Nanopores as the missing link to next generation protein sequencing

For sequencing DNA, this is an established technique called “Nanopore sequencing”. At its core, nanopore sequencing is based on a simple process: A DNA strand is threaded through a nanoscale pore and the experimenter monitors the changes in electrical current as individual bases pass through. This provides real-time, high-resolution data on the sequence, and can be scaled to process thousands of DNA strands. While this technology has been fully developed for DNA sequencing, there are three hurdles to overcome to apply it to full-length proteins. Namely, unfolding the protein, then threading it into a nanopore, and finally transporting it through.

We began with protein unfolding. Proteins possess intricate three-dimensional structures. The unique sequence of each protein governs how it folds into these complex structures, and these folded shapes are crucial for their specific functions within cells. However, when proteins must navigate the narrow confines of nanopores, they need to be carefully unfolded or denatured, temporarily losing their natural shape. To facilitate this, we employed a small molecule known as guanidinium, which disrupts many of the protein's interactions, allowing us to study their passage through the pore.

Having unfolded the protein, we then needed to thread it through the nanopore. The mechanism of threading DNA through a nanopore relies on the electric field and the negative charge along the DNA’s backbone. This charge under an electric field facilitates the controlled movement of DNA strands through the nanopore. Unlike DNA, however, proteins have a diverse array of amino acids with different charges, resulting in a less consistent charge distribution along the sequence. This makes the original electric field driven method ineffective. To thread the more intricate electric environment of a protein, we added a charged tail to the end of a protein using ten aspartic acid residues. We found that this charge tail not only facilitated threading, but also helps the protein to move smoothly. This slow transport is desirable because the signal readout has a finite time resolution; the more slowly a protein passes through the pore, the more time for sampling and thus obtaining a higher accuracy of readout.

Once the protein is unfolded and threaded, we then need it to move through the nanopore. Here we made a remarkable discovery. We found that conducting nanopore translocation experiments in electrolyte solutions containing moderate amounts of guanidinium chloride not only keeps the proteins unfolded, but also induces highly processive and unidirectional translocation of the unfolded proteins. To understand why this happens we carried out one of the longest molecular dynamics simulations ever conducted for nanopore sequencing. We determined that this rather surprising experimental observation is due to the binding of guanidinium ions to the inner surface of the nanopore, rendering it positively charged even when an unfolded protein is threaded through the nanopore. The positive surface charge produces an exceptionally strong “electro-osmotic” flow - the movement of water through the pore driven by an applied electric field. This phenomenon is a result of the interaction between the electric field and the charged particles (ions) within the water and the surface of the nanopore. We found that this huge electro-osmotic flow induced by guanidinium pushes the unfolded protein through the nanopore regardless of the local charge of the unfolded protein.

The discovery of strong guanidinium-induced electro-osmotic flow, capable of transporting unevenly charged polymers through nanopores, has opened up a realm of promising applications. In the field of protein sequencing, it could be used in conjunction with enzymes to precisely control the direction and rate of polypeptide translocation. Ongoing investigations will address the key questions of: first, whether the technique can be used for other pore systems, and second, whether we can find new ways to combine guanidinium-mediated transport with discrete enzyme-driven movement.