological processes in cancer cells.…”