It is shown that the SidSi dimers of the reconstructed Si(001) surface can react with the π bonds of unsaturated organic molecules to produce well-defined organic films with novel physical properties. Scanning tunneling microscopy (STM) studies show that the resulting layers are ordered both translationally and rotationally, with the SidSi dimers acting as a template for extending the translational and rotational order from the silicon substrate to the organic film. STM images and infrared spectroscopy experiments show that by using vicinal Si(001) surface having primarily double-height steps, the rotational order of the molecules can be preserved over macroscopic length scales, leading to measurable anisotropy in optical properties. It is proposed that this chemistry may provide a general method for formation of controlled organic films on Si(001) surfaces.
Two methods for patterning surfaces with supported lipid bilayers and immobilized protein are described. First, proteins are used to fabricate corrals for supported lipid bilayers. Poly(dimethylsiloxane) stamps are used to deposit arbitrarily shaped patterns of thin layers of immobilized protein onto glass surfaces. This is followed by vesicle fusion into the regions that are not coated with proteins. Second, supported bilayer membranes are blotted to remove patterned regions of the membrane, 1 and the blotted regions are filled in or caulked with protein from solution. In both cases, the lipid bilayer regions exhibit lateral fluidity, but each region is confined or corralled by the protein. These two methods can be combined and used iteratively to create arrays with increasing lateral complexity in both the fixed protein and mobilesupported membrane regions for biophysical studies or cell-based assays.
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.
The reactions of 1,3-dienes with the Si(001) surface have been investigated using scanning tunneling microscopy (STM) and Fourier transform infrared spectroscopy (FTIR), and the relative efficiencies of [2 + 2] and [4 + 2] reactions have been determined. STM and FTIR studies show that the 2,3-dimethyl-1,3-butadiene molecule has two bonding configurations; 80% of the molecules bond via a [4 + 2] reaction involving both alkene groups with the remaining 20% bonding via a [2 + 2] reaction involving only one alkene group. The molecule 1,3-cyclohaxadiene shows three separate bonding configurations in the STM, and the FTIR shows at least four separate peaks in the alkene stretching region. The [4 + 2] product is found to comprise 55% of the surface species, the [2 + 2] product 35%, and an unknown product 10%. The surface temperature is found to have little affect on the product distribution. The formation of multiple products and the lack of temperature effects indicate that the product distribution is controlled primarily by the kinetics of the adsorption process, not by the thermodynamics. Thus, although [4 + 2] reactions are predicted to be more stable, [2 + 2] reactions occur nearly as frequently.
DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences. Recently, we reported a singlemolecule 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 oligonucleotidebased 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 singlemolecule 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.single-molecule sequencing | nanopore | DNA sequencing by synthesis | polymer-tagged nucleotides | chip array T he importance of DNA sequencing has increased dramatically from its inception four decades ago. It is recognized as a crucial technology for most areas of biology and medicine and as the underpinning for the new paradigm of personalized and precision medicine. Information on individuals' genomes and epigenomes can help reveal their propensity for disease, clinical prognosis, and response to therapeutics, but routine application of genome sequencing in medicine will require comprehensive data delivered in a timely and cost-effective manner (1). Although 35 years of technological advances have improved sequence throughput and have reduced costs exponentially, genome analysis still takes several days and thousands of dollars to complete (1, 2). To realize the potential of personalized medicine fully, the speed and cost of sequencing must be brought down another order of magnitude while increasing sequencing accuracy and read length. Singlemolecule approaches are thought to be essential to meet these requirements and offer the additional benefit of eliminating amplification bias (3, 4). Although optical methods for singlemolecule sequencing have been achieved and commercialize...
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.
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