Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array.

DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences. Recently, we reported a single-molecule nanopore-based SBS strategy that accurately distinguishes four bases by electronically detecting and differentiating four different polymer tags attached to the 5'-phosphate of the nucleotides during their incorporation into a growing DNA strand catalyzed by DNA polymerase. Further developing this approach, we report here the use of nucleotides tagged at the terminal phosphate with oligonucleotide-based polymers to perform nanopore SBS on an α-hemolysin nanopore array platform. We designed and synthesized several polymer-tagged nucleotides using tags that produce different electrical current blockade levels and verified they are active substrates for DNA polymerase. A highly processive DNA polymerase was conjugated to the nanopore, and the conjugates were complexed with primer/template DNA and inserted into lipid bilayers over individually addressable electrodes of the nanopore chip. When an incoming complementary-tagged nucleotide forms a tight ternary complex with the primer/template and polymerase, the tag enters the pore, and the current blockade level is measured. The levels displayed by the four nucleotides tagged with four different polymers captured in the nanopore in such ternary complexes were clearly distinguishable and sequence-specific, enabling continuous sequence determination during the polymerase reaction. Thus, real-time single-molecule electronic DNA sequencing data with single-base resolution were obtained. The use of these polymer-tagged nucleotides, combined with polymerase tethering to nanopores and multiplexed nanopore sensors, should lead to new high-throughput sequencing methods.

Dynamic Reorganization and Correlation among Lipid Raft Components.

Lipid rafts are widely believed to be an essential organizational motif in cell membranes. However, direct evidence for interactions among lipid and/or protein components believed to be associated with rafts is quite limited owing, in part, to the small size and intrinsically dynamic interactions that lead to raft formation. Here, we exploit the single negative charge on the monosialoganglioside GM1, commonly associated with rafts, to create a gradient of GM1 in response to an electric field applied parallel to a patterned supported lipid bilayer. The composition of this gradient is visualized by imaging mass spectrometry using a NanoSIMS. Using this analytical method, added cholesterol and sphingomyelin, both neutral and not themselves displaced by the electric field, are observed to reorganize with GM1. This dynamic reorganization provides direct evidence for an attractive interaction among these raft components into some sort of cluster. At steady state we obtain an estimate for the composition of this cluster.

Clustering of alpha-synuclein on supported lipid bilayers: role of anionic lipid, protein, and divalent ion concentration.

Alpha-synuclein is the major component of Lewy body inclusions found in the brains of patients with Parkinson's disease. Several studies indicate that alpha-synuclein binds to negatively charged phospholipid bilayers. We examined the binding of alpha-synuclein to membranes containing different amounts of negatively charged lipids using supported lipid bilayers, epifluorescence microscopy, fluorescence recovery after photobleaching, and bulk fluorescence techniques. The membranes contained phosphatidylcholine and phosphatidylglycerol. In the absence of protein, these lipids mix uniformly. Our results show that the propensity of alpha-synuclein to cluster on the membrane increases as the concentration of anionic lipid and/or protein increases. Regions on the lipid bilayer where alpha-synuclein is clustered are enriched in phosphatidylglycerol. We also observe divalent metal ions stimulate protein cluster formation, primarily by promoting lipid demixing. The importance of protein structure, lipid demixing, and divalent ions, as well as the physiological implications, will be discussed. Because membrane-bound alpha-synuclein assemblies may play a role in neurotoxicity, it is of interest to determine how membranes can be used to tune the propensity of alpha-synuclein to aggregate.

Path dependence of three-phase or two-phase end points in fluid binary lipid mixtures.

The phase behavior of anionic/zwitterionic mixtures can be controlled by tuning the charge state of the anionic lipid. In the case of dioleoylphosphatidic acid (DOPA)/dioleoylphosphatidylcholine (DOPC) mixtures, demixing occurs either when DOPA is protonated or when DOPA(2-):Ca(2+) complexes form. Herein it will be shown that the final end point, a three-phase or two-phase system, depends on the order in which the charge state is manipulated. The facile accessibility of different end points is a clear demonstration of the inherent flexibility of biological systems.

Controlling the charge and organization of anionic lipid bilayers: effect of monovalent and divalent ions.

