DNA‐Templated Introduction of an Aldehyde Handle in Proteins

Abstract

Many medical and biotechnological applications rely on protein labeling, but a key challenge is the production of homogeneous and site-specific conjugates. This can rarely be achieved by simple residue-specific random labeling, but generally requires genetic engineering. Using site-selective DNA-templated reductive amination, we created DNA-protein conjugates with control over labeling stoichiometry and without genetic engineering. A guiding DNA strand with a metal-binding functionality facilitates site-selectivity by directing the coupling of a second reactive DNA strand in the vicinity of a protein metal-binding site. We demonstrate DNA-templated reductive amination for His6 -tagged proteins and metal-binding proteins, including IgG1 antibodies. We also used a cleavable linker between the DNA and the protein to remove the DNA and introduce a single aldehyde on the protein. This functions as a handle for further modifications with desired labels. In addition to directing the aldehyde positioning, the DNA provides a straightforward route for purification between reaction steps.

Considerations on Probe Design for Affinity‐Guided Protein Conjugation

“…We have shown that an aldehyde handle can be concealed during protein conjugation using a 1,2‐diol cleavable linker . The 1,2‐diol linker is cleaved rapidly (1 h) by using 1 m m NaIO 4 under slightly acidic conditions (NaOAc buffer 100 m m , pH 5.5).…”

“…In our studies, the concealed handle strategy allowed us to utilize the affinity motifs for purification of the modified proteins, which is nearly impossible in the direct handle‐transfer methods. Afterwards, the cleavable linker allowed removal of the affinity motifs leaving only the small reactive scar …”

Peptide‐Directed DNA‐Templated Protein Labelling for The Assembly of a Pseudo‐IgM

“…In previous work from our group, a tris nitrilotriacetic acid (NTA)‐modified oligonucleotide (ODN) was used for DNA‐templated reactions toward metal‐binding proteins. The tris NTA group showed very good coupling conversions for His‐tagged proteins and some proteins with natural metal‐binding sites, however, it is not ideal as a guiding motif when pursuing the labelling of IgG molecules, as the labelling results in yields of typically 13–23 % . Likewise, proteins without metal‐binding sites cannot be addressed using the tris NTA guiding group.…”

“…Reactions between the guiding and the reactive strands should thereby be prevented. Initially, we tested the concept with a Rituximab antibody (a humanized IgG1 commercially known as Mabthera ® ) employing a 22 nt benzaldehyde‐modified reactive strand …”

Peptide‐Directed DNA‐Templated Protein Labelling for The Assembly of a Pseudo‐IgM

“…In previous work from our group, [12] a trisnitrilotriacetic acid (NTA)-modified oligonucleotide (ODN) was used for DNA-templated [13] reactions toward metal-binding proteins. The trisNTAg roup showed very good coupling conversions for His-tagged proteins and some proteins with natural metalbinding sites,however, it is not ideal as aguiding motif when pursuing the labelling of IgG molecules,a st he labelling results in yields of typically 13-23 %.…”