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Hydrogels with spatial-temporal control over chemical and physical properties allow for the creation of cellular niches with controllable properties that better mimic tissue environments. Here, we present a protocol for synthesizing hydrogels incorporating photocaged oligonucleotides that can be activated with non-ultraviolet (UV) wavelengths. We detail the synthesis of bulk hydrogels and spatially defined hydrogels with different chemical functionalities that all share common photocaged DNA. Furthermore, we describe conditions for activating photocaged DNA to capture complementary oligonucleotides. For complete details on the use and execution of this protocol, please refer to Mathis et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The creation of complex cellular environments is critical to mimicking tissue environments that will play a critical role in next-generation tissue engineering, stem cell programming, and therapeutic screening. To address this growing need, techniques capable of manipulating cell-cell and cell-material interactions are required that span single-cell to 3D tissue architectures. DNA programmed assembly and placement of cells present a powerful technique for the bottom-up synthesis of living microtissues for probing key questions in cell-cell and cell-material-driven behaviors through its refined control over placement and architecture. This review examines the current state of the art in the programming of cellular interactions with DNA and its applications spanning tissue model building, fundamental cellular biology, and cell manipulation for measurements across a host of applications., (© 2024 Wiley‐VCH GmbH.)

The mechanical properties and forces of the extracellular environment modulate alveolar epithelial cell behavior. To model cancer/fibrosis associated stiffening and dynamic stretch, a biomimetic device was developed that imitates the active forces in the alveolus, while allowing control over the interstitial matrix stiffness. Alveolar epithelial A549 cancer cells were cultured on the devices and their transcriptome was profiled with RNA sequencing. Pathway analysis showed soft materials upregulated the expression of proteoglycans associated with cancer. Consequently, liposomes were modified with peptides targeting heparan sulfate and chondroitin sulfates of the cell surface glycocalyx. Chondroitin sulfate A targeting improved uptake in cells seeded on stiff biomimetic devices, which is attributed to increased chondroitin sulfate proteoglycan localization on cell surfaces in comparison to cells grown on soft devices. These results demonstrate the critical role that mechanical stiffness and stretch play in the alveolus and the importance of including these properties in nanotherapeutic design., Competing Interests: The authors declare no competing financial interest., (© 2024 The Author(s).)

Statistical analyses of homologous protein sequences can identify amino acid residue positions that co-evolve to generate family members with different properties. Based on the hypothesis that the coevolution of residue positions is necessary for maintaining protein structure, coevolutionary traits revealed by statistical models provide insight into residue-residue interactions that are important for understanding protein mechanisms at the molecular level. With the rapid expansion of genome sequencing databases that facilitate statistical analyses, this sequence-based approach has been used to study a broad range of protein families. An emerging application of this approach is to design hybrid transcriptional regulators as modular genetic sensors for novel wiring between input signals and genetic elements to control outputs. Among many allosterically regulated regulator families, the members contain structurally conserved and functionally independent protein domains, including a DNA-binding module (DBM) for interacting with a specific genetic element and a ligand-binding module (LBM) for sensing an input signal. By hybridizing a DBM and an LBM from two different family members, a hybrid regulator can be created with a new combination of signal-detection and DNA-recognition properties not present in natural systems. In this review, we present recent advances in the development of hybrid regulators and their applications in cellular engineering, especially focusing on the use of statistical analyses for characterizing DBM-LBM interactions and hybrid regulator design. Based on these studies, we then discuss the current limitations and potential directions for enhancing the impact of this sequence-based design approach.

Recreating tissue environments with precise control over mechanical, biochemical, and cellular organization is essential for next-generation tissue models for drug discovery, development studies, and the replication of disease environments. However, controlling these properties at cell-scale lengths remains challenging. Here, we report the development of printing approaches that leverage polyethylene glycol diacrylate (PEGDA) hydrogels containing photocaged oligonucleotides to spatially program material characteristics with non-destructive, non-ultraviolet light. We further integrate this system with a perfusion chamber to allow us to alter the composition of PEGDA hydrogels while retaining common light-activatable photocaged DNAs. We demonstrate that the hydrogels can capture DNA functionalized materials, including cells coated with complementary oligonucleotides with spatial control using biocompatible wavelengths. Overall, these materials open pathways to orthogonal capture of any DNA functionalized materials while not changing the sequences of the DNA.