It is shown that the organization of lipid bilayers containing phosphatidic acid (PA) and phosphatidlycholine (PC) can be controlled by altering the monovalent and divalent ion concentrations. At high pH and/or calcium concentration, 1:1 Ca(2+)-PA(2-) complexes form; these complexes demix, and PA-rich and PC-rich regions are observable with epifluorescence microscopy. The results are compared with predictions from electrostatic theory. It is noted that the complex formation correlates in a roughly linear fashion with the monovalent/divalent ion ratio, a parameter that cells adjust.

Using ionic strength to control liquid-ordered/liquid-disordered separation.

In this Letter, we will show that liquid-ordered/liquid-disordered separation can be controlled with ionic strength. Using this observation, a robust method was developed for creating visible, by fluorescence microscopy, liquid-ordered domains in supported lipid bilayers. The details of the method will be discussed.

Effect of salt concentration on membrane lysis pressure.

Cell membranes are capable of withstanding significant osmotic stress, the exact amount of which varies with the lipid composition. In this paper, we examine the effect that salt concentration has on the lysis pressure of membranes containing anionic lipids. Vesicles containing varying amounts of phosphatidylcholine and phosphatidylglycerol were osmotically stressed using NaCl as the osmolyte. The lysis pressure was observed to vary linearly with the Debye screening length and the extent of the variation was linear with anionic lipid content. The implications these results have for cells that frequently encounter low solute environments are discussed.

α-Synuclein-induced tubule formation in lipid bilayers.

α-Synuclein is a presynaptic protein that binds to phospholipid membranes and is involved in the pathogenesis of Parkinson's disease (PD). In this paper, we describe the effects of adding wild-type α-synuclein (WT) and three familial PD mutants (A53T, A30P, and E46K) to membranes containing 15-35 mol % anionic lipid. Tubules were observed to form in the membranes to an extent that depended on the α-synuclein variant, the anionic lipid content, and the protein concentration. For all four variants, tubule formation decreased with increasing anionic lipid content. Tubules were more readily observed with A30P and E46K than with WT or A53T. The results are consistent with a model wherein the helical content of α-synuclein increases with increasing anionic lipid content, and α-synuclein conformers with low helical content have a high propensity to induce tubule formation. This work, combined with previous work from our laboratory (Pandey et al. Biophys. J. 2009, 96, 540), shows that WT adsorption of the protein has deleterious effects on the membrane when the anionic lipid concentration is less than 30 mol % (tubule formation) or greater than 40 mol % (reorganization of the bilayer, clustering of protein).

Adsorption of alpha-synuclein on lipid bilayers: modulating the structure and stability of protein assemblies.

The interaction of alpha-synuclein with phospholipid membranes has been examined using supported lipid bilayers and epi-fluorescence microscopy. The membranes contained phosphatidylcholine (PC) and phosphatidic acid (PA), which mix at physiological pH. Upon protein adsorption, the lipids undergo fluid-fluid phase separation into PC-rich and PA-rich regions. The protein preferentially adsorbs to the PA-rich regions. The adsorption and subsequent aggregation of alpha-synuclein was probed by tuning several parameters: the charge on the lipids, the charge on the protein, and the screening environment. Conditions which promoted the greatest extent of adsorption resulted in structurally heterogeneous aggregates, while comparatively homogeneous aggregates were observed under conditions whereby adsorption did not occur as readily. Our observation that different alterations to the system lead to different degrees of aggregation and different aggregate structures poses a challenge for drug discovery. Namely, therapies aimed at neutralizing alpha-synuclein must target a broad range of potentially toxic, membrane-bound assemblies.

Formation of complex three-dimensional structures in supported lipid bilayers.

Cell membranes are continually undergoing a wide range of shape transformations. Here, we demonstrate the formation of several structures in supported bilayers, including tubules, caps, and giant multivesicular structures. The key elements required for these transformations are osmotic pressure imbalances, insertion of lipids with positive curvature, and lipids whose curvature is dependent on the screening environment. With these elements, a wide variety of transformations can be achieved in the absence of protein.

Formation of three-dimensional structures in supported lipid bilayers.

