We describe a novel single molecule nanopore-based sequencing by synthesis (Nano-SBS) strategy that can accurately distinguish four bases by detecting 4 different sized tags released from 5′-phosphate-modified nucleotides. The basic principle is as follows. As each nucleotide is incorporated into the growing DNA strand during the polymerase reaction, its tag is released and enters a nanopore in release order. This produces a unique ionic current blockade signature due to the tag's distinct chemical structure, thereby determining DNA sequence electronically at single molecule level with single base resolution. As proof of principle, we attached four different length PEG-coumarin tags to the terminal phosphate of 2′-deoxyguanosine-5′-tetraphosphate. We demonstrate efficient, accurate incorporation of the nucleotide analogs during the polymerase reaction, and excellent discrimination among the four tags based on nanopore ionic currents. This approach coupled with polymerase attached to the nanopores in an array format should yield a single-molecule electronic Nano-SBS platform.
Solid-binding peptides (SBPs) recognizing inorganic and synthetic interfaces have enabled a broad range of materials science applications and hold promise as adhesive or morphogenetic control units that can be genetically encoded within desirable or designed protein frameworks. To date, the underlying relationships governing both SBP–surface and SBP–SBP interactions and how they give rise to different adsorption mechanisms remain unclear. Here, we combine protein engineering, surface plasmon resonance characterization, and molecular dynamics (MD) simulations initiated from Rosetta predictions to gain insights on the interplay of amino acid composition, structure, self-association, and adhesion modality in a panel of variants of the Car9 silica-binding peptide (DSARGFKKPGKR) fused to the C-terminus of superfolder green fluorescent protein (sfGFP). Analysis of kinetics, energetics, and MD-predicted structures shows that the high-affinity binding of Car9 to the silanol-rich surface of silica is dominated by electrostatic contributions and a spectrum of several persistent interactions that, along with a high surface population of bound molecules, promote cooperative interactions between neighboring SBPs and higher order structure formation. Transition from cooperative to Langmuir adhesion in sfGFP-Car9 variants occurs in concert with a reduction of stable surface interactions and self-association, as confirmed by atomic force microscopy imaging of proteins exhibiting the two different binding behaviors. We discuss the implications of these results for the de novo design of SBP–surface binding systems.
At-will tailoring of the formation and reconfiguration of hierarchical structures is a key goal of modern nanomaterial design. Bioinspired systems comprising biomacromolecules and inorganic nanoparticles have potential for new functional material structures. Yet, consequential challenges remain because we lack a detailed understanding of the temporal and spatial interplay between participants when it is mediated by fundamental physicochemical interactions over a wide range of scales. Motivated by a system in which silica nanoparticles are reversibly and repeatedly assembled using a homobifunctional solid-binding protein and single-unit pH changes under near-neutral solution conditions, we develop a theoretical framework where interactions at the molecular and macroscopic scales are rigorously coupled based on colloidal theory and atomistic molecular dynamics simulations. We integrate these interactions into a predictive coarse-grained model that captures the pH-dependent reversibility and accurately matches small-angle X-ray scattering experiments at collective scales. The framework lays a foundation to connect microscopic details with the macroscopic behavior of complex bioinspired material systems and to control their behavior through an understanding of both equilibrium and nonequilibrium characteristics.
Combinatorially selected solid-binding peptides (SBPs) provide a versatile route for synthesizing advanced materials and devices, especially when they are installed within structurally or functionally useful protein scaffolds. However, their promise has not been fully realized because we lack a predictive understanding of SBP-material interactions. Thermodynamic and kinetic binding parameters obtained by fitting quartz crystal microbalance and surface plasmon resonance (SPR) data with the Langmuir model whose assumptions are rarely satisfied provide limited information on underpinning molecular interactions. Using SPR, we show here that a technologically useful SBP called Car9 confers proteins to which is fused a sigmoidal adsorption behavior modulated by partner identity, quaternary structure, and ionic strength. We develop a two-step cooperative model that accurately captures the kinetics of silica binding and provides insights into how SBP−SBP interactions, fused scaffold, and solution conditions modulate adsorption. Because cooperative binding can be converted to Langmuir adhesion by mutagenesis, our approach offers a path to identify and to better understand and design practically useful SBPs.
We investigated the effect of two commonly studied surfactants, sodium dodecyl sulfate (SDS) and dodecyl trimethylammonium bromide (C12TAB), on skin barrier properties. Using skin conductivity, FT-IR of stratum corneum samples, and penetration of radiolabelled SDS, we determined that addition of C12TAB lowers the ability of SDS to perturb skin’s barrier properties. Ultrafiltration experiments revealed that addition of C12TAB serves to decrease the concentration of monomers and sub-micellar aggregates. None of the measured skin properties including enhancement of skin conductivity, perturbation of lipid structure and skin concentration of SDS correlated with the total SDS concentration in the donor compartment (i.e., the total SDS concentration). However, all these parameters correlated well against the concentration of monomers and sub-micellar aggregates. These findings provide the evidence of the importance of monomer and sub-micellar components in altering skin barrier properties.
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