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An illustration depicting pores on a membrane
An illustration depicting pores on a membrane

DNA Nanopore Sequencing Adapted for Protein Sequence Comparisons

Researchers link a stretch of DNA to a peptide of interest and measure current changes as the molecule is pulled by a helicase through a nanopore.

Headshot of Sophie Fessl
Sophie Fessl

Sophie Fessl is a freelance science journalist. She has a PhD in developmental neurobiology from King’s College London and a degree in biology from the University of Oxford.

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Since the 1990s, nanopores have been used for sequencing strands of DNA. A voltage is applied across the nanopore, which is embedded in a thin lipid membrane, causing a stretch of DNA to thread through the pore. A helicase enzyme then methodically pulls the molecule back through. As this happens, the nitrogenous bases that make up the DNA affect the ion current flowing through the pore, and by measuring these current changes, researchers can decode the DNA sequence. Now, biophysicist Cees Dekker of Delft University of Technology in the Netherlands and colleagues have repurposed this technology for deciphering amino acid differences among peptides (Science, 374:1509–13, 2021).

Dekker’s team starts by linking a synthetic peptide with the 5’ end of a single strand of DNA. After a zap of voltage sends the conjugated molecule through the nanopore, the Hel308 helicase walks on the DNA section, pulling both the DNA and the attached peptide back through the nanopore. As with DNA sequencing, ratcheting the peptide through the nanopore changes the ion current, and the researchers can link the changes to a specific sequence of amino acids in their designed peptide. The target peptide is read in this way multiple times, threading back through the pore as the helicase falls off and being pulled back through again by another, improving the technique’s fidelity. In a proof-of-principle study, the researchers were able to distinguish three different 26-amino-acid-long peptides that only varied by a single amino acid.

The method cannot be used to decode protein sequences without a known reference for comparison, however. That’s because not only does the amino acid at the pore’s entrance affect the ion current, but the eight surrounding amino acids do as well. “Right now, it is not yet a full de novo sequencing tool,” Dekker writes in an email to The Scientist. “Yet it is very powerful since we showed that by changing even a single amino acid within the chain, we observed dramatic differences in the current step signals.” The new method therefore could be useful for detecting amino acid mutations or identifying the presence of a specific peptide of interest within a mixture of proteins, he says.  

PULL AND READ: In a proof-of-concept study, researchers show that nanopore sequencing techniques can be used to interrogate the sequence of a peptide of interest. First they link the peptide to a stretch of DNA and apply a voltage to feed the conjugated molecule through a nanopore embedded in a thin membrane. A helicase molecule then walks along the DNA strand, effectively pulling the DNA and attached peptide back through the pore. As the peptide passes through, changes in the current across the membrane can be measured, providing clues to the amino acid composition of that stretch of the peptide. WEB | PDF

In theory, this method is “perfect” for analyzing proteins, says Giovanni Maglia, a chemical biologist at the University of Groningen who recently published a proteasome-nanopore that can unfold proteins for sequencing. The helicase is already known to work for DNA sequencing, he notes, and it pulls the DNA through the pore in a controlled way. Maglia points out that the approach is limited to peptides that are 26 amino acids or shorter, however. This is because the helicase sits on top of the pore and can only pull the molecule by its DNA tail.

Dekker acknowledges this limitation but notes that this read length is enough to discriminate all proteins in the human proteome if they are broken into pieces. Also, the nanopore-based approach requires smaller samples than does mass spectrometry—a commonly used protein analysis approach—and would be able to detect rare variants, something mass spec can’t, Dekker says.

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