Smaller Gold Nanoparticles Release DNA More Efficiently During fs Laser Pulsed Optical Heating.

This work investigates the effect of plasmonic gold nanoparticle (AuNP) size on the rate of thermal release of single-stranded oligonucleotides under femtosecond (fs)-pulsed laser irradiation sources. Contrary to the theoretical predictions that larger AuNPs (50-60 nm diameter) would produce the most solution heating and fastest DNA release, it is found that smaller AuNP diameters (25 nm) lead to faster dsDNA denaturation rates. Controlling for the pulse energy fluence, AuNP concentration, DNA loading density, and the distance from the AuNP surface finds the same result. These results imply that the solution temperature increases around the AuNP during fs laser pulse optical heating may not be the only significant influence on dsDNA denaturation, suggesting that direct energy transfer from the AuNP to the DNA (phonon-phonon coupling), which is increased as AuNPs decrease in size, may play a significant role.

Multivalent Display of Erythropoietin on Quantum Dots Enhances Aquaporin-4 Expression and Water Transport in Human Astrocytes In Vitro.

In mammalian cells, growth factor-induced intracellular signaling and protein synthesis play a critical role in cellular physiology and homeostasis. In the brain's glymphatic system (GS), the water-conducting activity of aquaporin-4 (AQPN-4) membrane channels (expressed in polarized fashion on astrocyte end-feet) mediates the clearance of wastes through the convective transport of fluid and solutes through the perivascular space. The glycoprotein erythropoietin (EPO) has been shown to induce the astrocyte expression of AQPN-4 via signaling through the EPO receptor and the JAK/STAT signaling pathway. Here, we self-assemble EPO in a multivalent fashion onto the surface of semiconductor quantum dots (QDs) (driven by polyhistidine-based self-assembly) to drive the interaction of the bioconjugates with EPOR on human astrocytes (HA). This results in a 2-fold augmentation of JAK/STAT signaling activity and a 1.8-fold enhancement in the expression of AQPN-4 in cultured primary HA compared to free EPO. This translates into a 2-fold increase in the water transport rate in HA cells as measured by the calcein AM water transport assay. Importantly, EPO-QD-induced augmented AQPN-4 expression does not elicit any deleterious effect on the astrocyte viability. We discuss our results in the context of the implications of EPO-nanoparticle (NP) bioconjugates for use as research tools to understand the GS and their potential as therapeutics for the modulation of GS function. More generally, our results illustrate the utility of NP bioconjugates for the controlled modulation of growth factor-induced intracellular signaling.

Enzyme assembly on nanoparticle scaffolds enhances cofactor recycling and improves coupled reaction kinetics.

Enzyme activity can be many times enhanced in configurations where they are displayed on a nanoparticle (NP) and this same format sometimes even provides access to channeling phenomena within multienzyme cascades. Here, we demonstrate that such enhancement phenomena can be expanded to enzymatic cofactor recycling along with the coupled enzymatic processes that they are associated with. We begin by showing that the efficiency of glucose driven reduction of nicotinamide adenine dinucleotide (NAD+ → NADH) by glucose dehydrogenase (GDH) is enhanced ca. 5-fold when the enzyme is displayed on nanocrystalline semiconductor quantum dots (QDs) which are utilized as prototypical NP materials in our experimental assays. Coupling this enzymatic step with NADH-dependent lactate dehydrogenase (LDH) conversion of lactate to pyruvate also increases the latter's rate by a similar amount when both enzymes were jointly incorporated into self-assembled QD-based nanoclusters. Detailed agarose gel mobility assays and transmission electron microscopy imaging studies confirm that both tetrameric enzymes assemble to and crosslink the QDs into structured nanoclusters via their multiple-pendant terminal (His)6 sequences. Unexpectedly, control experiments utilizing blocking peptides to prevent enzyme-crosslinking of QDs resulted in even further enhancement of individual enzyme on-QD kinetic activity. This activity was also probed revealing that 200-fold excess peptide/QD addition enhanced individual GDH and LDH on-QD kcat a further 2- and 1.5×, respectively, above that seen just by QD display to a maximum of ∼10-fold GDH enhancement. The potential implications for how these enzyme kinetics-enhancing phenomena can be applied to single and multi-enzyme cascaded reactions in the context of cofactor recycling and cell-free synthetic biology are discussed.