Determining the mechanistic causes of lung diseases, developing new treatments thereof, and assessing toxicity whether from chemical exposures or engineered nanomaterials would benefit significantly from a preclinical human lung alveolar interstitium model of physiological relevance. The existing preclinical models have limitations because they fail to replicate the key anatomical and physiological characteristics of human alveoli. Thus, a human lung alveolar interstitium chip was developed to imitate key alveolar microenvironmental factors including an electrospun nanofibrous membrane as the analogue of the basement membrane for co-culture of epithelial cells with fibroblasts embedded in 3D collagenous gels, physiologically relevant interstitial matrix stiffness, interstitial fluid flow, and 3D breathing-like mechanical stretch. The biomimetic chip substantially improved the epithelial barrier function compared to transwell models. Moreover, the chip having a gel made of a collagen I-fibrin blend as the interstitial matrix sustained the interstitium integrity and further enhanced the epithelial barrier, resulting in a longevity that extended beyond eight weeks. The assessment of multiwalled carbon nanotube toxicity on the chip was in line with the animal study.

The interactions between heterogeneous cell populations play important roles in dictating various cell behaviors. Cell-cell contact mediates communication through the exchange of signaling molecules, electrical coupling, and direct membrane-linked ligand-receptor interactions. In vitro culturing of multiple cell types with control over their specific arrangement is difficult, especially in three-dimensional (3D) systems. While techniques that allow one to control the arrangement of cells and direct contact between different cell types have been developed that expand upon simple co-culture methods, specific control over heterojunctions that form between cells is not easily accomplished with current methods, such as 3D cell-printing. In this article, DNA-mediated cell interactions are combined with cell-compatible photolithographic approaches to control cell assembly. Specifically, cells are coated with oligonucleotides containing DNA nucleobases that are protected with photocleavable moieties; this coating facilitated light-controlled cell assembly when these cells were mixed with cells coated with complementary oligonucleotides. By combining this technology with digital micromirror devices mounted on a microscope, selective activation of specific cell populations for interactions with other cells was achieved. Importantly, this technique is rapid and uses non-UV light sources. Taken together, this technique opens new pathways for on-demand programming of complex cell structures., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Regulating cell behavior using nanotopography has been widely implemented. To facilitate cell adhesion, physical nanotopography is usually coated with adhesive proteins such as fibronectin (FN). However, the confounding effects of physical and biochemical cues of nanotopography hinder the understanding of nanotopography in regulating cell behavior, which ultimately limits the biomedical applications of nanotopography. To delineate the roles of the physical and biochemical cues in cell regulation, we fabricate substrates that have either the same physical nanotopography but different biochemical (FN) nanopatterns or identical FN nanopatterns but different physical nanotopographies. We then examine the influences of physical and biochemical cues of nanotopography on spreading, nuclear deformation, mechanotransduction, and function of human mesenchymal stem cells (hMSCs). Our results reveal that physical topographies, especially nanogratings, dominantly control cell spreading, YAP localization, proliferation, and differentiation of hMSCs. However, biochemical FN nanopatterns affect hMSC elongation, YAP intracellular localization, and lamin a/c (LAMAC) expression. Furthermore, we find that physical nanogratings induce nanoscale curvature of nuclei at the basal side, which attenuates the osteogenic differentiation of hMSCs. Collectively, our study highlights the dominant effect of physical nanotopography in regulating stem cell functions, while suggesting that fine-tuning of cell behavior can be achieved through altering the presentation of biochemical cues on substrate surfaces.

