Joonil Seog

Surface Induced nanofiber growth by self-assembly of a silk-elastin-like protein polymer.

Many synthetic and natural peptides are known to self-assemble to form various nanostructures. During the self-assembling process, environmental conditions such as salt concentration, pH, temperature, and surface characteristics play a critical role by influencing intermolecular interactions, and hence the process of self-assembly. Here we studied the self-assembly of a genetically engineered protein polymer composed of silk-like and elastin-like repeats on a mica surface. Silk-elastin-like protein polymers (SELPs) consist of tandem repeats of Gly-Ala-Gly-Ala-Gly-Ser from Bombyx mori (silkworm) and Gly-Val-Gly-Val-Pro from mammalian elastin. At a very low polymer concentration of 1 mug/mL, SELPs self-assembled into nanofibrous structures on a mica surface. Examination using atomic force microscopy (AFM) and dynamic light scattering techniques showed that SELPs self-assembled into nanofibers in the presence of the mica surface but not in the bulk state. Ionic strength had a significant influence on nanofiber growth, indicating the importance of electrostatic interactions between the polymer and the mica surface. At low ionic strength, the kinetics of nanofiber growth showed that the mica surface effectively removed a lag phase by providing nucleating sites, facilitating nanofiber self-assembly of SELPs. Furthermore, self-assembly on additional substrates such as silicon and a hydrophobic pyrolytic carbon surface revealed that the charged hydrophilic surface provides the optimal surface to facilitate self-assembly of SELPs.

Atmospheric pressure plasma treatment of lipopolysaccharide in a controlled environment

The atmospheric pressure plasma jet (APPJ) has been widely investigated for sterilization of surfaces, but studies on surface chemical changes of model compounds in controlled environments have been lacking. We present measurements on lipopolysaccharide (LPS) using x-ray photoelectron spectroscopy after 1% O2 in Ar APPJ treatments in controlled ambients composed of N2/Ar mixtures. By varying the N2 concentration from 20% to 100%, we find that the interaction of the jet with the environment plays a major role in modifying surface reactions. This is due to the plasma exciting N2, which quenches reactive oxygen species (ROS) that would otherwise modify the film surface. By minimizing the interaction of the APPJ with the environment, e.g. by changing the APPJ geometry, we show that surface modifications increase even when the plasma itself is removed farther from the LPS surface. Measurements on the biological activity, optical emission, and ozone production of the jet using O2, N2 and O2/N2 admixtures all demonstrate that ROS are readily quenched by N2 species excited by the plasma. These results clearly reveal the importance of considering plasma–environment interactions for APPJ treatments of surfaces.

Nanomechanical stimulus accelerates and directs the self-assembly of silk-elastin-like nanofibers.

One-dimensional nanostructures are ideal building blocks for functional nanoscale assembly. Peptide-based nanofibers have great potential in building smart hierarchical structures due to their tunable structures at the single residue level and their ability to reconfigure themselves in response to environmental stimuli. We observed that pre-adsorbed silk-elastin-based protein polymers self-assemble into nanofibers through conformational changes on a mica substrate. Furthermore, we demonstrate that the rate of self-assembly was significantly enhanced by applying a nanomechanical stimulus using atomic force microscopy. The orientation of the newly grown nanofibers was mostly perpendicular to the scanning direction, implying that the new fiber assembly was locally activated with directional control. Our method provides a novel way to prepare nanofiber patterned substrates using a bottom-up approach.

Enhanced silencing and stabilization of siRNA polyplexes by histidine-mediated hydrogen bonds

