Single-molecule analysis of phospholipid scrambling by TMEM16F.

Transmembrane protein 16F (TMEM16F) is a Ca2+-dependent phospholipid scramblase that translocates phospholipids bidirectionally between the leaflets of the plasma membrane. Phospholipid scrambling of TMEM16F causes exposure of phosphatidylserine in activated platelets to induce blood clotting and in differentiated osteoblasts to promote bone mineralization. Despite the importance of TMEM16F-mediated phospholipid scrambling in various biological reactions, the fundamental features of the scrambling reaction remain elusive due to technical difficulties in the preparation of a platform for assaying scramblase activity in vitro. Here, we established a method to express and purify mouse TMEM16F as a dimeric molecule by constructing a stable cell line and developed a microarray containing membrane bilayers with asymmetrically distributed phospholipids as a platform for single-molecule scramblase assays. The purified TMEM16F was integrated into the microarray, and monitoring of phospholipid translocation showed that a single TMEM16F molecule transported phospholipids nonspecifically between the membrane bilayers in a Ca2+-dependent manner. Thermodynamic analysis of the reaction indicated that TMEM16F transported 4.5 × 104 lipids per second at 25 °C, with an activation free energy of 47 kJ/mol. These biophysical features were similar to those observed with channels, which transport substrates by facilitating diffusion, and supported the stepping-stone model for the TMEM16F phospholipid scramblase.

Single-molecule Imaging Analysis of Binding, Processive Movement, and Dissociation of Cellobiohydrolase Trichoderma reesei Cel6A and Its Domains on Crystalline Cellulose.

Trichoderma reesei Cel6A (TrCel6A) is a cellobiohydrolase that hydrolyzes crystalline cellulose into cellobiose. Here we directly observed the reaction cycle (binding, surface movement, and dissociation) of single-molecule intact TrCel6A, isolated catalytic domain (CD), cellulose-binding module (CBM), and CBM and linker (CBM-linker) on crystalline cellulose Iα The CBM-linker showed a binding rate constant almost half that of intact TrCel6A, whereas those of the CD and CBM were only one-tenth of intact TrCel6A. These results indicate that the glycosylated linker region largely contributes to initial binding on crystalline cellulose. After binding, all samples showed slow and fast dissociations, likely caused by the two different bound states due to the heterogeneity of cellulose surface. The CBM showed much higher specificity to the high affinity site than to the low affinity site, whereas the CD did not, suggesting that the CBM leads the CD to the hydrophobic surface of crystalline cellulose. On the cellulose surface, intact molecules showed slow processive movements (8.8 ± 5.5 nm/s) and fast diffusional movements (30-40 nm/s), whereas the CBM-Linker, CD, and a catalytically inactive full-length mutant showed only fast diffusional movements. These results suggest that both direct binding and surface diffusion contribute to searching of the hydrolysable point of cellulose chains. The duration time constant for the processive movement was 7.7 s, and processivity was estimated as 68 ± 42. Our results reveal the role of each domain in the elementary steps of the reaction cycle and provide the first direct evidence of the processive movement of TrCel6A on crystalline cellulose.

Single-molecule imaging analysis of elementary reaction steps of Trichoderma reesei cellobiohydrolase I (Cel7A) hydrolyzing crystalline cellulose Iα and IIII.

Trichoderma reesei cellobiohydrolase I (TrCel7A) is a molecular motor that directly hydrolyzes crystalline celluloses into water-soluble cellobioses. It has recently drawn attention as a tool that could be used to convert cellulosic materials into biofuel. However, detailed mechanisms of action, including elementary reaction steps such as binding, processive hydrolysis, and dissociation, have not been thoroughly explored because of the inherent challenges associated with monitoring reactions occurring at the solid/liquid interface. The crystalline cellulose Iα and IIII were previously reported as substrates with different crystalline forms and different susceptibilities to hydrolysis by TrCel7A. In this study, we observed that different susceptibilities of cellulose Iα and IIII are highly dependent on enzyme concentration, and at nanomolar enzyme concentration, TrCel7A shows similar rates of hydrolysis against cellulose Iα and IIII. Using single-molecule fluorescence microscopy and high speed atomic force microscopy, we also determined kinetic constants of the elementary reaction steps for TrCel7A against cellulose Iα and IIII. These measurements were performed at picomolar enzyme concentration in which density of TrCel7A on crystalline cellulose was very low. Under this condition, TrCel7A displayed similar binding and dissociation rate constants for cellulose Iα and IIII and similar fractions of productive binding on cellulose Iα and IIII. Furthermore, once productively bound, TrCel7A processively hydrolyzes and moves along cellulose Iα and IIII with similar translational rates. With structural models of cellulose Iα and IIII, we propose that different susceptibilities at high TrCel7A concentration arise from surface properties of substrate, including ratio of hydrophobic surface and number of available lanes.