Exploring the performance of Escherichia coli outer membrane vesicles as a tool for vaccine development against Chagas disease.

BACKGROUND: Vaccine development is a laborious craftwork in which at least two main components must be defined: a highly immunogenic antigen and a suitable delivery method. Hence, the interplay of these elements could elicit the required immune response to cope with the targeted pathogen with a long-lasting protective capacity. OBJECTIVES: Here we evaluate the properties of Escherichia coli spherical proteoliposomes - known as outer membrane vesicles (OMVs) - as particles with natural adjuvant capacities and as antigen-carrier structures to assemble an innovative prophylactic vaccine for Chagas disease. METHODS: To achieve this, genetic manipulation was carried out on E. coli using an engineered plasmid containing the Tc24 Trypanosoma cruzi antigen. The goal was to induce the release of OMVs displaying the parasite protein on their surface. FINDINGS: As a proof of principle, we observed that native OMVs - as well as those carrying the T. cruzi antigen - were able to trigger a slight, but functional humoral response at low immunization doses. Of note, compared to the non-immunized group, native OMVs-vaccinated animals survived the lethal challenge and showed minor parasitemia values, suggesting a possible involvement of innate trained immunity mechanism. MAIN CONCLUSION: These results open the range for further research on the design of new carrier strategies focused on innate immunity activation as an additional immunization target and venture to seek for alternative forms in which OMVs could be used for optimizing vaccine development.

Self assembling nanoparticle enzyme clusters provide access to substrate channeling in multienzymatic cascades.

Access to efficient enzymatic channeling is desired for improving all manner of designer biocatalysis. We demonstrate that enzymes constituting a multistep cascade can self-assemble with nanoparticle scaffolds into nanoclusters that access substrate channeling and improve catalytic flux by orders of magnitude. Utilizing saccharification and glycolytic enzymes with quantum dots (QDs) as a model system, nanoclustered-cascades incorporating from 4 to 10 enzymatic steps are prototyped. Along with confirming channeling using classical experiments, its efficiency is enhanced several fold more by optimizing enzymatic stoichiometry with numerical simulations, switching from spherical QDs to 2-D planar nanoplatelets, and by ordering the enzyme assembly. Detailed analyses characterize assembly formation and clarify structure-function properties. For extended cascades with unfavorable kinetics, channeled activity is maintained by splitting at a critical step, purifying end-product from the upstream sub-cascade, and feeding it as a concentrated substrate to the downstream sub-cascade. Generalized applicability is verified by extending to assemblies incorporating other hard and soft nanoparticles. Such self-assembled biocatalytic nanoclusters offer many benefits towards enabling minimalist cell-free synthetic biology.

Photothermal-Enhanced Modulation of Cellular Membrane Potential Using Long-Wavelength-Activated Gold Nanoflowers.

In mammalian cells, plasma membrane potential plays vital roles in both physiology and pathology and it is controlled by a network of membrane-resident ion channels. There is considerable interest in the use of nanoparticles (NPs) to control biological functions, including the modulation of membrane potential. The photoexcitation of gold NPs (AuNPs) tethered close to the plasma membrane has been shown to induce membrane depolarization via localized heating of the AuNP surface coupled with the opening of voltage-gated sodium channels. Previous work has employed spherical AuNPs (AuNS) with absorption in the 500-600 nm range for this purpose. However, AuNP materials with absorption at longer wavelengths [e.g., near-infrared (NIR)] would enable greater tissue penetration depth in vivo. We show here the use of new anisotropic-shaped AuNPs [gold nanoflowers (AuNFs)] with broad absorption spanning into the NIR part of the spectrum (∼650-1000 nm). The AuNFs are directly synthesized with bidentate thiolate ligands, which preserves the AuNF's shape and colloidal stability, while facilitating conjugation to biomolecules. We describe the characterization of the AuNF particles and demonstrate that they adhere to the plasma membrane when bioconjugated to PEGylated cholesterol (PEG-Chol) moieties. The AuNF-PEG-Chol mediated the depolarization of rat adrenal medulla pheochromocytoma (PC-12) neuron-like cells more effectively than AuNS-PEG-Chol and unconjugated AuNS and AuNF when photoexcited at ∼561 or ∼640 nm. Importantly, AuNF induction of depolarization had no impact on cellular viability. This work demonstrates anisotropic AuNFs as an enabling nanomaterial for use in cellular depolarization and the spatiotemporal control of cellular activity.

