Using nanopores to detect epigenetic changes faster

In recent years, nanopores have become a widely applicable tool for the analysis of molecules. Due to their special properties, they allow the structure of molecules to be analyzed in fractions of a second: as cylindrically arranged proteins, nanopores form tiny channels only a few millionths of a millimeter (nanometer) in diameter that can be incorporated into biomembranes. “For experiments, we apply a constant voltage across the membrane so that ions from the surrounding medium pass through the pore. This creates a constant and precisely measurable electric current,” says Professor Jan C. Behrends of the Faculty of Medicine at the University of Freiburg, in whose laboratory the now published experiments took place. However, when a molecule migrates into the pore, the current is blocked: the larger the molecule, the more strongly it is blocked as well.

A protein in the research spotlight: H4

Within the framework of the now published experiments, the scientists from Freiburg devoted themselves to the study of the so-called histone H4 protein. This protein is firmly associated with DNA in all cells with a nucleus and is one of the best-studied targets of epigenetic modifications. A region at the N-terminal end of the protein is particularly affected by these modifications. “The protein sequence contains the amino acid lysine several times over,” explains Behrends. Acetyl or methyl groups, for example, can be attached to these lysines, which are designated K8, K12 and K16 according to their position in the protein chain, as part of epigenetic modifications. Which chemical change takes place at which position of lysine is definitely of medical importance, as the Freiburg physiologist points out. “Acetylation at K16, for example, is important for human development, while methylation at K12 plays a role in the development of certain prostate and lung tumors, according to the latest results from the Medical Center – University of Friborg . »

Detecting changes using a nanopore

In their experiments, Behrends and his team were now able to clearly distinguish H4 fragments with or without acetylation, as well as fragments with one, two or three acetylations. Moreover, they managed to demonstrate that the nanopore they used was also sensitive to the acetylation site: histone fragments with an acetyl group at K8 blocked the current through the pore more strongly than those acetylated at K12, and these in turn more strongly than those with K16 acetylation. “This kind of sensitivity is surprising since these fragments are identical in terms of mass and total volume,” says Behrends. Thus, the porous current appears to be sensitive not only to the size, but also to the shape of the molecule. It was also easy to distinguish the different variants of doubly acetylated histone fragments – K8 and K12, K8 and K16, and K12 and K16 – again, despite the identical mass. H4 moieties methylated to different degrees and at different positions also blocked current through the pore to different degrees, but not as clearly as the acetylated variants.

“We were able to show for the first time through our experiments that the analysis of nanopores allows us to distinguish molecules not only by their size, but also by their shape”, summarizes the leader of the Behrends study. Molecular dynamics simulations carried out by the research group led by Aleksei Aksimentiev of the University of Illinois in the United States – also involved in the study – show that a highly inhomogeneous electric field inside the pore plays a key role in this effect.

Future vision: optimized medical diagnostics

While DNA sequencing using nanopores is already established and commercialized, the development of nanopore-based protein analysis is just beginning, Behrends points out. “The difficulty with protein sequencing is that they are molecules with very non-uniform charge patterns. While DNA, which is negatively charged, migrates directionally in the electric field and can thus be pulled through pores on a base-by-base basis, proteins are made up of building blocks made up of amino acids with different charges. As a result, directed movement in the electric field and “scanning” amino acid by amino acid is not possible. The Freiburg scientists therefore relied on a different approach for their experiments. Instead of a pore with a short constriction, as used in DNA sequencing, they used a custom-made pore with some sort of molecular trap. “This made it possible to capture the entire protein fragment at once,” says Behrends.

It is not yet clear up to what fragment size this type of analysis can be used. However, additional experiments show that the method will also be suitable for the analysis of H4 fragments previously used in epigenetic research. These contain 14 amino acids instead of the ten used here, and are currently being studied for epigenetic modifications with tandem mass spectrometry, a very sophisticated technique. The researchers hope that the nanopores will make the analysis much simpler, faster and more cost-effective, and that it can be performed close to the patient.

The further development of protein nanopore analysis for medical diagnostics and its implementation in concrete products and services is also one of the central projects of the recently approved BMBF Cluster4Future nanodiagBW, which Behrends leads together with Prof. Dr. Felix von Stetten of Hahn-Schickard-Gesellschaft, who is the leader of this project.