The introduction of a ring of arginine residues near the constriction in the transmembrane beta barrel of the staphylococcal alpha-hemolysin heptamer yielded a pore that could be almost completely blocked by phosphate anions at pH 7.5. Block did not occur with other oxyanions, including nitrate, sulfate, perchlorate, and citrate. Based on this finding, additional pores were engineered with high affinities for important cell signaling molecules, such as the Ca(2+)-mobilizing second messenger inositol 1,4,5-trisphosphate (IP(3)), that contain phosphate groups. One of these engineered pores, P(RR-2), provides a ring of fourteen arginines that project into the lumen of the transmembrane barrel. Remarkably, P(RR-2) bound IP(3) with low nanomolar affinity while failing to bind another second messenger, adenosine 3', 5'-cyclic monophosphate (cAMP). The engineered alpha-hemolysin pores may be useful as components of stochastic sensors for cell signaling molecules.

Engineered protein pores have several potential applications in biotechnology: as sensor elements in stochastic detection and ultrarapid DNA sequencing, as nanoreactors to observe singlemolecule chemistry, and in the construction of nano-and microdevices. One important class of pores contains molecular adapters, which provide internal binding sites for small molecules. Mutants of the α-hemolysin (αHL) pore that bind the adapter β-cyclodextrin (βCD) ∼10 4 times more tightly than the wild type have been obtained. We now use single-channel electrical recording, protein engineering including unnatural amino acid mutagenesis, and highresolution x-ray crystallography to provide definitive structural information on these engineered protein nanopores in unparalleled detail.alpha-hemolysin | single molecule | stochastic sensing | structure | unnatural amino acid M any research groups have used protein engineering to obtain enzymes and antibodies with new properties suited for specific tasks (1-6). Fewer groups have taken on the difficult problem of engineering membrane proteins (7). We have engineered the α-hemolysin protein pore, mindful of several potential applications in biotechnology, including its ability to act as a detector in stochastic sensing (8) and ultrarapid DNA sequencing (9), to serve as a nanoreactor for the observation of singlemolecule chemistry (10) and to act as a component for the construction of nano-and microdevices (11).An important breakthrough in this area, which enabled the stochastic sensing of organic molecules including the detection of DNA bases in the form of nucleoside monophosphates (12, 13), was the discovery of internal molecular adapters, a form of noncovalent protein modification (14). Most useful have been cyclodextrin (CD) adapters, which have until now been used in the absence of detailed structural information about how they work. The present paper is a definitive investigation, which provides such information through the application of a wide variety of technical approaches: single-channel electrical recording, protein engineering including unnatural amino acid mutagenesis, and x-ray crystallography. The studies employing mutagenesis show that the striking interactions seen in the crystal structures also occur in individual pores in lipid bilayers.We reveal that the tight-binding αHL mutants (15) M113N 7 and M113F 7 bind βCD in different orientations within the heptameric pore. In the case of M113N 7 , the top (primary hydroxyls) of the CD ring faces the trans entrance of the pore. In the case of M113F 7 , the bottom (secondary hydroxyls) of the CD ring faces the trans entrance, while the top of the ring is bonded to the pore through remarkable CH-π interactions. Another tight-binding mutant, M113V 7 , can bind the CD in both orientations. These results illustrate the exquisite level of engineering that can be achieved with protein nanopores, which is, for example, far beyond what is possible with solid-state pores. The work also provides information valuable for the design of ...

Engineered protein channels have many potential applications in biosensing at the single molecule level. A future generation of biosensor could be an array of target-specific ion channels, where each protein pore acts as a sensor element. An important step toward this goal is to create a portable, durable, single protein channel-integrated chip device. Here we report a versatile, modular chip that contains a single ion channel for single molecular biosensing. The core of the device is a long-lived lipid membrane that has been sandwiched between two air-insulated agarose layers which gel in situ. A single protein pore embedded in the membrane serves as the sensor element. The modular device is highly portable, allowing a single ion channel to continuously function following detachment of the chip from the instrument and independent transportation of the device. The chip also exhibits high durability, which is evidenced from long-duration continuous observation of single channel dynamics. Once engineered protein pores are installed, the chip becomes a robust stochastic sensor for real-time targeting such as detection of the second messenger IP3. This pluggable biochip could be incorporated with many applicable devices, such as a micro-fluidic system, and be made into a micro-array for both biomedical detection and membrane protein research.

The motion of polarizable particles in a non-uniform electric field, i.e., dielectrophoresis, has been extensively used for concentration, separation, sorting, and transport of biological particles, from cancer cells and viruses to biomolecules such as DNAs and proteins. However, current approaches to dielectrophoretic manipulation are not sensitive enough to selectively target individual molecular species. Here we describe the application of the dielectrophoretic principle for selective detection of DNA and RNA molecules using an engineered biological nanopore. The key element of our approach is a synthetic polycationic nanocarrier that selectively binds to the target biomolecules, dramatically increasing their dielectrophoretic response to the electric field gradient generated by the nanopore. The dielectrophoretic capture of the nanocarrier-target complexes is detected as a transient blockade of the nanopore ionic current while any non-target nucleic acids are repelled from the nanopore by electrophoresis and thus do not interfere with the signal produced by the target’s capture. Strikingly, we show that even modestly charged nanocarriers can be used to capture DNA or RNA molecules of any length or secondary structure and simultaneously detect several molecular targets. Such selective, multiplex molecular detection technology would be highly desirable for real-time analysis of complex clinical samples.

Cancer driver mutations are clinically significant biomarkers. In precision medicine, accurate detection of these oncogenic changes in patients would enable early diagnostics of cancer, individually tailored targeted therapy, and precise monitoring of treatment response. Here we investigated a novel nanolock−nanopore method for single-molecule detection of a serine/threonine protein kinase gene BRAF V600E mutation in tumor tissues of thyroid cancer patients. The method lies in a noncovalent, mutation sequence-specific nanolock. We found that the nanolock formed on the mutant allele/probe duplex can separate the duplex dehybridization procedure into two sequential steps in the nanopore. Remarkably, this stepwise unzipping kinetics can produce a unique nanopore electric marker, with which a single DNA molecule of the cancer mutant allele can be unmistakably identified in various backgrounds of the normal wild-type allele. The single-molecule sensitivity for mutant allele enables both binary diagnostics and quantitative analysis of mutation occurrence. In the current configuration, the method can detect the BRAF V600E mutant DNA lower than 1% in the tumor tissues. The nanolock−nanopore method can be adapted to detect a broad spectrum of both transversion and transition DNA mutations, with applications from diagnostics to targeted therapy.