Branched peptides containing histidines and lysines (HK) have been shown to be effective carriers for DNA and siRNA. We anticipate that elucidation of the binding mechanism of HK with siRNA will provide greater insight into the self-assembly and delivery of the HK:siRNA polyplex. Non-covalent bonds between histidine residues and nucleic acids may enhance the stability of siRNA polyplexes. We first compared the polyplex biophysical properties of a branched HK with those of branched asparagine-lysine peptide (NK). Consistent with siRNA silencing experiments, gel electrophoresis demonstrated that the HK siRNA polyplex maintained its integrity with prolonged incubation in serum, whereas siRNA in complex with NK was degraded in a time-dependent manner. Isothermal titration calorimetry of various peptides binding to siRNA at pH 7.3 showed that branched polylysine, interacted with siRNA was initially endothermic, whereas branched HK exhibited an exothermic reaction at initial binding. The exothermic interaction indicates formation of non-ionic bonds between histidines and siRNA; purely electrostatic interaction is entropy-driven and endothermic. To investigate the type of non-ionic bond, we studied the protonation state of imidazole rings of a selectively (15)N labeled branched HK by heteronuclear single quantum coherence NMR. The peak of Nδ1-H tautomers of imidazole shifted downfield (in the direction of deprotonation) by 0.5-1.0 ppm with addition of siRNA, providing direct evidence that histidines formed hydrogen bonds with siRNA at physiological pH. These results establish that histidine-rich peptides form hydrogen bonds with siRNA, thereby enhancing the stability and biological activity of the polyplex in vitro and in vivo.

Deactivation of lipopolysaccharide by Ar and H 2 inductively coupled low-pressure plasma

Using an inductively coupled plasma system, we study the effects of direct plasma, plasma-generated high-energy photons in the ultraviolet and vacuum ultraviolet (UV/VUV), and radical treatments on lipopolysaccharide (LPS). LPS is a biomolecule found in the outer membrane of Gram-negative bacteria and a potent stimulator of the immune system composed of polysaccharide and lipid A, which contains six aliphatic chains. LPS film thickness spun on silicon was monitored by ellipsometry while the surface chemistry was characterized before and after treatments by x-ray photoelectron spectroscopy (XPS). Additionally, biological activity was measured using an enzyme-linked immunosorbent assay under (a) a sensitive regime (sub-µM concentrations of LPS) and (b) a bulk regime (above µM concentrations of LPS) after plasma treatments. Direct plasma treatment causes rapid etching and deactivation of LPS in both Ar and H2 feed gases. To examine the effect of UV/VUV photons, a long-pass filter with a cut-off wavelength of 112nm was placed over the sample. H2 UV/VUV treatment causes material removal and deactivation due to atomic and molecular UV/VUV emission while Ar UV/VUV treatment shows minimal effects as Ar plasma does not emit UV/VUV photons in the transmitted wavelength range explored. Interestingly, radical treatments remove negligible material but cause deactivation. Based on the amphiphilic structure of LPS, we expect a lipid A rich surface layer to form at the air‐water interface during sample preparation with polysaccharide layers underneath. XPS shows that H2 plasma treatment under direct and UV/VUV conditions causes oxygen depletion through removal of C‐O and O‐C=O bonds in the films, which does not occur in Ar treatments. Damage to these groups can remove aliphatic chains that contribute to the pyrogenicity of LPS. Radical treatments from both Ar and H2 plasmas remove aliphatic carbon from the near-surface, demonstrating the important role of neutral species.

Directed patterning of the self-assembled silk-elastin-like nanofibers using a nanomechanical stimulus

We investigate the effects of the frequency and density of a nanomechanical stimulus on nucleation and growth of silk-elastin-like protein polymer (SELP) nanofibers. Repetitive tappings are crucial to create nucleation areas and a potential molecular level mechanism was proposed. Using this technique mechanically guided nanofiber patterns were successfully created.

Utilization of simple scaling laws for modulating tip-sample peak forces in atomic force microscopy characterization in liquid environments

The dynamics of atomic force microscopy (AFM) microcantilevers in liquid environments have been previously shown to be extremely complex and nonlinear, exhibiting phenomena such as momentary excitation of higher eigenmodes, fluid-borne excitation, mass loading, and the emergence of sub-harmonic responses. It has also been shown that the signals acquired by the instrument can differ significantly between tip- and base-excited cantilevers in highly damped environments, such that it can be difficult for users to modulate the peak impact forces for base-excited AFM systems that are not equipped with advanced force spectroscopy tools. Despite the dynamic complexity, we show that through understanding of simple scaling laws based on the damped harmonic oscillator model and operation at high amplitude setpoints, it is possible to modulate the tip-sample forces, which could be useful in studies that require experiments involving different but controllable peak force levels.