Exploring the performance of Escherichia coli outer membrane vesicles as a tool for vaccine development against Chagas disease

BACKGROUND Vaccine development is a laborious craftwork in which at least two main components must be defined: a highly immunogenic antigen and a suitable delivery method. Hence, the interplay of these elements could elicit the required immune response to cope with the targeted pathogen with a long-lasting protective capacity. OBJECTIVES Here we evaluate the properties of Escherichia coli spherical proteoliposomes - known as outer membrane vesicles (OMVs) - as particles with natural adjuvant capacities and as antigen-carrier structures to assemble an innovative prophylactic vaccine for Chagas disease. METHODS To achieve this, genetic manipulation was carried out on E. coli using an engineered plasmid containing the Tc24 Trypanosoma cruzi antigen. The goal was to induce the release of OMVs displaying the parasite protein on their surface. FINDINGS As a proof of principle, we observed that native OMVs - as well as those carrying the T. cruzi antigen - were able to trigger a slight, but functional humoral response at low immunization doses. Of note, compared to the non-immunized group, native OMVs-vaccinated animals survived the lethal challenge and showed minor parasitemia values, suggesting a possible involvement of innate trained immunity mechanism. MAIN CONCLUSION These results open the range for further research on the design of new carrier strategies focused on innate immunity activation as an additional immunization target and venture to seek for alternative forms in which OMVs could be used for optimizing vaccine development.

Nanoparticle-based delivery of nitric oxide for therapeutic applications.

Nitric oxide (NO), a low molecular weight signaling molecule, plays critical roles in both cellular health and disease. There is continued interest in new modalities for the controlled therapeutic delivery of NO to cells and tissues. The physicochemical properties of NO (including its short half-life and on-demand synthesis at the point of function), however, pose considerable challenges for its specific and efficient delivery. Recently, a number of nanoparticle (NP)-based systems are described that address some of these issues by taking advantage of the unique attributes of the NP carrier to effect efficient NO delivery. This review highlights the progress that has been made over the past 5 years in the use of various constructs for the therapeutic delivery of NO.

This review details progress made over the past 5 years in the implementation of various nanoparticle (NP) bioconjugates for the therapeutic delivery of nitric oxide. Various NP formulations including liposomes, polymeric NPs, and hard NPs such as AuNPs and upconversion NPs are covered and we discuss the inherent advantages and challenges in using these materials for the controlled delivery of nitric oxide to cells and tissues.

Hybrid Nucleic Acid-Quantum Dot Assemblies as Multiplexed Reporter Platforms for Cell-Free Transcription Translation-Based Biosensors.

Cell-free synthetic biology has emerged as a valuable tool for the development of rapid, portable biosensors that can be readily transported in the freeze-dried form to the point of need eliminating cold chain requirements. One of the challenges associated with cell-free sensors is the ability to simultaneously detect multiple analytes within a single reaction due to the availability of a limited set of fluorescent and colorimetric reporters. To potentially provide multiplexing capabilities to cell-free biosensors, we designed a modular semiconductor quantum dot (QD)-based reporter platform that is plugged in downstream of the transcription-translation functionality in the cell-free reaction and which converts enzymatic activity in the reaction into distinct optical signals. We demonstrate proof of concept by converting restriction enzyme activity, utilized as our prototypical sensing output, into optical changes across several distinct spectral output channels that all use a common excitation wavelength. These hybrid Förster resonance energy transfer (FRET)-based QD peptide PNA-DNA-Dye reporters (QD-PDDs) are completely self-assembled and consist of differentially emissive QD donors paired to a dye-acceptor displayed on a unique DNA encoding a given enzyme's cleavage site. Three QD-based PDDs, independently activated by the enzymes BamHI, EcoRI, and NcoI, were prototyped in mixed enzyme assays where all three demonstrated the ability to convert enzymatic activity into fluorescent output. Simultaneous monitoring of each of the three paired QD-donor dye-acceptor spectral channels in cell-free biosensing reactions supplemented with added linear genes encoding each enzyme confirmed robust multiplexing capabilities for at least two enzymes when co-expressed. The modular QD-PDDs are easily adapted to respond to other restriction enzymes or even proteases if desired.