The plasma membrane and the actomyosin cytoskeleton play key roles in controlling how cells sense and interact with their surrounding environment. Myosin, a force-generating actin network-associated protein, is a major regulator of plasma membrane tension, which helps control endocytosis. Despite the important link between plasma membranes and actomyosin (the actin-myosin complex), little is known about how the actomyosin arrangement regulates endocytosis. Here, nanoscopic ligand arrangements defined by polymer pen lithography (PPL) are used to control actomyosin contractility and examine cell uptake. Confocal microscopy, atomic force microscopy, and flow cytometry suggest that the cytoskeletal tension imposed by the nanoscopic ligand arrangement can actively regulate cellular uptake through clathrin- and caveolin-mediated pathways. Specifically, ligand arrangements that increase cytoskeletal tension tend to reduce the cellular uptakes of cholera toxin (CTX) and spherical nucleic acids (SNAs) by regulating endocytic budding and limiting the formation of clathrin- and caveolae-coated pits. Collectively, this work demonstrates how the cell endocytic fate is regulated by actomyosin mechanical forces, which can be tuned by subcellular cues defined by PPL., Competing Interests: The authors declare the following competing financial interest(s): C.A.M. has financial interests in TERA-print LLC, which could potentially benefit from the outcomes of this research. The other authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Maximizing the tissue-targeting efficiency of nanomaterials while also protecting them from rapid clearance from the bloodstream and limiting their immunogenicity remains a central problem in the field of systemic-administered nanomedicine. Herein, we introduce a generalizable strategy to simultaneously increase tumor accumulation, prolong blood circulation, and limit nonspecific immune activation of nanomaterials via peptide-based, tumor-responsive, "sheddable" coatings. Spherical nucleic acids (SNAs) were designed and synthesized to contain an exterior coating composed of zwitterionic polypeptides with recognition sequences for tumor-associated proteases. In the presence of matrix metalloproteinases (MMPs), the polypetide coating is rapidly cleaved, leading to increased cellular uptake of these SNAs, relative to SNAs containing nonsheddable shells. Moreover, the zwitterionic nature of the polypeptide shell shields the SNAs from immune system recognition, which extends their blood circulation time and improves tumor accumulation and in vivo cellular uptake relative to control SNAs with no protective coating. Taken together, these results indicate that this strategy is a viable method for increasing nanoparticle tumor accumulation and can have utility for the systemic delivery of oligonucleotides and nanomaterials to target cells in vivo with low immunogenicity.

Abnormal scarring is a consequence of dysregulation in the wound healing process, with limited options for effective and noninvasive therapies. Given the ability of spherical nucleic acids (SNAs) to penetrate skin and regulate gene expression within, we investigated whether gold-core SNAs (AuSNAs) and liposome-core SNAs (LSNAs) bearing antisense oligonucleotides targeting transforming growth factor beta 1 (TGF- β 1) can function as a topical therapy for scarring. Importantly, both SNA constructs appreciably downregulated TGF- β 1 protein expression in primary hypertrophic and keloid scar fibroblasts in vitro. In vivo, topically applied AuSNAs and LSNAs downregulated TGF- β 1 protein expression levels and improved scar histology as determined by the scar elevation index. These data underscore the potential of SNAs as a localized, self-manageable treatment for skin-related diseases and disorders that are driven by increased gene expression., Competing Interests: The authors declare the following competing financial interest(s): C.A.M. has financial interests in Exicure, Inc, which could potentially benefit from the outcomes of this research.

A novel method for synthesizing arrays of uniform sub-2 nm particles on substrates is described. Such particles are made by (i) using dip-pen nanolithography to prepare nanoreactors consisting of metal-coordinated polymers; (ii) designing polymers with only one metal atom attached to each polymer chain; (iii) systematically controlling nanoreactor volume down to the yoctoliter scale; and (iv) transforming each nanoreactor into a metal nanoparticle through thermal annealing. Polymer design in this study is crucial, since it allows one to tightly control nanoparticle size by tuning the volume of the polymer reactors, which correlates with the number of polymer chains and, therefore, metal atoms. Mixtures of different metal-functionalized polymers were used to synthesize ultrasmall alloy particles. The technique and results described herein point toward a way of using these novel polymers to systematically explore the properties and uses of this important class of nanomaterials in many fields.