On-Chip Enrichment System for Digital Bioassay Based on Aqueous Two-Phase System.

We developed an on-chip enrichment method based on an aqueous two-phase system of dextran/polyethylene glycol mix, DEX/PEG ATPS, for digital bioassay. Accordingly, we prepared an array device of femtoliter reactors that displays millions of uniformly shaped DEX-rich droplets under a PEG-rich medium. The DEX-rich droplets effectively enriched DNA molecules from the PEG-rich medium. To quantify the enrichment power of the system, we performed a digital bioassay of alkaline phosphatase (ALP). Upon genetically tagging ALP molecules with the DEX-binding domain (DBD) derived from dextransucrase, the ALP molecules were enriched 59-fold in the DEX droplets in comparison to that in a conventional digital bioassay. Subsequently, we performed a Cas13-based digital SARS-CoV-2 RNA detection assay to evaluate the performance of this system for a more practical assay. In this assay, the target RNA molecules bound to the DBD-tagged Cas13 molecules were effectively enriched in the DEX droplets. Consequently, an enrichment factor of 31 was achieved. Enrichment experiments for nonlabeled proteins were also performed to test the expandability of this technique. The model protein, nontagged ß-galactosidase, was enriched in DEX droplets containing DBD-tagged antibody, with an enrichment factor of over 100. Thus, this system enabled effective on-chip enrichment of target molecules to enhance the detection sensitivity of digital bioassays without using external instruments or an external power source, which would be applicable for on-site bioassays or portable diagnostic tests.

Ion permeation by a folded multiblock amphiphilic oligomer achieved by hierarchical construction of self-assembled nanopores.

A multiblock amphiphilic molecule 1, with a tetrameric alternating sequence of hydrophilic and hydrophobic units, adopts a folded structure in a liposomal membrane like a multipass transmembrane protein, and is able to transport alkali metal cations through the membrane. Hill's analysis and conductance measurements, analyzed by the Hille equation, revealed that the tetrameric assembly of 1 forms a 0.53 nm channel allowing for permeation of cations. Since neither 3, bearing flexible hydrophobic units and forming no stacked structures in the membrane, nor 2, a monomeric version of 1, is able to transport cations, the folded conformation of 1 in the membrane is likely essential for realizing its function. Thus, function and hierarchically formed higher-order structures of 1, is strongly correlated with each other like proteins and other biological macromolecules.

A microreactor sealing method using adhesive tape for digital bioassays.

Digital assays using microreactors fabricated on solid substrates are useful for carrying out sensitive assays of infectious diseases and other biological tests. However, sealing of the microchambers using fluid oil is difficult for non-experts, and thus hinders the widespread use of digital microreactor assays. Here, we propose the physical isolation of tiny reactors with adhesive tape (PITAT) using simple, commercially available pressure-sensitive adhesive (PSA) tape as a separator of the microreactors. We confirmed that PSA tape can effectively seal the microreactors and prevent molecules from diffusing out. By testing several types of adhesive tape, we found that rubber-based adhesives are the most suitable for this purpose. In addition, we demonstrated that single-molecule enzyme assays can be successfully performed inside microreactors sealed with PSA tape. The results obtained using PITAT are quantitatively comparable to conventional oil sealing, although it is quick and cost-effective. Finally, we demonstrated that single-particle virus counting of the influenza virus can be achieved using PITAT. Collectively, our results suggest that PITAT may be suitable for use in the design of sensitive tests for infectious diseases at the point of care, where no sophisticated equipment or machines are available.

Ultrafast water permeation through nanochannels with a densely fluorous interior surface.

Ultrafast water permeation in aquaporins is promoted by their hydrophobic interior surface. Polytetrafluoroethylene has a dense fluorine surface, leading to its strong water repellence. We report a series of fluorous oligoamide nanorings with interior diameters ranging from 0.9 to 1.9 nanometers. These nanorings undergo supramolecular polymerization in phospholipid bilayer membranes to form fluorous nanochannels, the interior walls of which are densely covered with fluorine atoms. The nanochannel with the smallest diameter exhibits a water permeation flux that is two orders of magnitude greater than those of aquaporins and carbon nanotubes. The proposed nanochannel exhibits negligible chloride ion (Cl-) permeability caused by a powerful electrostatic barrier provided by the electrostatically negative fluorous interior surface. Thus, this nanochannel is expected to show nearly perfect salt reflectance for desalination.

