Lipid bilayer membrane technologies: A review on single-molecule studies of DNA sequencing by using membrane nanopores

Nanopores based on α-hemolysin and MspA represent attractive sensing platforms due to easy production and operation with relatively low background noise. Such characteristics make them highly favorable for sequencing nucleic acids. Artificial lipid bilayer membranes, also referred to as black lipid membranes, in conjunction with membrane nanopores, can be applied to both the detection and highly efficient sequencing of DNA on a single-molecule level. However, the inherently weak physical properties of the membrane have impeded progress in these areas. Current issues impeding the ultimate recognition of the artificial lipid bilayer as a viable platform for detection and sequencing of DNA include membrane stability, lifespan, and automation. This review (with 105 references) highlights attempts to improve the attributes of the artificial lipid bilayer membrane starting with an overview on the present state and limitations. The first main section covers lipid bilayer membranes (BLM) in general. The following section reviews the various kinds of lipid bilayer membrane platforms with subsections on polymer membranes, solid-supported membranes, hydrogel-encapsulated membranes, shippable and storable membrane platforms, and droplet interface bilayers. A further section covers engineered biological nanopore sensor applications using BLMs with subsections offering a comparative view of different DNA sequencing methods, a detailed look at DNA Sequencing by synthesis using alpha-hemolysin nanopores, sequencing by synthesis using the MspA nanopore and quadromer map, and on limitations of sequencing based on synthesis technology. We present an outlook at the end that discusses current research trends on single-molecule sequencing to highlight the significance of this technology and its potential in the medical and environmental fields.

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Graphical abstract Sequencing by Synthesis, a novel method of sequencing DNA, uses the αHL biological nanopore and the artificial lipid bilayer membrane platform. Polymer tags attached to nucleotides bind to the polymerase-primer–template complex and are characterized by the nanopore upon release.

     

Fluorogenic Granzyme A Substrates Enable Real-Time Imaging of Adaptive Immune Cell Activity

Cytotoxic immune cells, including T lymphocytes (CTLs) and natural killer (NK) cells, are essential components of the host response against tumors. CTLs and NK cells secrete granzyme A (GzmA) upon recognition of cancer cells; however, there are very few tools that can detect physiological levels of active GzmA with high spatiotemporal resolution. Herein, we report the rational design of the near-infrared fluorogenic substrates for human GzmA and mouse GzmA. These activity-based probes display very high catalytic efficiency and selectivity over other granzymes, as shown in tissue lysates from wild-type and GzmA knock-out mice. Furthermore, we demonstrate that the probes can image how adaptive immune cells respond to antigen-driven recognition of cancer cells in real time.     

Synthesis and structure of stilbene crowns.

Syntheses of $\text{E}$- and $\text{Z}$-[a,e]-dibenzo-7,10,13-trioxacyclotrideca-1,3,5-triene, $\text{1}$ (n=1) and $\text{E}$- and $\text{Z}$-[e,a]-dibenzo-7,10,13,16-tetraoxacyclohexadeca-1,3,5-triene, $\text{1}$ (n=2) are described and the X-ray structure of E-$\text{1}$ (n=1) is reported.     

Classifying relative equilibria. III

We announce several theorems on the evolution of relative equilibria classes in the planar n-body problem. In an earlier paper [1] we announced a partial classification of relative equilibria of four equal masses. In [2] we described these new relative equilibria classes and showed the way in which a degeneracy arose in the four body problem. These results point the way toward classifying relative equilibria for any n > 4.Research supported in part by NSF grant MPS-73-03735 A04.     

Folding of small proteins using a single continuous potential

Extensive Monte Carlo folding simulations for four proteins of various structural classes are carried out, using a single continuous potential (united-residue force field). In all cases, collapse occurs at a very early stage, and proteins fold into their nativelike conformations at appropriate temperatures. We also observe that glassy transitions occur at low temperatures. The simulation results demonstrate that the folding mechanism is controlled not only by thermodynamic factors but also by kinetic factors: The way a protein folds into its native structure is also determined by the convergence point of early folding trajectories, which cannot be obtained by the free energy surface.     

Simple curves in and Ahlfors' Schwarzian derivative

We derive sharp injectivity criteria for mappings in terms of Ahlfors' definition of the Schwarzian derivative for such mappings.

     

Fiber interferometer for simultaneous multiwavelength phase measurement with a broadband femtosecond laser

We present a fiber interferometer for the simultaneous measurement of phase at multiple wavelengths from a single broadband femtosecond laser. Narrow-bandwidth fiber Bragg gratings isolate a particular frequency from the broad-bandwidth laser pulse produced. The multiwavelength phase data permit the unambiguous measurement range to be significantly increased compared with the wavelengths used in the interferometer. Preliminary experimental results are presented for a two-frequency sensor with an absolute range of 0.13 mm and associated dynamic range of 43,000:1.     

A photo-responsive organogel

A photo-responsive organogel has been made by addition of a novel stilbene-containing photo-surfactant to toluene: exposure to UV light led to a gel-to-sol transition with spatial control.