Spherical nucleic acids (SNAs) are nanomaterials typically consisting of a nanoparticle core and a functional, dense, and highly oriented oligonucleotide shell with unusual biological properties that make them appealing for many applications, including sequence-specific gene silencing, mRNA quantification, and immunostimulation. When placed in biological fluids, SNAs readily interact with serum proteins, leading to the formation of ill-defined protein coronae on the surface, which can influence the targeting capabilities of the conjugate. In this work, SNAs were designed and synthesized with functional proteins, such as antibodies and serum albumin, deliberately adsorbed onto their surfaces. These particles exhibit increased resistance to protease degradation compared with native SNAs but still remain functional, as they can engage in hybridization with complementary oligonucleotides. SNAs with adsorbed targeting antibodies exhibit improved cellular selectivity within mixed cell populations. Similarly, SNAs coated with the dysopsonizing protein serum albumin show reduced macrophage uptake, providing a strategy for tailoring selective SNA delivery. Importantly, the protein coronae remain stable on the SNAs in human serum, exhibiting a less than 45% loss of protein through exchange after 12 h at 37 °C. Taken together, these results show that protein-SNA complexes and the method used to prepare them provide a new avenue for enhancing SNA stability, targeting, and biodistribution., Competing Interests: The authors declare no competing financial interest., (Copyright © 2019 American Chemical Society.)

Although microscale patterning techniques have been used to control cell morphology and shape, they only provide indirect control over the formation of the subcellular cytoskeletal elements that determine contractility. This paper addresses the hypotheses that nanoscale anisotropic features of a patterned matrix can direct the alignment of internal cytoskeletal actin fibers within a confined shape with an unbiased aspect ratio, and that this enhanced control over cytoskeletal architecture directs programmed cell behaviors. Here, large-area polymer pen lithography is used to pattern substrates with nanoscale extracellular matrix protein features and to identify cues that can be used to direct cytoskeletal organization in human mesenchymal stem cells. This nanopatterning approach is used to identify how anisotropic focal adhesions around the periphery of symmetric patterns yield an organized and contractile actin cytoskeleton. This work reports the important finding that anisotropic cues that increase cell contractility within a circular shape redirect cell differentiation from an adipogenic to an osteogenic fate. Together, these experiments introduce a programmable approach for using subcellular spatial cues to control cell behavior within defined geometries.

Nanomaterials that form as heterostructures have applications in catalysis, plasmonics, and electronics. Multielement nanoparticles can now be synthesized through a variety of routes, but how thermodynamic phases form in such structures and how specific interfaces between them can be designed and synthesized are still poorly understood. We explored how palladium-tin alloys form mixed-composition phases with metals with known but complex miscibilities. Nanoparticles with up to seven elements were synthesized, and many form triphase heterostructures consisting of either three-interface or two-interface architectures. Density functional theory calculations and experimental work were used to determine the balance between the surface and interfacial energies of the observed phases. From these observations, design rules have been established for making polyelemental systems with specific heterostructures, including tetraphase nanoparticles with as many as six junctions., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The nanomaterial landscape is so vast that a high-throughput combinatorial approach is required to understand structure-function relationships. To address this challenge, an approach for the synthesis and screening of megalibraries of unique nanoscale features (>10,000,000) with tailorable location, size, and composition has been developed. Polymer pen lithography, a parallel lithographic technique, is combined with an ink spray-coating method to create pen arrays, where each pen has a different but deliberately chosen quantity and composition of ink. With this technique, gradients of Au-Cu bimetallic nanoparticles have been synthesized and then screened for activity by in situ Raman spectroscopy with respect to single-walled carbon nanotube (SWNT) growth. Au 3 Cu, a composition not previously known to catalyze SWNT growth, has been identified as the most active composition., Competing Interests: Conflict of interest statement: K.A.B. and C.A.M. have financial interests in TERA-print, LLC, which could potentially benefit from the outcomes of this research. All other authors declare no competing interests.

The interactions between nanoparticles and solvents play a critical role in the formation of complex, metastable nanostructures. However, direct observation of such interactions with high spatial and temporal resolution is challenging with conventional liquid-cell transmission electron microscopy (TEM) experiments. Here, a windowless system consisting of polymer nanoreactors deposited via scanning probe block copolymer lithography (SPBCL) on an amorphous carbon film is used to investigate the coarsening of ultrafine (1-3 nm) Au-Pt bimetallic nanoparticles as a function of solvent evaporation. In such reactors, homogeneous Au-Pt nanoparticles are synthesized from metal-ion precursors in situ under electron irradiation. The nonuniform evaporation of the thin polymer film not only concentrates the nanoparticles but also accelerates the coalescence kinetics at the receding polymer edges. Qualitative analysis of the particle forces influencing coalescence suggests that capillary dragging by the polymer edges plays a significant role in accelerating this process. Taken together, this work (1) provides fundamental insight into the role of solvents in the chemistry and coarsening behavior of nanoparticles during the synthesis of polyelemental nanostructures, (2) provides insight into how particles form via the SPBCL process, and (3) shows how SPBCL-generated domes, instead of liquid cells, can be used to study nanoparticle formation. More generally, it shows why conventional models of particle coarsening, which do not take into account solvent evaporation, cannot be used to describe what is occurring in thin film, liquid-based syntheses of nanostructures.

