The expression of genes tells us a lot about what happens in biology, but genes code for proteins (which are made from sequences of amino acids that are folded into three-dimensional structures), and it's also crucial to understand protein activity for a more complete picture of what happens in cells. Researchers have now created a way to use nanopores to identify the amino acids that make up a protein, which can help scientists learn more about biology. The work has been reported in Nature Biotechnology.
There is a straightforward technique to learn the sequence of DNA that has been used for decades. DNA is made of nucleotide bases, and there are only four bases. A gene sequence is processed by cells to make proteins. Three nucleotide bases code for twenty different amino acids and protein sequences can be predicted from the gene sequence, but once the gene sequence is transcribed into an intermediary molecule called RNA, the cell can cut or modify it in different ways before translating it into proteins. There is no method for easily finding the sequence of proteins.
"DNA codes for many things that can happen; it tells us what is potentially possible. The actual product that comes out - the proteins that do the work in the cell - you can't tell from the DNA alone," said University of Illinois at Urbana-Champaign physics professor Aleksei Aksimentiev, a study co-leader. "Many modifications happen along the way during the process of making protein from DNA. The proteins are spliced, chemically modified, folded, and more."
"Many amino acids are very similar," Aksimentiev said. "For example, if you look at leucine and isoleucine, they have the same atoms, the same molecular weight, and the only difference is that the atoms are connected in a slightly different order."
A cell membrane is needed to maintain structure, but stuff like ions must be able to pass through it. Nanopores are channels that can be used by molecules as they go through the membrane. Nanopores have been used in DNA sequencing, and this research used a bacterial membrane channel called aerolysin to show that they can be used for sequencing amino acids that compose proteins. Proteins that had been chopped up were carried through the nanopore with a chemical carrier. Each amino acid was kept in the pore while an electrical signature was registered.
"This is a proof-of-concept study showing that we can identify the different amino acids," explained Abdelghani Oukhaled, a professor of biophysics at Cergy-Pontoise University in France. "The current method for protein characterization is mass spectrometry, but that does not determine the sequence; it compares a sample to what's already in the database. Its ability to characterize new variations or mutations is limited. With nanopores, we finally could look at those modifications which have not yet been studied."
Different forms of modified amino acids could be differentiated with this tool by using sensitive equipment or with a chemical treatment. Hundreds of modifications could be detected this way, said Aksimentiev.
Other uses for this technology are being considered as well. "One potential application would be to combine this with immunoassays to fish out proteins of interest and then sequence them. Sequencing them will tell us whether they're modified or not, and that could lead to a clinical diagnostic tool," added Aksimentiev.
Learn more about how nanopores are used to sequence DNA from the video above.