Sculpting conducting nanopore size and shape through de novo protein design

Abstract Transmembrane β-barrels (TMBs) are widely used for single molecule DNA and RNA sequencing and have considerable potential for a broad range of sensing and sequencing applications. Current engineering approaches for nanopore sensors are limited to naturally occurring channels such as CsgG, which have evolved to carry out functions very different from sensing, and hence provide sub-optimal starting points. In contrast, de novo protein design can in principle create an unlimited number of new nanopores with any desired properties. Here we describe a general approach to the design of transmembrane β-barrel pores with different diameter and pore geometry. NMR and crystallographic characterization shows that the designs are stably folded with structures close to the design models. We report the first examples of de novo designed TMBs with 10, 12 and 14 stranded β-barrels. The designs have distinct conductances that correlate with their pore diameter, ranging from 110 pS (∼0.5 nm pore diameter) to 430 pS (∼1.1 nm pore diameter), and can be converted into sensitive small-molecule sensors with high signal to noise ratio. The capability to generate on demand β-barrel pores of defined geometry opens up fundamentally new opportunities for custom engineering of sequencing and sensing technologies. One sentence summary De novo design enables the generation of stable and quite transmembrane beta-barrel nanopores with tailored sizes, shapes and properties.