Liposomal spherical nucleic acids (LSNAs) are an attractive therapeutic platform for gene regulation and immunomodulation due to their biocompatibility, chemically tunable structures, and ability to enter cells rapidly without the need for ancillary transfection agents. Such structures consist of small (<100 nm) liposomal cores functionalized with a dense, highly oriented nucleic acid shell, both of which are key components in facilitating their biological activity. Here, the properties of LSNAs synthesized using conventional methods, anchoring cholesterol terminated oligonucleotides into a liposomal core, are compared to LSNAs made by directly modifying the surface of a liposomal core containing azide-functionalized lipids with dibenzocyclooctyl-terminated oligonucleotides. The surface densities of the oligonucleotides are measured for both types of LSNAs, with the lipid-modified structures having approximately twice the oligonucleotide surface coverage. The stabilities and cellular uptake properties of these structures are also evaluated. The higher density, lipid-functionalized structures are markedly more stable than conventional cholesterol-based structures in the presence of other unmodified liposomes and serum proteins as evidenced by fluorescence assays. Significantly, this new form of LSNA exhibits more rapid cellular uptake and increased sequence-specific toll-like receptor activation in immune reporter cell lines, making it a promising candidate for immunotherapy., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A high-throughput, solution-based, scanning-probe photochemical nanopatterning approach, which does not require the use of probes with subwavelength apertures, is reported. Specifically, pyramid arrays made from high-refractive-index polymeric materials were constructed and studied as patterning tools in a conventional liquid-phase beam pen lithography experiment. Two versions of the arrays were explored with either metal-coated or metal-free tips. Importantly, light can be channeled through both types of tips and the appropriate solution phase (e.g., H 2 O or CH 3 OH) and focused on subwavelength regions of a substrate to effect a photoreaction in solution that results in localized patterning of a self-assembled monolayer (SAM)-coated Au thin film substrate. Arrays with as many as 4500 pyramid-shaped probes were used to simultaneously initiate thousands of localized free-radical photoreactions (decomposition of a lithium acylphosphinate photoinitiator in an aqueous solution) that result in oxidative removal of the SAM. The technique is attractive since it allows one to rapidly generate features less than 200 nm in diameter, and the metal-free tips afford more than 10-fold higher intensity than the tips with nanoapertures over a micrometer propagation length. In principle, this mask-free method can be utilized as a versatile tool for performing a wide variety of photochemistries across multiple scales that may be important in high-throughput combinatorial screening applications related to chemistry, biology, and materials science.

Recent developments in scanning probe block copolymer lithography (SPBCL) enable the confinement of multiple metal precursors in a polymer nanoreactor and their subsequent transformation into a single multimetallic heterostructured nanoparticle through thermal annealing. However, the process by which multimetallic nanoparticles form in SPBCL-patterned nanoreactors remains unclear. Here, we utilize the combination of PEO-b-P2VP and Au, Ag, and Cu salts as a model three-component system to investigate this process. The data suggest that the formation of single-component Au, Ag, or Cu nanoparticles within polymer nanoreactors consists of two stages: (I) nucleation, growth, and coarsening of the particles to yield a single particle in each reactor; (II) continued particle growth by depletion of the remaining precursor in the reactor until the particle reaches a stable size. Also, different aggregation rates are observed for single-component particle formation (Au > Ag > Cu). This behavior is also observed for two-component systems, where nucleation sites have greater Au content than the other metals. This information can be used to trap nanoparticles with kinetic structures. High-temperature treatment ultimately facilitates the structural evolution of the kinetic particle into a particle with a fixed structure. Therefore, with multicomponent systems, a third stage that involves elemental redistribution within the particle must be part of the description of the synthetic process. This work not only provides a glimpse at the mechanism underlying multicomponent nanoparticle formation in SPBCL-generated nanoreactors but also illustrates, for the first time, the utility of SPBCL as a platform for controlling the architectural evolution of multimetallic nanoparticles in general.

