Engineered transmembrane pores

Abstract

Today, hundreds of researchers are working on nanopores, making an impact in both basic science and biotechnology. Proteins remain the most versatile sources of nanopores, based on our ability to engineer them with sub-nanometer precision. Recent work aimed at the construction and discovery of novel pores has included unnatural amino acid mutagenesis and the application of selection techniques. The diversity of structures has now been increased through the development of helix-based pores as well as the better-known β barrels. New developments also include truncated pores, which pierce bilayers through lipid rearrangement, and hybrid pores, which do away with bilayers altogether. Pore dimers, which span two lipid bilayers, have been constructed and pores based on DNA nanostructures are gaining in importance. While nanopore DNA sequencing has received enthusiastic attention, protein pores have a wider range of potential applications, requiring specifications that will require engineering efforts to continue for years to come.

Controlling Interactions of Cyclic Oligosaccharides with Hetero‐Oligomeric Nanopores: Kinetics of Binding and Release at the Single‐Molecule Level

“…Although single molecule sensing through a nanopore is a powerful technique, several limitations still persist including the need for a high resolution to detect the fast translocation events of the analytes . The limited pore size and long β‐barrel conformation of alpha hemolysin restricts its application in the analysis of nucleic acids or small molecules . Further, MspA pore improves spatial resolution but still we observe rapid translocation rates of nucleic acids .…”

“…Single molecule electrical sensing with engineered protein nanopores has been developed over the recent years to characterize the size, chemical composition, and structural conformation of macromolecules . A wide range of molecules such as metal ions, organic molecules, and nucleic acids have been detected using this methodology . Most of the single molecule nanopore studies have been focused on nucleic acid sequencing based on the ability to detect single‐stranded DNA and RNA translocations .…”

“…Here, charged analytes are electrophoretically threaded through a range of biological pores under an applied transmembrane potential, generating an ion current signal reflecting the identity of the molecule . Alpha hemolysin from Staphylococcus aureus and MspA from the Mycobacterium smegmatis are the two major protein nanopores that have been specifically engineered for sequencing nucleic acids . Notably, Oxford Nanopore Technologies have developed a portable universal serial bus (USB) device, which can sequence DNA applied to a chip that supports hundreds of protein pores .…”

“…Notably, Oxford Nanopore Technologies have developed a portable universal serial bus (USB) device, which can sequence DNA applied to a chip that supports hundreds of protein pores . Recent work on protein nanopores has been expanded for the broader scope of single molecule sensing of peptides, proteins, and oligosaccharides including the detection of unique chemical reactions and complex synthetic biopolymers . Notably, cyclodextrins (CD) with different symmetries have been used to block protein pores such as heptameric alpha‐hemolysin, octameric polysaccharide transporter Wza, and the outer membrane protein CymA .…”

“…Solid‐state nanopores were introduced as a potential alternative to protein pores, but controlled transport of biopolymers was not achieved as they cannot be assembled with sub‐nm precision . However, the geometry of the protein pore turned out to be the most sensitive parameter to distinguish different analytes and its identity . Engineered membrane pores such as ClyA, aerolysin, FraC, and phi29 exhibit structural properties that improve their efficiency in single‐molecule sensing of oligonucleotides .…”