Imidazolinium-based Multiblock Amphiphile as Transmembrane Anion Transporter.

Transmembrane anion transport is an important biological process in maintaining cellular functions. Thus, synthetic anion transporters are widely developed for their biological applications. Imidazolinium was introduced as anion recognition site to a multiblock amphiphilic structure that consists of octa(ethylene glycol) and aromatic units. Ion transport assay using halide-sensitive lucigenin and pH-sensitive 8-hydroxypyrene-1,3,6-trisulfonate (HPTS) revealed that imidazolinium-based multiblock amphiphile (IMA) transports anions and showed high selectivity for nitrate, which plays crucial roles in many biological events. Temperature-dependent ion transport assay using 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) indicated that IMA works as a mobile carrier. 1 H NMR titration experiments indicated that the C2 proton of the imidazolinium ring recognizes anions via a (C-H)+ ⋅⋅⋅X- hydrogen bond. Furthermore, all-atom molecular dynamics simulations revealed a dynamic feature of IMA within the membranes during ion transportation.

A single-molecule enzymatic assay in a directly accessible femtoliter droplet array.

The enzyme assay in a femtoliter chamber array is a simple and efficient method for concentrating the reaction product; it greatly improves the detection sensitivity down to the single-molecule level. However, in previous methods, controlling the initiation and termination of the reaction in each chamber is difficult once enclosed. Furthermore, the recovery of the enzyme and product is also difficult. To overcome these drawbacks, we developed a femtoliter droplet array in which the individual droplets are fixed on the substrate and are directly accessible from outside. A hydrophilic-in-hydrophobic micropatterned surface was used for the preparation of the droplets. When the aqueous solution on the surface is exchanged with oil, the hydrophilic surface retains the aqueous solution, and more than 10(6) dome-shaped droplets that are usable for further assay can be prepared simultaneously. The curvature radius of the droplet obeys the Young-Laplace equation, and the volume can be precisely controlled by the micropipette, which applies pressure into the droplet. Changing the pressure makes the addition, collection, and exchange of the aqueous content for individual droplets possible. Using these advantages, we successfully measured the kinetic parameters of the single-molecule enzyme ß-galactosidase and rotary motor protein F(1)-ATPase enclosed in a droplet.

Simultaneous optical and electrical single channel recordings on a PEG glass.

Single molecule imaging of working ion-channels is much more difficult than that of water-soluble proteins because of the fragile nature of membranes and lateral diffusion of particles in the membranes, which does not allow fluorescent contamination for optical single channel recording. In this report, we reconstituted maxi-potassium channels from porcine uterine smooth muscle into artificial planar bilayers formed on poly(ethylene glycol) (PEG) modified glass and performed simultaneous optical and electrical recording of the single channels. The channels were immobilized in the membranes by anchoring to PEG molecules on the glass. The technique developed in this study should pave the way for single molecule pharmacology of ion-channels.

Monodisperse Liposomes with Femtoliter Volume Enable Quantitative Digital Bioassays of Membrane Transporters and Cell-Free Gene Expression.

Digital bioassays have emerged as a new category of bioanalysis. However, digital bioassays for membrane transporter proteins have not been well established yet despite high demands in molecular physiology and molecular pharmacology due to the lack of biologically functional monodisperse liposomes with femtoliter volumes. Here, we established a simple and robust method to produce femtoliter-sized liposomes (femto-liposomes). We prepared 106 monodispersed water-in-oil droplets stabilized by a lipid monolayer using a polyethylene glycol-coated femtoliter reactor array device. Droplets were subjected to the optimized emulsion transfer process for femto-liposome production. Liposomes were monodispersed (coefficient of variation = 5-15%) and had suitable diameter (0.6-5.3 µm) and uniform volumes of subfemtoliter or a few femtoliters; thus, they were termed uniform femto-liposomes. The unilamellarity of uniform femto-liposomes allowed quantitative single-molecule analysis of passive and active transporter proteins: α-hemolysin and FoF1-ATPase. Digital gene expression in uniform femto-liposomes (cell-free transcription and translation from single DNA molecules) was also demonstrated, showing the versatility of digital assays for membrane transporter proteins and cell-free synthetic biology.