Multicomponent nanoparticles can be synthesized with either homogeneous or phase-segregated architectures depending on the synthesis conditions and elements incorporated. To understand the parameters that determine their structural fate, multicomponent metal-oxide nanoparticles consisting of combinations of Co, Ni, and Cu were synthesized by using scanning probe block copolymer lithography and characterized using correlated electron microscopy. These studies revealed that the miscibility, ratio of the metallic components, and the synthesis temperature determine the crystal structure and architecture of the nanoparticles. A Co-Ni-O system forms a rock salt structure largely owing to the miscibility of CoO and NiO, while Cu-Ni-O, which has large miscibility gaps, forms either homogeneous oxides, heterojunctions, or alloys depending on the annealing temperature and composition. Moreover, a higher-ordered structure, Co-Ni-Cu-O, was found to follow the behavior of lower ordered systems., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A one-pot synthesis of micellar spherical nucleic acid (SNA) nanostructures using Pluronic F127 as a thermoresponsive template is reported. These novel constructs are synthesized in a chemically straightforward process that involves intercalation of the lipid tails of DNA amphiphiles (CpG motifs for TLR-9 stimulation) into the hydrophobic regions of Pluronic F127 micelles, followed by chemical cross-linking and subsequent removal of non-cross-linked structures. The dense nucleic acid shell of the resulting cross-linked micellar SNA enhances their stability in physiological media and facilitates their rapid cellular internalization, making them effective TLR-9 immunomodulatory agents. These constructs underscore the potential of SNAs in regulating immune response and address the relative lack of stability of noncovalent constructs.

DNA can be manipulated to design nano-machines through specific sequence recognition. We report a switchable DNA carrier for repeatable capture and release of a single stranded DNA. The activity of the carrier was regulated by the interactions among a double-stranded actuator, single stranded target, fuel, and anti-fuel DNA strands. Inosine was used to maintain a stable triple-stranded complex when the actuator's conformation was switched between open (capture) and closed (release) configurations. Time lapse fluorescence measurements show repeatable capture and release of target strands. TEM images also show visible capture of target DNA strands when gold nanoparticles were attached to the DNA carrier and the target DNA strand. The carrier activity was controlled by length of toeholds, number of mismatches, and inosine substitutions. Significantly, unlike in previously published work that reported the devices functioned only when there is a perfect match between the interacting DNA strands, the present device works only when there are mismatches in the fuel strand and the best performance is achieved for 1-3 mismatches. The device was used to successfully capture and release gold nanoparticles when linked to the target single-stranded DNA. In general, this type of devices can be used for transport and delivery of theranostic molecules.

Connexin proteins form hexameric assemblies known as hemichannels. When docked to form gap junction (GJ) channels, hemichannels play a critical role in cell-cell communication and cellular homeostasis, but often are functional entities on their own in unapposed cell membranes. Defects in the Connexin26 (Cx26) gene are the major cause of hereditary deafness arising from dysfunctional hemichannels in the cochlea. Structural studies of Cx26 hemichannels properly trafficked and inserted in plasma membranes, including their clustering that forms a plaque-like feature in whole gap junctions, are limited. We used atomic force microscopy (AFM) to study the surface topography of Cx26 hemichannels using two different membrane preparations. Rat Cx26 containing appended carboxy terminal V5 and hexahistidine tags were expressed in baculovirus/Sf9 cell systems. The expressed Cx26 proteins form hemichannels in situ in Sf9 cells that were then purified either as (1) Sf9 membrane fragments containing Cx26 hemichannels or (2) solubilized hemichannels. The latter were subsequently reconstituted in liposomes. AFM images of purified membrane fragments showed clusters of protein macromolecular structures in the membrane that at higher magnification corresponded to Cx26 hemichannels. Hemichannels reconstituted into DOPC bilayers displayed two populations of channel heights likely resulting from differences in orientations of inserted hemichannels. Hemichannels in the protein rich portions of purified membranes also showed a reduced channel height above the bilayer compared to membranes with reconstituted hemichannels perhaps due to reduced AFM probe access to the lipid bilayer. These preparations of purified membranes enriched for connexin hemichannels that have been properly trafficked and inserted in membranes provide a platform for high-resolution AFM imaging of the structure, interconnexon interactions, and cooperativity of properly trafficked and inserted noncrystalline connexin hemichannels.

