Artificial protein-DNA conjugates: Increasing the versatility of proteins with the help of DNA

Hey there! This is Lorena, and in this post I will talk about protein-DNA conjugates, as part of the lecture course 🙂 I have recently been working quite a lot with them, so I am very excited to cover some of their aspects

Proteins have incredible properties nobody else in the bio-world has. Their highly complex tertiary structure and the richness of their chemical groups allows them to bind molecules with high affinity and in a very selective way. They are also able to act as very efficient catalysts, showing high substrate turnover ratios. Thus, it is no surprise that proteins attract a great deal of attention and are in high demand for a lot of applications.

However, the cool features that proteins provide come at the expense of having to deal with the huge variability that exists between one another, and unpredictable structures. The latter is actually one of the biggest and long-standing challenges for the past 50 years: the protein structure prediction problem. Figuring out which shape a protein folds into is very important, as the structure of a protein defines what it can do, its function. While computational biologists have been trying to find out ways to predict and connect the amino acid sequence of proteins to their final 3D structure (with very recent exciting findings! See here), some biotechnologists have focused their efforts on making proteins more user friendly and versatile. And guess how: with DNA!

Obviously, I could not skip this post without mentioning the main character of our PhD: the DNA molecule. Very opposite to proteins, the structure of DNA can be easily and very accurately predicted by the sequence of bases, which makes it programmable and convenient to use. Then, one might think (or at least some scientists did) that a way to make a new generation of well-behaved proteins that might outperform their natural counterparts could be by attaching some DNA into them, so that we can enjoy the best of both worlds; the binding specificity and catalytic activity of proteins and the programmability of DNA. These are better known as protein-DNA conjugates.

Figure 1. Conjugation of DNA to a proteina

These hybrids have found numerous applications in diagnostics, therapeutics, and as a tool for molecular biology, significantly expanding our knowledge. Here a brief history about them:

  • One of the first reports using protein-DNA conjugates came up in the 1980s for diagnostic applications, where the conjugates were used to detect nucleic acid targets1.
  • Important diagnostic tools such as immuno-PCR2 and proximity ligation assay (PLA)3, which use an antibody for target recognition and DNA as a signal reporter, were developed in 1992 and 2002, respectively.
  • The structural organization of proteins by means of DNA sequences was firstly reported in 19944 and since the early 2000s, different types of DNA nanostructures, such as DNA tiles or DNA origami, have been employed to position proteins onto a DNA network with a high spatial control5, 6.
Figure 2. Scheme of the immuno-PCR assayb

And how are these artificial protein-DNA conjugates synthesized?

It can be through the covalent or non-covalent attachment of DNA into the protein. As we would expect, covalent coupling is preferred, as it results in much stronger bonds compared to non-covalent interactions, and therefore more stable conjugates for its use in physiological conditions.

Among the methods for the covalent conjugation of DNA to proteins, we find non-specific and site-specific ones. The first type makes use of reactants that target the native amino acids of the protein, normally lysines and cysteines. Whereas it is a straightforward method, it lacks selectivity, and thus many of the chemical groups of the protein can end up randomly conjugated to DNA, resulting in a multi-labelled protein (which might not always be desired). Also the activity of the protein can be affected if active epitopes are chemically modified, resulting in the inhibition of the catalytic activity.

To address this issue, site-specific methods were developed, where the DNA is attached into a very specific site in the protein and therefore it allows the control of the stoichiometry and orientation of the DNA in the protein. This can be achieved through mutagenesis, i.e. the introduction of mutated aminoacids in the protein that showcase with unique chemical groups that can be further targeted in a specific way7. Also, specific residues can be modified using biorthogonal chemistry (which uses chemical groups that are not present in the native protein and thus they don’t cross-react, such as azide or alkyne groups)8.