Polystyrene as a model system to probe the impact of ambient gas chemistry on polymer surface modifications using remote atmospheric pressure plasma under well-controlled conditions

An atmospheric pressure plasma jet (APPJ) was used to treat polystyrene (PS) films under remote conditions where neither the plume nor visible afterglow interacts with the film surface. Carefully controlled conditions were achieved by mounting the APPJ inside a vacuum chamber interfaced to a UHV surface analysis system. PS was chosen as a model system as it contains neither oxygen nor nitrogen, has been extensively studied, and provides insight into how the aromatic structures widespread in biological systems are modified by atmospheric plasma. These remote treatments cause negligible etching and surface roughening, which is promising for treatment of sensitive materials. The surface chemistry was measured by X-ray photoelectron spectroscopy to evaluate how ambient chemistry, feed gas chemistry, and plasma-ambient interaction impact the formation of specific moieties. A variety of oxidized carbon species and low concentrations of NOx species were measured after APPJ treatment. In the remote conditions used in this work, modifications are not attributed to short-lived species, e.g., O atoms. It was found that O3 does not correlate with modifications, suggesting that other long-lived species such as singlet delta oxygen or NOx are important. Indeed, surface-bound NO3 was observed after treatment, which must originate from gas phase NOx as neither N nor O are found in the pristine film. By varying the ambient and feed gas chemistry to produce O-rich and O-poor conditions, a possible correlation between the oxygen and nitrogen composition was established. When oxygen is present in the feed gas or ambient, high levels of oxidation with low concentrations of NO3 on the surface were observed. For O-poor conditions, NO and NO2 were measured, suggesting that these species contribute to the oxidation process, but are easily oxidized when oxygen is present. That is, surface oxidation limits and competes with surface nitridation. Overall, surface oxidation takes place easily, but nitridation only occurs under specific conditions with the overall nitrogen content never exceeding 3%. Possible mechanisms for these processes are discussed. This work demonstrates the need to control plasma-ambient interactions and indicates a potential to take advantage of plasma-ambient interactions to fine-tune the reactive species output of APP sources, which is required for specialized applications, including polymer surface modifications and plasma medicine.

Direct Observation of Dynamic Mechanical Regulation of DNA Condensation by Environmental Stimuli

Gene delivery is a promising way to treat hereditary diseases and cancer; however, there is little understanding of DNA:carrier complex mechanical properties, which may be critical for the protection and release of nucleic acids. We applied optical tweezers to directly measure single-molecule mechanical properties of DNA condensed using 19-mer poly-L-lysine (PLL) or branched histidine-lysine (HK) peptides. Force-extension profiles indicate that both carriers condense DNA actively, showing force plateaus during stretching and relaxation cycles. As the environment such as carrier concentration, pH, and the presence of zinc ions changes, DNA:HK complexes showed dynamically regulated mechanical properties at multiple force levels. The fundamental knowledge from this study can be applied to design a mechanically tailored complex which may enhance transfection efficiency by controlling the stability of the complex temporally and spatially.

Folding behavior of a T-shaped, ribosome-binding translation enhancer implicated in a wide-spread conformational switch

Turnip crinkle virus contains a T-shaped, ribosome-binding, translation enhancer (TSS) in its 3’UTR that serves as a hub for interactions throughout the region. The viral RNA-dependent RNA polymerase (RdRp) causes the TSS/surrounding region to undergo a conformational shift postulated to inhibit translation. Using optical tweezers (OT) and steered molecular dynamic simulations (SMD), we found that the unusual stability of pseudoknotted element H4a/Ψ3 required five upstream adenylates, and H4a/Ψ3 was necessary for cooperative association of two other hairpins (H5/H4b) in Mg2+. SMD recapitulated the TSS unfolding order in the absence of Mg2+, showed dependence of the resistance to pulling on the 3D orientation and gave structural insights into the measured contour lengths of the TSS structure elements. Adenylate mutations eliminated one-site RdRp binding to the 3’UTR, suggesting that RdRp binding to the adenylates disrupts H4a/Ψ3, leading to loss of H5/H4b interaction and promoting a conformational switch interrupting translation and promoting replication. DOI: http://dx.doi.org/10.7554/eLife.22883.001