Current work in tuning DNA kinetics has focused on changing toehold lengths and DNA concentrations. However, kinetics can also be improved by enhancing the completion probability of the strand displacement process. Here, we execute this strategy by creating a toehold DNA motor device with the inclusion of a synthetic nucleotide, inosine, at selected sites. Furthermore, we found that the energetic bias can be tuned such that the device can stay in a stable partially displaced state. This work demonstrates the utility of energetic biases to change DNA strand displacement kinetics and introduces a complementary strategy to the existing designs.

With aging, the menisci become more susceptible to degeneration due to sustained mechanical stress accompanied by age-related changes in the extracellular matrix (ECM). However, the mechanistic relationship between age-related meniscal degeneration and osteoarthritis (OA) development is not yet fully understood. We have examined the nanomechanical properties of the ECM of normal, aged, and degenerated human menisci using atomic force microscopy (AFM). Elasticity maps of the ECM revealed a unique differential qualitative nanomechanical profile of healthy young tissue: prominent unimodal peaks in the elastic moduli distribution in each region (outer, middle, and inner). Healthy aged tissue showed similar regional elasticity but with both unimodal and bimodal distributions that included higher elastic moduli. In contrast, degenerated OA tissue showed the broadest distribution without prominent peaks indicative of substantially increased mechanical heterogeneity in the ECM. AFM analysis reveals distinct regional nanomechanical profiles that underlie aging-dependent tissue degeneration and OA., From the Clinical Editor: The authors of this study used atomic force microscopy to determine the nanomechanical properties of the extracellular matrix in normal and degenerated human menisci, as well as in menisci undergoing healthy aging. Comparison of these properties help to understand the relationship between healthy ageing, and age-dependent joint degeneration and osteoarthritis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Bacterial systems are being extensively studied and modified for energy, sensors, and industrial chemistry; yet, their molecular scale structure and activity are poorly understood. Designing efficient bioengineered bacteria requires cellular understanding of enzyme expression and activity. An atomic force microscope (AFM) was modified to detect and analyze the activity of redox active enzymes expressed on the surface of E. coli. An insulated gold-coated metal microwire with only the tip conducting was used as an AFM cantilever and a working electrode in a three-electrode electrochemical cell. Bacteria were engineered such that alcohol dehydrogenase II (ADHII) was surface displayed. A quinone, an electron transfer mediator, was covalently attached site specifically to the displayed ADHII. The AFM probe was used to lift a single bacterium off the surface for electrochemical analysis in a redox-free buffer. An electrochemical comparison between two quinone containing mutants with different distances from the NAD(+) binding site in alcohol dehydrogenase II was performed. Electron transfer in redox active proteins showed increased efficiency when mediators are present closer to the NAD(+) binding site. This study suggests that an integrated conducting AFM used for single cell electrochemical analysis would allow detailed understanding of enzyme electron transfer processes to electrodes, the processes integral to creating efficiently engineered biosensors and biofuel cells.

Accurately defining the nanoporous structure and sensing the ionic flow across nanoscale pores in thin films and membranes has a wide range of applications, including characterization of biological ion channels and receptors, DNA sequencing, molecule separation by nanoparticle films, sensing by block co-polymers films, and catalysis through metal-organic frameworks. Ionic conductance through nanopores is often regulated by their 3D structures, a relationship that can be accurately determined only by their simultaneous measurements. However, defining their structure-function relationships directly by any existing techniques is still not possible. Atomic force microscopy (AFM) can image the structures of these pores at high resolution in an aqueous environment, and electrophysiological techniques can measure ion flow through individual nanoscale pores. Combining these techniques is limited by the lack of nanoscale interfaces. We have designed a graphene-based single-nanopore support (∼5 nm thick with ∼20 nm pore diameter) and have integrated AFM imaging and ionic conductance recording using our newly designed double-chamber recording system to study an overlaid thin film. The functionality of this integrated system is demonstrated by electrical recording (<10 pS conductance) of suspended lipid bilayers spanning a nanopore and simultaneous AFM imaging of the bilayer.