The creation of three-dimensional structures in supported lipid bilayers has been examined. In bilayers, shape transformations can be triggered by adjusting a variety of parameters. Here, it is shown that bilayers composed of phosphatidylcholine and phosphatidic acid can be induced to reversibly form cap structures when exposed to an asymmetry in ionic strength. The structures that form depend on the asymmetry in the ionic strength and the amount of anionic lipid. Other factors that may be of importance in the creation of the structures, expansion forces, osmotic forces, and the bilayer-support interaction are discussed. The cap structures have the potential to be of considerable utility in examining the effect that curvature has on membrane processes.

Effect of ions on the organization of phosphatidylcholine/phosphatidic acid bilayers.

Lipid bilayers are two-dimensional fluids. Here, the effect of monovalent ion concentration on the mixing, and consequently the organization, of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) bilayers has been examined. Epifluorescence microscopy was used to visualize the organization. Fluorescence recovery after photobleaching and attenuated total reflection-Fourier transform infrared spectroscopy were used to assess the fluidity of the lipids. At high ionic strength the DOPC and DOPA lipids appear uniformly mixed. Upon lowering the ionic strength, rapid separation is observed. The DOPA-rich regions appear fractal-like and exhibit hysteresis in their properties. The lipids freely exchange between the two regions. These experiments clearly demonstrate the significant effect that electrostatics can have on membrane organization.

Effect of surface treatment on diffusion and domain formation in supported lipid bilayers.

Supported lipid bilayers are widely used as model systems due to their robustness. Due to the solid support, the properties of supported lipid bilayers are different from those of freestanding bilayers. In this article, we examine whether different surface treatments affect the properties of supported lipid bilayers. It will be shown that depending on the treatment method, the diffusion of the lipids can be adjusted approximately threefold without altering the composition. Additionally, as the bilayer-support interaction decreases, it becomes easier to form coexisting liquid-ordered and liquid-disordered domains. The physical/chemical alterations that result from the different treatment methods will be discussed.

Influence of lipid chemistry on membrane fluidity: tail and headgroup interactions.

Membrane fluidity plays an important role in cell function and may, in many instances, be adjusted to facilitate specific cellular processes. To understand better the effect that lipid chemistry has on membrane fluidity the inclusion of three different lipids into egg phosphatidylcholine (eggPC) bilayers has been examined; the three lipids are egg phosphatidylethanolamine ((eggPE) made by transphosphatidylation of eggPC in the presence of ethanolamine), lyso-phosphatidylcholine (LPC), and lyso-phosphatidylethanolamine (LPE). The fluidity of the membranes was determined using fluorescence recovery after photobleaching and the intermolecular interactions were examined using attenuated total reflection Fourier transform infrared spectroscopy. It was observed that both headgroup and tail chemistry can significantly modulate lipid diffusion. Specifically, the inclusion of LPC and eggPE significantly altered the lipid diffusion, increased and decreased, respectively, whereas the inclusion of LPE had an intermediate effect, a slight decrease in diffusion. Strong evidence for the formation of hydrogen-bonds between the phosphate group and the amine group in eggPE and LPE was observed with infrared spectroscopy. The biological implications of these results are discussed.

Infrared spectroscopy of fluid lipid bilayers.

Infrared spectroscopy is a powerful technique for examining lipid bilayers; however, it says little about the fluidity of the bilayer-a key physical aspect. It is shown here that it is possible to both acquire spectroscopic data of supported lipid bilayer samples and make measurements of the membrane fluidity. Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FT-IR) is used to obtain the spectroscopic information and fluorescence recovery after photobleaching (FRAP) is used to determine the fluidity of the samples. In the infrared spectra of lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, the following major peaks were observed; nu(as)(CH3) 2954 cm(-1), nu(s)(CH3) 2870 cm(-1), nu(as)(CH2) 2924 cm(-1), nu(s)(CH2) 2852 cm(-1), nu(C=O) 1734 cm(-1), delta(CH2) 1463-1473 cm(-1), nu(as)(PO2-) 1226 cm(-1), nu(s)(PO2-) 1084 cm(-1), and nu(as)(N+(CH3)3) 973 cm(-1). The diffusion coefficient of the same lipid bilayer was measured to be 3.5 +/- 0.5 micom(2)/s with visual recovery also noted through use of epifluorescence microscopy. FRAP and visual data confirm the formation of a uniform, mobile supported lipid bilayer. The combination of ATR-FT-IR and FRAP provides complementary data giving a more complete picture of fully hydrated model membrane systems.