A humanized nanobody phage display library yields potent binders of SARS CoV-2 spike.

Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants.

Polyhistidine-Tag-Enabled Conjugation of Quantum Dots and Enzymes to DNA Nanostructures.

DNA nanostructures self-assemble into almost any arbitrary architecture, and when combined with their capability to precisely position and orient dyes, nanoparticles, and biological moieties, the technology reaches its potential. We present a simple yet multifaceted conjugation strategy based on metal coordination by a multi-histidine peptide tag (Histag). The versatility of the Histag as a means to conjugate to DNA nanostructures is shown by using Histags to capture semiconductor quantum dots (QDs) with numerical and positional precision onto a DNA origami breadboard. Additionally, Histag-expressing enzymes, such as the bioluminescent luciferase, can also be captured to the DNA origami breadboard with similar precision. DNA nanostructure conjugation of the QDs or luciferase is confirmed through imaging and/or energy transfer to organic dyes integrated into the DNA nanostructure.

Liquid Crystal Nanoparticle Conjugates for Scavenging Reactive Oxygen Species in Live Cells.

The elevated intracellular production of or extracellular exposure to reactive oxygen species (ROS) causes oxidative stress to cells, resulting in deleterious irreversible biomolecular reactions (e.g., lipid peroxidation) and disease progression. The use of low-molecular weight antioxidants, such as 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), as ROS scavengers fails to achieve the desired efficacy because of their poor or uncontrolled cellular uptake and off-target effects, such as dysfunction of essential redox homeostasis. In this study, we fabricated a liquid crystal nanoparticle (LCNP) conjugate system with the fluorescent dye perylene (PY) loaded in the interior and poly (ethylene glycol) (PEG) decorated on the surface along with multiple molecules of TEMPO (PY-LCNP-PEG/TEMPO). PY-LCNP-PEG/TEMPO exhibit enhanced cellular uptake, and efficient ROS-scavenging activity in live cells. On average, the 120 nm diameter PY-LCNPs were conjugated with >1800 molecules of TEMPO moieties on their surface. PY-LCNP-PEG/TEMPO showed significantly greater reduction in ROS activity and lipid peroxidation compared to free TEMPO when the cells were challenged with ROS generating agents, such as hydrogen peroxide (H2O2). We suggest that this is due to the increased local concentration of TEMPO molecules on the surface of the PY-LCNP-PEG/TEMPO NPs, which efficiently bind to the plasma membrane and enter cells. Overall, these results demonstrate the enhanced capability of TEMPO-conjugated LCNPs to protect live cells from oxidative stress by effectively scavenging ROS and reducing lipid peroxidation.

High-throughput Confocal Imaging of Quantum Dot-Conjugated SARS-CoV-2 Spike Trimers to Track Binding and Endocytosis in HEK293T Cells.

The development of new technologies for cellular fluorescence microscopy has facilitated high-throughput screening methods for drug discovery. Quantum dots are fluorescent nanoparticles with excellent photophysical properties imbued with bright and stable photoluminescence as well as narrow emission bands. Quantum dots are spherical in shape, and with the proper modification of the surface chemistry, can be used to conjugate biomolecules for cellular applications. These optical properties, combined with the ability to functionalize them with biomolecules, make them an excellent tool for investigating receptor-ligand interactions and cellular trafficking. Here, we present a method that uses quantum dots to track the binding and endocytosis of SARS-CoV-2 spike protein. This protocol can be used as a guide for experimentalists looking to utilize quantum dots to study protein-protein interactions and trafficking in the context of cellular physiology.

Self-assembled nanoparticle-enzyme aggregates enhance functional protein production in pure transcription-translation systems.