Figure 3. Site-specific conjugation of DNA to the Fc region of antibodies and their assembly into a DNA nanostructurec

At this point I would like to mention some of the research that takes place at Kurt Gothelf’s lab at Aarhus University, where I do my secondment. There is extensive experience on protein-DNA conjugates in the lab, and they address all the aspects; from the organic synthesis of the labeling reagents to the protein bio-conjugation itself, forming a very nice tandem. They develop new organic molecules with definite properties that allows them to react only with specific amino acid residues, therefore achieving the so-called site-specific conjugation of proteins. This approach is actually very ambitious, as it aims to target endogenous residues (the natural amino acids) in the proteins in a selective way, without the need to insert non-canonical amino acids.

The properties of the molecules that allow for targeted conjugation can be related, for instance, to their molecular size or to specific interactions that the molecule might have with the protein. In the case of the former, the size of a molecule can be tuned so that it is accommodated in very specific cavities of the protein, where it can later react. For the latter, the specific positioning of the molecule with respect to the protein can be guided through interactions in between both, resulting in labelling only occurring in a restricted area in the vicinity of the place of interaction.

But the strategies are not limited to these; you can get as creative as you like regarding the ways to direct a labeling molecule to a specific microenvironment of the protein so that it only reacts there.

Here I leave some of the magic molecules designed at Kurt’s lab that are able to achieve site-specific conjugation:

  • Märcher, A., Palmfeldt, J., Nisavic, M., & Gothelf, K. V. (2021). A Reagent for Amine-Directed Conjugation to IgG1 Antibodies. Angewandte Chemie – International Edition, 60(12), 6539–6544.
  • Nielsen, T., Märcher, A., Drobňáková, Z., Hučko, M., Štengl, M., Balšánek, V., … Cló, E. (2020). Disulphide-mediated site-directed modification of proteins. Organic and Biomolecular Chemistry, 18(25), 4717–4722.


  1. Jablonski E, Moomaw EW, Tullis RH, Ruth JL (1986) Preparation of oligodeoxynucleotide alkaline phosphatase conjugates and their use as hybridization probes. Nucleic Acids Res 14(15):6115–6128
  2. Sano T, Smith CL, Cantor CR (1992) Immuno-PCR: very sensitive antigen detection by means of specific antibody–DNA conjugates. Science 258(5079):120–122
  3. Fredriksson S, Gullberg M, Jarvius J, Olsson C, Pietras K, Gustafsdottir SM, Ostman A, Landegren U (2002) Protein detection using proximity-dependent DNA ligation assays. Nat Biotechnol 20(5):473–477
  4. Niemeyer CM, Sano T, Smith CL, Cantor CR (1994) Oligonucleotide-directed self-assembly of proteins: semisynthetic DNA—streptavidin hybrid molecules as connectors for the generation of macroscopic arrays
  5. Dong YC, Mao YD (2019) DNA origami as scaffolds for self-assembly of lipids and proteins. ChemBioChem 20(19):2422–2431
  6. Bujold KE, Lacroix A, Sleiman HF (2018) DNA nanostructures at the interface with biology. Chem 4(3):495–521
  7. Kim, C. H., Axup, J. Y., & Schultz, P. G. (2013, June). Protein conjugation with genetically encoded unnatural amino acids. Current Opinion in Chemical Biology, Vol. 17, pp. 412–419.
  8. Synakewicz M, Bauer D, Rief M, Itzhaki LS (2019) Bioorthogonal protein–DNA conjugation methods for force spectroscopy. Sci Rep-Uk 20:9

Figure References

a. Trads, J. B., Tørring, T., & Gothelf, K. V. (2017). Site-Selective Conjugation of Native Proteins with DNA.

b. Ultrasensitive ELISA and ImmunoPCR – Novatein Biosciences. (n.d.). Retrieved June 21, 2021, from

c. Rosier, B. J. H. M., Cremers, G. A. O., Engelen, W., Merkx, M., Brunsveld, L., & De Greef, T. F. A. (2017). Incorporation of native antibodies and Fc-fusion proteins on DNA nanostructures via a modular conjugation strategy. Chemical Communications, 53(53), 7393–7396.

This post was written by PhD student Lorena Baranda from Francesco Ricci lab at Tor Vergata 

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