Using nanopore analysis, researchers have succeeded in characterizing the chemical changes in proteins that are usual for epigenetic modifications.

As specified in a Phys.org report, changes identified as epigenetic modifications play a vital role in cancer development.

Being able to examine them fast and dependably could substantially contribute to the further development of customized therapy.

In recent years, nanopores have become a widely applicable mechanism for analyzing molecules.

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Nanopore Sequencing
(Photo: Wikimedia Commons/National Human Genome Research Institute (NHGRI) from Bethesda, MD, USA )
Nanopore-based DNA sequencing concepts generally entail one of the DNA strands passing through the nanopore sensor, where the individual nucleotides (DNA building blocks) are distinguished from each other.


Nanometer Implanted in Biomembranes

Because of their special properties enable the molecules' structure to be examined within fractions of a second.

Relatively, as cylindrically arranged proteins, nanopores are forming small channels, only a few million of a millimeter or nanometer in diameter, that can be implanted in biomembranes.

The research team from the Institute of Physiology at the University of Freiburg has published their study findings in the Journal of the American Chemical Society.

For the experiments, the researchers said they apply a constant voltage throughout the membrane so that the ions from the surrounding medium flow across the pore.

This then produces a constant, accurately measurable electric current, explains Professor Dr. Jan Behrends from the Faculty of Medicine at the University of Freiburg, whose laboratory currently-published experiments were carried out.

Nonetheless, the current is blocked when a molecule migrates into the pore. This then means that the larger the molecule, the more robustly it is blocked, as well.

Changes Detected Through Nanopore

In their study, Behrends and his team could clearly distinguish between H4 fragments with or without acetylation and fragments with one, two, or three acetylations.

Additionally, they illustrated that the nanopore they used is also sensitive to the site of the acetylation.

These histone fragments that have an acetyl group at K8 blocked current through the pore more robustly compared to those acetylated at K12, and these, in turn, are more strongly compared to those with K16 acetylation.

Behrends explained that this kind of sensitivity is surprising because such fragments are identical in their mass and total volume.

DNA Sequencing Using Nanopores

While DNA sequencing using nanopores is already established on commercialized, the development of an analysis of proteins based on nanopores is just starting, said Behrends.

The struggle with sequencing proteins is that these molecules have very non-uniform charge patterns, ScienceDaily in a related report.

Furthermore, while DNA, which is negatively charged, directionally migrates in the electric field and can therefore be pulled through the pore "base by base," proteins comprise building blocks made of amino acids with different charges, the researchers explained.

Consequently, directed movement in the electric field, scanning amino acid by amino acid, is impossible. The scientists at Freiburg thus depended on a different approach for their experiments.

Instead of a pore that has a short constriction, as used in DNA sequencing, the researchers used a tailor-made pore with a molecular trap type. This enabled the whole protein fragment to be captured at once.

Related information about nanopore sequencing is shown on Quick Biochemistry Basics' YouTube video below:

 

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