Nanopores (otherwise known as transmembrane β-barrel pores (TMBs)) are perhaps best known for their role in the sequencing of nucleic acids—both DNA and RNA. They can also be used to sequence peptides. Nanopores present enormous benefits in their ability to miniaturize sensing and sequencing applications into portable—USB-size—devices and point-of-care technologies.

Current approaches to engineering nanopores are limited to naturally occurring proteins, which are less than ideal starting points for sensor development. Indeed, these proteins have complex requirements.

Now, research led by the VIB-VUB Center for Structural Biology in Belgium and the University of Washington School of Medicine in Seattle describes a general approach to designing TMB pores from scratch—with a range of geometries ranging from oval to square to triangular and with different diameters and pore geometries—with the ultimate goal of controlling the shape and chemistry on a molecular level. This work opens up new opportunities for single-molecule analytics.

The findings are published in Science in the article, “Sculpting conducting nanopore size and shape through de novo protein design.”

“This collaboration is a great example of what’s possible with protein design,” noted David Baker, PhD, professor at the University of Washington School of Medicine and HHMI investigator. “Rather than repurposing biomolecules from nature, we can now create the functions we want from first principles.”

With the help of computational design, the researchers developed methods to design stable nanopore channels with tunable pore shapes, sizes, and conductance. Characterized by nuclear magnetic resonance and crystallography, the pores were shown to be stable, opening the door to designing nanopore channels de novo that are suitable for many applications of interest in research and industry. More specifically, the designs “have distinct conductances that correlate with their pore diameter, ranging from 110 picosiemens (~0.5 nanometer pore diameter) to 430 picosiemens (~1.1 nanometer pore diameter).” The summary noted that one important feature of the work was the incorporation of breaks in the secondary structure to prevent aggregation and to facilitate folding into the membrane.

“These developments are very exciting,” said Anastassia Vorobieva, PhD, group leader at the VIB-VUB Center for Structural Biology. “When we started with this idea a few years ago, many people thought it was impossible, because the design and folding of β-sheets is incredibly complex, let alone in lipid membranes. Now we have shown that we can successfully design nanopores with a high success rate, which have stable and reproducible conductance.”

A team from the Baker lab successfully designed new proteins that can specifically bind small-molecule metabolites. They split the proteins into three parts and fused the parts into the loops of a TMB pore. They found that they could directly detect single-molecule binding events using the constructs.

The authors noted that “de novo protein design can in principle create an unlimited number of new nanopores with any desired properties.” This work advances the idea that portable devices, with different nanopores, could sense a range of metabolites, proteins, and small molecules.