Cell-free protein synthesis systems (CFPS) utilize cellular transcription and translation (TX-TL) machinery to synthesize proteins in vitro. These systems are useful for multiple applications including production of difficult proteins, as high-throughput tools for genetic circuit screening, and as systems for biosensor development. Though rapidly evolving, CFPS suffer from some disadvantages such as limited reaction rates due to longer diffusion times, significant cost per assay when using commercially sourced materials, and reduced reagent stability over prolonged periods. To address some of these challenges, we conducted a series of proof-of-concept experiments to demonstrate enhancement of CFPS productivity via nanoparticle assembly driven nanoaggregation of its constituent proteins. We combined a commercially available CFPS that utilizes purified polyhistidine-tagged (His-tag) TX-TL machinery with CdSe/CdS/ZnS core/shell/shell quantum dots (QDs) known to readily coordinate His-tagged proteins in an oriented fashion. We show that nanoparticle scaffolding of the CFPS cross-links the QDs into nanoaggregate structures while enhancing the production of functional recombinant super-folder green fluorescent protein and phosphotriesterase, an organophosphate hydrolase; the latter by up to 12-fold. This enhancement, which occurs by an undetermined mechanism, has the potential to improve CFPS in general and specifically CFPS-based biosensors (faster response time) while also enabling rapid detoxification/bioremediation through point-of-concern synthesis of similar catalytic enzymes. We further show that such nanoaggregates improve production in diluted CFPS reactions, which can help to save money and extend the amount of these costly reagents. The results are discussed in the context of what may contribute mechanistically to the enhancement and how this can be applied to other CFPS application scenarios.

Mechanistic Understanding of DNA Denaturation in Nanoscale Thermal Gradients Created by Femtosecond Excitation of Gold Nanoparticles.

There is significant interest in developing photothermal systems that can precisely control the structure and function of biomolecules through local temperature modulation. One specific application is the denaturation of double-stranded (ds) DNA through femtosecond (fs) laser pulse optical heating of gold nanoparticles (AuNPs); however, the mechanism of DNA melting in these systems is not fully understood. Here, we utilize 55 nm AuNPs with surface-tethered dsDNA, which are locally heated using fs laser pulses to induce DNA melting. By varying the dsDNA distance from the AuNP surface and the laser pulse energy fluence, this system is used to study how the nanosecond duration temperature increase and the steep temperature gradient around the AuNP affect dsDNA dehybridization. Through modifying the distance between the dsDNA and AuNP surface by 3.8 nm in total and the pulse energy fluence from 7.1 to 14.1 J/m2, the dehybridization rates ranged from 0.002 to 0.05 DNA per pulse, and the total amount of DNA released into solution was controlled over a range of 26-93% in only 100 s of irradiation. By shifting the dsDNA position as little as ∼1.1 nm, the average dsDNA dehybridization rate is altered up to 30 ± 2%, providing a high level of control over DNA melting and release. By comparing the theoretical temperature around the dsDNA to the experimentally derived temperature, we find that maximum or peak temperatures have a greater influence on the dehybridization rate when the dsDNA is closer to the AuNP surface and when lower laser pulse fluences are used. Furthermore, molecular dynamics simulations mimicking the photothermal heat pulse around a AuNP provide mechanistic insight into the stochastic nature of dehybridization and demonstrate increased base pair separation near the AuNP surface during laser pulse heating when compared to steady-state heating. Understanding how biological materials respond to the short-lived and non-uniform temperature increases innate to fs laser pulse optical heating of AuNPs is critical to improving the functionality and precision of this technique so that it may be implemented into more complex biological systems.

A humanized nanobody phage display library yields potent binders of SARS CoV-2 spike.

Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants.

Fluorescent quantum dots enable SARS-CoV-2 antiviral drug discovery and development.

INTRODUCTION: SARS-CoV-2 is a highly infectious and deadly coronavirus whose study requires the use of a biosafety level 3 (BSL-3) containment facility to investigate viral biology and pathogenesis, which limits the study of live virus and slows progress toward finding suitable treatments for infection. While vaccines from several companies have proven very effective in combating the virus, few treatments exist for those who do succumb to the viral-induced systemic disease called COVID-19. AREAS COVERED: This short review focuses on fluorescent quantum dot-based modeling of SARS-CoV-2. New BSL-2 viral models are essential for finding small molecules and biologics that may be effective in stopping viral infection, as well as treating already infected individuals. Nanoparticles are invaluable tools for biological research as they can be used to both model pathogens and serve as a platform for developing vaccines. EXPERT OPINION: Visualizing viral activity with fluorescent quantum dots enables both biochemical and cell-based assays to detect virus-host receptor interactions, cellular activity after binding to the cell plasma membrane, screening for interventions using small-molecule drug repurposing, and testing of novel biologics. Quantum dots can also be used for diagnostic assays, vaccine development, and importantly, pan-antiviral drugs to address variants that may escape the immune response.