Design of Sealable Custom-Shaped Cell Mimicries Based on Self-Assembled Monolayers on CYTOP Polymer.

In bottom-up synthetic biology, one of the major methodological challenges is to provide reaction spaces that mimic biological systems with regard to topology and surface functionality. Of particular interest are cell- or organelle-shaped membrane compartments, as many protein functions unfold at lipid interfaces. However, shaping artificial cell systems using materials with non-intrusive physicochemical properties, while maintaining flexible lipid interfaces relevant to the reconstituted protein systems, is not straightforward. Herein, we develop micropatterned chambers from CYTOP, a less commonly used polymer with good chemical resistance and a refractive index matching that of water. By forming a self-assembled lipid monolayer on the polymer surface, we dramatically increased the biocompatibility of CYTOP-fabricated systems. The phospholipid interface provides an excellent passivation layer to prevent protein adhesion to the hydrophobic surface, and we succeeded in cell-free protein synthesis inside the chambers. Importantly, the chambers could be sealed after loading by a lipid monolayer, providing a novel platform to study encapsulated systems. We successfully reconstituted pole-to-pole oscillations of the Escherichia coli MinDE system, which responds dramatically to compartment geometry. Furthermore, we present a simplified fabrication of our artificial cell compartments via replica molding, making it a readily accessible technique for standard cleanroom facilities.

Electrophysiological recordings of single ion channels in planar lipid bilayers using a polymethyl methacrylate microfluidic chip.

Planar lipid bilayers are used for functional studies of ion channel proteins using electrophysiological techniques. We have been developing a plastic micro-fluidic device for the reconstitution of planar lipid bilayers and electrophysiological recordings toward a "membrane protein chip" for high-throughput screening. In the previous report [Suzuki, H., Tabata, K.V., Noji, H., Takeuchi, S., 2006. Highly reproducible method of planar lipid bilayer reconstitution in polymethyl methacrylate microfluidic chip. Langmuir 22 (4), 1937-1942], we presented the method and device in which the reproducibility of planar lipid bilayers reached 90%, and multiple bilayers were formed simultaneously. In this communication, we show that our device has excellent electric properties suitable for ion channel analysis down to single molecular level. Additional aspects on the optical accessibility and controllability on lipid bilayer formation are also presented.

Micro patterning of active proteins with perforated PDMS sheets (PDMS sieve).

We propose a novel technique for patterning active proteins on a glass substrate using a perforated polydimethylsiloxane (PDMS) sheet-sieve. The sieve, which has tapering holes, is fabricated by spin-coating PDMS on a pyramidal-shaped mold. By means of this sieve, FITC (fluorescent isothiocyanate, bovine)-albumin was successfully spotted in a 5 x 5 microm(2) area in an array. The patterned spots were perfectly isolated, which eliminates the problem of non-specific binding of proteins to undesired areas. To show that proteins maintained their activity after the patterning, we used F(1)-ATPase biomolecular motors; their activity can easily be verified by observing their rotary motion after patterning. Selective patterning with three kinds of fluorescent micro beads indicated the possibility of patterning of different proteins on the same substrate by using the sieve.

Planar lipid bilayer reconstitution with a micro-fluidic system.

A planar lipid bilayer which is widely used for the electrophysiological study of membrane proteins in laboratories is reconstituted using a micro-fluidic system, in a manner that is suitable for automated processing. We fabricated micro-channels on both sides of the substrate, which are connected through a 100-200 microm aperture, and showed that the bilayer can be formed at the aperture by flowing the lipid solution and buffer, alternately. Parylene coating is found to be suitable for both bilayer formation and electric noise reduction. Future applications include a high-sensitivity ion sensor chip and a high-throughput drug screening device.

Large-scale femtoliter droplet array for digital counting of single biomolecules.

