HYBRIPORE · Hybrid DNA-protein nanopores with large and uniform pore sizes

Hybrid DNA-protein nanopores with large and uniform pore sizes

Every living cell transports thousands of molecules per second across its membrane and into different compartments. Proteins actively and selectively regulate this transport in the majority of cases and they are vital to cell functioning. Over the past years some of the pore-forming proteins involved in transport have been isolated or expressed in vitro to develop biosensors for heavy metals and neurotransmitters, and DNA sequencing technologies with a high selectivity and extreme sensitivity. An important bottleneck in technologies based on these protein nanopores is the inflexibility of the self-assembly of the pore-forming proteins. As a direct consequence, it is currently impossible to reliably make smaller or larger pores than the size that is dictated by the protein itself: a heptameric protein like haemolysin will always assemble into a heptameric pore. A second consequence is that the relative order of these seven subunits cannot be imposed. This limits the development of novel sensors and artificial enzymes that require more than one altered subunit. To overcome these limitations our key objective is to design a molecular scaffold that can template both the number and the relative order of protein subunits that later form a nanopore (Fig. 1). Our scaffold is made of DNA and can be constructed accurately and reproducibly in a single step. It can be modified with proteins or parts of proteins that should be brought together in a nanopore, and it can be detached if required after the correct nanopore has formed. Nanopore scaffolds will not only allow us to construct more advanced biosensors and DNA sequencing tools, but we can also use them to learn more about pore-forming proteins, about how they work and why they are vital to cells. We can use them to understand what pore sizes are preferred by various proteins and why, and we could discover which parts of a protein are essential for it to make a pore. Ultimately, these insights can help improve existing antibacterial therapeutics that target transport proteins and stimulate the discovery of new antibiotics.

Data: CORDIS, © European Union