Nanopore sequencing has the potential to become a direct, fast, and inexpensive DNA sequencing technology. The simplest form of nanopore DNA sequencing utilizes the hypothesis that individual nucleotides of single-stranded DNA passing through a nanopore will uniquely modulate an ionic current flowing through the pore, allowing the record of the current to yield the DNA sequence. We demonstrate that the ionic current through the engineered Mycobacterium smegmatis porin A, MspA, has the ability to distinguish all four DNA nucleotides and resolve single-nucleotides in single-stranded DNA when double-stranded DNA temporarily holds the nucleotides in the pore constriction. Passing DNA with a series of double-stranded sections through MspA provides proof of principle of a simple DNA sequencing method using a nanopore. These findings highlight the importance of MspA in the future of nanopore sequencing.bionanotechnology | next generation sequencing | single-molecule | stochastic sensing | protein pore T he information encoded in DNA is of paramount importance to medicine and to the life sciences. The mapping of the human genome is revolutionizing the understanding of genetic disorders and the prediction of disease and will aid in developing therapies as in refs. 1-3. The ability to sequence DNA quickly and inexpensively is essential to individualized medicine and to scientific research and has prompted the development of new sequencing techniques beyond the original Sanger sequencing (4-7). Nanopore DNA sequencing represents one of the approaches being developed to rapidly sequence a human genome for under $1,000 (www.genome.gov/12513210).In the most elementary form of nanopore DNA sequencing, a nanometer-scale pore provides the sole pathway for an ionic current. Single-stranded DNA (ssDNA) is electrophoretically driven through the pore, and as the ssDNA passes through, it reduces the ionic current through the pore. If each passing nucleotide yields a characteristic residual ionic current then the record of the current will correspond to the DNA sequence. This simple and reagent-free sequencing technique holds the promise to inexpensively read long lengths of DNA molecules at intrinsically fast rates (8). Due to its inherently small size, this system is amenable to parallelization (9).Lately, nanopore sequencing techniques have progressed substantially. This progress and the remaining challenges in nanopore DNA sequencing are summarized in a review article by Branton et al. (8). While nanotechnology usually involves materials such as Si and SiN, nanopore DNA sequencing first evolved using the well-studied protein porin α-hemolysin (10). In contrast to pores made from inorganic materials (11, 12), protein pores can be easily modified and produced with repeatable subnanometer accuracy. Stoddart (13) and Purnell (14) demonstrated that several locations within the beta barrel of α-hemolysin exhibit nucleotide-specific sensitivity with immobilized ssDNA (13). However, α-hemolysin's 5 nm-long cylindrical beta barrel presents...

Membranes containing a wide variety of ternary mixtures of high chain-melting temperature lipids, low chain-melting temperature lipids, and cholesterol undergo lateral phase separation into coexisting liquid phases at a miscibility transition. When membranes are prepared from a ternary lipid mixture at a critical composition, they pass through a miscibility critical point at the transition temperature. Since the critical temperature is typically on the order of room temperature, membranes provide an unusual opportunity in which to perform a quantitative study of biophysical systems that exhibit critical phenomena in the two-dimensional Ising universality class. As a critical point is approached from either high or low temperature, the scale of fluctuations in lipid composition, set by the correlation length, diverges. In addition, as a critical point is approached from low temperature, the line tension between coexisting phases decreases to zero. Here we quantitatively evaluate the temperature dependence of line tension between liquid domains and of fluctuation correlation lengths in lipid membranes to extract a critical exponent, nu. We obtain nu = 1.2 +/- 0.2, consistent with the Ising model prediction nu = 1. We also evaluate the probability distributions of pixel intensities in fluorescence images of membranes. From the temperature dependence of these distributions above the critical temperature, we extract an independent critical exponent of beta = 0.124 +/- 0.03, which is consistent with the Ising prediction of beta = 1/8.

Plasma membranes of cells are asymmetric in both lipid and protein composition. The mechanism by which proteins on both sides of the membrane colocalize during signaling events is unknown but may be due to the induction of inner leaflet domains by the outer leaflet. Here we show that liquid domains form in asymmetric Montal-Mueller planar bilayers in which one leaflet's composition would phase-separate in a symmetric bilayer and the other's would not. Equally important, by tuning the lipid composition of the second leaflet, we are able to suppress domains in the first leaflet. When domains are present in asymmetric membranes, each leaflet contains regions of three distinct lipid compositions, implying strong interleaflet interactions. Our results show that mechanisms of domain induction between the outer and inner leaflets of cell plasma membranes do not necessarily require the participation of membrane proteins. Based on these findings, we suggest mechanisms by which cells could actively regulate protein function by modulating local lipid composition or interleaflet interactions.cholesterol ͉ induction ͉ raft ͉ membrane ͉ phase C ell membranes are asymmetric in lipid composition between the inner and outer leaflet (1). Lipids of the two leaflets are assumed to also differ in their ability to separate into domains enriched in particular lipids and proteins. Model membranes composed with the goal to mimic the lipid mixture of the outer leaflet of a cell membrane separate into two liquid phases (2-4), whereas at least one inner leaflet mixture does not (5). To explain colocalization of inner and outer leaflet proteins during signaling events, it has been hypothesized that domains in the outer leaflet induce domains in the inner leaflet (6-8). However, domain induction across membrane leaflets is controversial. Some researchers argue that only proteins should be able to induce domains across leaflets (9). Other groups, including our own, have observed what seems to be domain induction across leaflets of protein-free membranes deposited on solid surfaces, but generally these efforts have been frustrated by experimental difficulties (7, 10-12).Here we construct asymmetric, protein-free, planar bilayers in water. One leaflet's composition would phase-separate in a symmetric bilayer and the other's would not. We show that liquid domains form in both leaflets of the asymmetric bilayer. One leaflet induces phase separation in the other. Equally important, we show that changing the lipid composition of the second leaflet suppresses domain formation in the original leaflet. We find that leaflets are strongly coupled. Our results imply that cells could tune membrane composition to create or annihilate domains. Because induction of domains occurs in planar membranes devoid of membrane proteins, induction of domains in cell membranes need not rely on membrane curvature or protein coupling (9,13,14).We construct membranes of diphytanoylphosphatidylcholine (DiPhyPC), dipalmitoylphosphatidylcholine (DPPC), and cholesterol (Chol)...

Formation of a water-expelling nonpolar core is the paradigm of protein folding and stability. Although experiment largely confirms this picture, water buried in “hydrophobic” cavities is required for the function of some proteins. Hydration of the protein core has also been suggested as the mechanism of pressure-induced unfolding. We therefore are led to ask whether even the most nonpolar protein core is truly hydrophobic (i.e., water-repelling). To answer this question we probed the hydration of an ≈160-Å 3 , highly hydrophobic cavity created by mutation in T4 lysozyme by using high-pressure crystallography and molecular dynamics simulation. We show that application of modest pressure causes approximately four water molecules to enter the cavity while the protein itself remains essentially unchanged. The highly cooperative filling is primarily due to a small change in bulk water activity, which implies that changing solvent conditions or, equivalently, cavity polarity can dramatically affect interior hydration of proteins and thereby influence both protein activity and folding.