Microbial survival and growth on non-corrodible conductive materials.

Biofilms growing aerobically on conductive substrates are often correlated with a positive, sustained shift in their redox potential. This phenomenon has a beneficial impact on microbial fuel cells by increasing their overall power output but can be detrimental when occurring on stainless steel by enhancing corrosion. The biological mechanism behind this potential shift is unresolved and a metabolic benefit to cells has not been demonstrated. Here, biofilms containing the electroautotroph 'Candidatus Tenderia electrophaga' catalysed a shift in the open circuit potential of graphite and indium tin oxide electrodes by >100 mV. Biofilms on open circuit electrodes had increased biomass and a significantly higher proportion of 'Ca. Tenderia electrophaga' compared to those on plain glass. Addition of metabolic inhibitors showed that living cells were required to maintain the more positive potential. We propose a model to describe these observations, in which 'Ca. Tenderia electrophaga' drives the shift in open circuit potential through electron uptake for oxygen reduction and CO2 fixation. We further propose that the electrode is continuously recharged by oxidation of trace redox-active molecules in the medium at the more positive potential. A similar phenomenon is possible on natural conductive substrates in the environment.

Direct and Efficient Conjugation of Quantum Dots to DNA Nanostructures with Peptide-PNA.

DNA nanotechnology has proven to be a powerful strategy for the bottom-up preparation of colloidal nanoparticle (NP) superstructures, enabling the coordination of multiple NPs with orientation and separation approaching nanometer precision. To do this, NPs are often conjugated with chemically modified, single-stranded (ss) DNA that can recognize complementary ssDNA on the DNA nanostructure. The limitation is that many NPs cannot be easily conjugated with ssDNA, and other conjugation strategies are expensive, inefficient, or reduce the specificity and/or precision with which NPs can be placed. As an alternative, the conjugation of nanoparticle-binding peptides and peptide nucleic acids (PNA) can produce peptide-PNA with distinct NP-binding and DNA-binding domains. Here, we demonstrate a simple application of this method to conjugate semiconductor quantum dots (QDs) directly to DNA nanostructures by means of a peptide-PNA with a six-histidine peptide motif that binds to the QD surface. With this method, we achieved greater than 90% capture efficiency for multiple QDs on a single DNA nanostructure while preserving both site specificity and precise spatial control of QD placement. Additionally, we investigated the effects of peptide-PNA charge on the efficacy of QD immobilization in suboptimal conditions. The results validate peptide-PNA as a viable alternative to ssDNA conjugation of NPs and warrant studies of other NP-binding peptides for peptide-PNA conjugation.

The SARS-CoV-2 Cytopathic Effect Is Blocked by Lysosome Alkalizing Small Molecules.

Understanding the SARS-CoV-2 virus' pathways of infection, virus-host-protein interactions, and mechanisms of virus-induced cytopathic effects will greatly aid in the discovery and design of new therapeutics to treat COVID-19. Chloroquine and hydroxychloroquine, extensively explored as clinical agents for COVID-19, have multiple cellular effects including alkalizing lysosomes and blocking autophagy as well as exhibiting dose-limiting toxicities in patients. Therefore, we evaluated additional lysosomotropic compounds to identify an alternative lysosome-based drug repurposing opportunity. We found that six of these compounds blocked the cytopathic effect of SARS-CoV-2 in Vero E6 cells with half-maximal effective concentration (EC50) values ranging from 2.0 to 13 µM and selectivity indices (SIs; SI = CC50/EC50) ranging from 1.5- to >10-fold. The compounds (1) blocked lysosome functioning and autophagy, (2) prevented pseudotyped particle entry, (3) increased lysosomal pH, and (4) reduced (ROC-325) viral titers in the EpiAirway 3D tissue model. Consistent with these findings, the siRNA knockdown of ATP6V0D1 blocked the HCoV-NL63 cytopathic effect in LLC-MK2 cells. Moreover, an analysis of SARS-CoV-2 infected Vero E6 cell lysate revealed significant dysregulation of autophagy and lysosomal function, suggesting a contribution of the lysosome to the life cycle of SARS-CoV-2. Our findings suggest the lysosome as a potential host cell target to combat SARS-CoV-2 infections and inhibitors of lysosomal function could become an important component of drug combination therapies aimed at improving treatment and outcomes for COVID-19.