Biological membranes contain ion channels, which are nanoscale pores allowing controlled ionic transport and mediating key biological functions underlying normal/abnormal living. Synthetic membranes with defined pores are being developed to control various processes, including filtration of pollutants, charge transport for energy storage, and separation of fluids and molecules. Although ionic transport (currents) can be measured with single channel resolution, imaging their structure and ionic currents simultaneously is difficult. Atomic force microscopy enables high resolution imaging of nanoscale structures and can be modified to measure ionic currents simultaneously. Moreover, the ionic currents can also be used to image structures. A simple method for fabricating conducting AFM cantilevers to image pore structures at high resolution is reported. Tungsten microwires with nanoscale tips are insulated except at the apex. This allows simultaneous imaging via cantilever deflections in normal AFM force feedback mode as well as measuring localized ionic currents. These novel probes measure ionic currents as small as picoampere while providing nanoscale spatial resolution surface topography and is suitable for measuring ionic currents and conductance of biological ion channels.

A DNA four-way junction device capable of junction expansion and contraction cycles using an inosine-based partial strand displacement scheme is reported. These nanoscale positioning capabilities are used to provide on-demand activation and deactivation of a pair of split E6 DNAzymes on the device. The device also demonstrates a combined catalytic rate significantly higher than the original E6 DNAzyme under similar operational conditions. This approach can provide structural organization and spatially control other multicomponent molecular complexes.

In this study we employ atomic force microscopy, supported by finite element analysis and fluorescence microscopy, to characterize the elastic properties accompanying cytoskeletal structural rearrangements of lung microvascular endothelial cells in response to barrier altering stimuli. Statistical analysis of elasticity data obtained from multiple cells demonstrates a heterogeneous cellular elastic response to barrier-enhancing and barrier-disrupting agents; sphingosine 1-phosphate (S1P) and thrombin, respectively. A small but detectable (10%) increase in the average elastic modulus of all cells is observed for S1P, which is accompanied by a corresponding significant decrease in cell thickness. Variable effects of thrombin on these parameters were observed. To account for possible substrate effects in our elasticity analysis, we analyzed only the low-force sections of the force-displacement curves and utilized a finite-thickness correction to the Hertzian model. Our finite element analysis results substantiate this approach. The heterogeneous elastic behavior correlates with differential cytoskeletal rearrangements observed with fluorescence microscopy., From the Clinical Editor: This team of investigators employed atomic force microscopy coupled with finite element analysis and fluorescence microscopy to characterize the elastic properties accompanying cytoskeletal structural rearrangements of lung microvascular endothelial cells in response to barrier altering stimuli, demonstrating the validity of their approach., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Interactions between amphiphilic block copolymers and lipids are of medical interest for applications such as drug delivery and the restoration of damaged cell membranes. A series of monodisperse poly(ethylene oxide)-poly(butylene oxide) (EOBO) block copolymers were obtained with two ratios of hydrophilic/hydrophobic block lengths. We have explored the surface activity of EOBO at a clean interface and under 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayers as a simple cell membrane model. At the same subphase concentration, EOBO achieved higher equilibrium surface pressures under DPPC compared to a bare interface, and the surface activity was improved with longer poly(butylene oxide) blocks. Further investigation of the DPPC/EOBO monolayers showed that combined films exhibited similar surface rheology compared to pure DPPC at the same surface pressures. DPPC/EOBO phase separation was observed in fluorescently doped monolayers, and within the liquid-expanded liquid-condensed coexistence region for DPPC, EOBO did not drastically alter the liquid-condensed domain shapes. Grazing incidence X-ray diffraction (GIXD) and X-ray reflectivity (XRR) quantitatively confirmed that the lattice spacings and tilt of DPPC in lipid-rich regions of the monolayer were nearly equivalent to those of a pure DPPC monolayer at the same surface pressures., (© 2011 American Chemical Society)