We present a novel device employing one million femtoliter droplets immobilized on a substrate for the quantitative detection of extremely low concentrations of biomolecules in a sample. Surface-modified polystyrene beads carrying either zero or a single biomolecule-reporter enzyme complex are efficiently isolated into femtoliter droplets formed on hydrophilic-in-hydrophobic surfaces. Using a conventional micropipette, this is achieved by sequential injection first with an aqueous solution containing beads, and then with fluorinated oil. The concentration of target biomolecules is estimated from the ratio of the number of signal-emitting droplets to the total number of trapped beads (digital counting). The performance of our digital counting device was demonstrated by detecting a streptavidin-ß-galactosidase conjugate with a limit of detection (LOD) of 10 zM. The sensitivity of our device was >20-fold higher than that noted in previous studies where a smaller number of reactors (fifty thousand reactors) were used. Such a low LOD was achieved because of the large number of droplets in an array, allowing simultaneous examination of a large number of beads. When combined with bead-based enzyme-linked immunosorbent assay (digital ELISA), the LOD for the detection of prostate specific antigen reached 2 aM. This value, again, was improved over that noted in a previous study, because of the decreased coefficient of variance of the background measurement determined by the Poisson noise. Our digital counting device using one million droplets has great potential as a highly sensitive, portable immunoassay device that could be used to diagnose diseases.

Mechanism of inhibition by C-terminal alpha-helices of the epsilon subunit of Escherichia coli FoF1-ATP synthase.

The epsilon subunit of bacterial FoF1-ATP synthase (FoF1), a rotary motor protein, is known to inhibit the ATP hydrolysis reaction of this enzyme. The inhibitory effect is modulated by the conformation of the C-terminal alpha-helices of epsilon, and the "extended" but not "hairpin-folded" state is responsible for inhibition. Although the inhibition of ATP hydrolysis by the C-terminal domain of epsilon has been extensively studied, the effect on ATP synthesis is not fully understood. In this study, we generated an Escherichia coli FoF1 (EFoF1) mutant in which the epsilon subunit lacked the C-terminal domain (FoF1epsilonDeltaC), and ATP synthesis driven by acid-base transition (DeltapH) and the K+-valinomycin diffusion potential (DeltaPsi) was compared in detail with that of the wild-type enzyme (FoF1epsilonWT). The turnover numbers (kcat) of FoF1epsilonWT were severalfold lower than those of FoF1epsilonDeltaC. FoF1epsilonWT showed higher Michaelis constants (Km). The dependence of the activities of FoF1epsilonWT and FoF1epsilonDeltaC on various combinations of DeltapH and DeltaPsi was similar, suggesting that the rate-limiting step in ATP synthesis was unaltered by the C-terminal domain of epsilon. Solubilized FoF1epsilonWT also showed lower kcat and higher Km values for ATP hydrolysis than the corresponding values of FoF1epsilonDeltaC. These results suggest that the C-terminal domain of the epsilon subunit of EFoF1 slows multiple elementary steps in both the ATP synthesis/hydrolysis reactions by restricting the rotation of the gamma subunit.

Lipid bilayer microarray for parallel recording of transmembrane ion currents.

This paper describes a multiwell biochip for simultaneous parallel recording of ion current through transmembrane pores reconstituted in planar lipid bilayer arrays. Use of a thin poly(p-xylylene) (parylene) film having micrometer-sized apertures (phi=15-50 microm, t=20 microm) led to formation of highly stable bilayer lipid membranes (BLMs) for incorporation of transmembrane pores; thus, a large number of BLMs could be arrayed without any skillful technique. We optically confirmed the simultaneous formation of BLMs in a 5x5 matrix, and in our durability test, the BLM lasted more than 15 h. Simultaneous parallel recording of alamethicin and gramicidin transmembrane pores in multiple contiguous recording sites demonstrated the feasibility of high-throughput screening of transmembrane ion currents in artificial lipid bilayers.

Loop-mediated isothermal amplification of a single DNA molecule in polyacrylamide gel-based microchamber.

Loop-mediated isothermal amplification (LAMP) is an original nucleic acid amplification method established by Notomi et al. LAMP is performed under isothermal condition, employing only a basic reaction protocol and minimal supporting electronics. These requirements prove to be viable for exploring the avenues to down-scale this biological reaction for Lab-on-a-chip application. Hence here, we developed a novel technique for fluorescent imaging of LAMP at a single molecule level. The experiment was conducted in a polyacrylamide (PAA) gel-based microchamber where a single DNA template, freely suspended in a solution containing primers and polymerase was initially encapsulated. In order to activate the amplification reaction, a microheater regulated by an automatic computerized feedback system was used for localized heating. This microchamber-based approach for LAMP demonstrated the effective exploitation of minute amount of templates and primers, and the overall reduction in LAMP detection time. An average efficiency of 80% was evaluated for conducting DNA amplification after 50 min of incubation at 65 degrees C. As the total time for reaction including detection can be completed in less than 1 h, this one-step, direct observation method displays the potential as a simple alternative to conventional techniques for genetic analysis and diagnosis in the clinical laboratory.