Modeling identifies variability in SARS-CoV-2 uptake and eclipse phase by infected cells as principal drivers of extreme variability in nasal viral load in the 48 h post infection.

The SARS-CoV-2 coronavirus continues to evolve with scores of mutations of the spike, membrane, envelope, and nucleocapsid structural proteins that impact pathogenesis. Infection data from nasal swabs, nasal PCR assays, upper respiratory samples, ex vivo cell cultures and nasal epithelial organoids reveal extreme variabilities in SARS-CoV-2 RNA titers within and between the variants. Some variabilities are naturally prone to clinical testing protocols and experimental controls. Here we focus on nasal viral load sensitivity arising from the timing of sample collection relative to onset of infection and from heterogeneity in the kinetics of cellular infection, uptake, replication, and shedding of viral RNA copies. The sources of between-variant variability are likely due to SARS-CoV-2 structural protein mutations, whereas within-variant population variability is likely due to heterogeneity in cellular response to that particular variant. With the physiologically faithful, agent-based mechanistic model of inhaled exposure and infection from (Chen et al., 2022), we perform statistical sensitivity analyses of the progression of nasal viral titers in the first 0-48 h post infection, focusing on three kinetic mechanisms. Model simulations reveal shorter latency times of infected cells (including cellular uptake, viral RNA replication, until the onset of viral RNA shedding) exponentially accelerate nasal viral load. Further, the rate of infectious RNA copies shed per day has a proportional influence on nasal viral load. Finally, there is a very weak, negative correlation of viral load with the probability of infection per virus-cell encounter, the model proxy for spike-receptor binding affinity.

The power of weak, transient interactions across biology: A paradigm of emergent behavior.

A growing list of diverse biological systems and their equally diverse functionalities provides realizations of a paradigm of emergent behavior. In each of these biological systems, pervasive ensembles of weak, short-lived, spatially local interactions act autonomously to convey functionalities at larger spatial and temporal scales. In this article, a range of diverse systems and functionalities are presented in a cursory manner with literature citations for further details. Then two systems and their properties are discussed in more detail: yeast chromosome biology and human respiratory mucus.

Modeling insights into SARS-CoV-2 respiratory tract infections prior to immune protection.

Mechanistic insights into human respiratory tract (RT) infections from SARS-CoV-2 can inform public awareness as well as guide medical prevention and treatment for COVID-19 disease. Yet the complexity of the RT and the inability to access diverse regions pose fundamental roadblocks to evaluation of potential mechanisms for the onset and progression of infection (and transmission). We present a model that incorporates detailed RT anatomy and physiology, including airway geometry, physical dimensions, thicknesses of airway surface liquids (ASLs), and mucus layer transport by cilia. The model further incorporates SARS-CoV-2 diffusivity in ASLs and best-known data for epithelial cell infection probabilities, and, once infected, duration of eclipse and replication phases, and replication rate of infectious virions. We apply this baseline model in the absence of immune protection to explore immediate, short-term outcomes from novel SARS-CoV-2 depositions onto the air-ASL interface. For each RT location, we compute probability to clear versus infect; per infected cell, we compute dynamics of viral load and cell infection. Results reveal that nasal infections are highly likely within 1-2 days from minimal exposure, and alveolar pneumonia occurs only if infectious virions are deposited directly into alveolar ducts and sacs, not via retrograde propagation to the deep lung. Furthermore, to infect just 1% of the 140 m2 of alveolar surface area within 1 week, either 103 boluses each with 106 infectious virions or 106 aerosols with one infectious virion, all physically separated, must be directly deposited. These results strongly suggest that COVID-19 disease occurs in stages: a nasal/upper RT infection, followed by self-transmission of infection to the deep lung. Two mechanisms of self-transmission are persistent aspiration of infected nasal boluses that drain to the deep lung and repeated rupture of nasal aerosols from infected mucosal membranes by speaking, singing, or cheering that are partially inhaled, exhaled, and re-inhaled, to the deep lung.

Hierarchical Assembly of Nucleic Acid/Coiled-Coil Peptide Nanostructures.

DNA and peptides are two of the most commonly used biomolecules for building self-assembling materials, but few examples exist of hybrid nanostructures that contain both components. Here we report the modification of two peptides that comprise a coiled-coil heterodimer pair with unique DNA handles in order to link DNA origami nanostructures bearing complementary strands into micrometer-long one-dimensional arrays. We probed the effect of number of coils on self-assembly and demonstrated the formation of  structures through multiple routes: one-pot assembly, formation of dimers and trimers and an alternating copolymer of two different origami structures, and stepwise assembly of purified structures with coiled-coil conjugates. Our results demonstrate the successful merging of two distinct self-assembly modes to create hybrid bionanomaterials expected to have a range of potential applications in the future.

Supramolecular Exchange among Assemblies of Opposite Charge Leads to Hierarchical Structures.

Hierarchical assemblies of proteins into fibrillar structures occur in both physiologic and pathologic extracellular spaces and often involve interactions between oppositely charged peptide domains. However, the interplay between tertiary structure dynamics and quaternary hierarchical structure formation remains unclear. In this work, we investigate supramolecular mimics of these systems by mixing one-dimensional assemblies of small alkylated peptides bearing opposite charge and varying in peptide sequence. We found that assemblies with weak cohesive interactions readily create fibrous superstructures of bundled filaments as molecules redistribute upon mixing. Low cohesion allows molecules to escape from the original assemblies and exchange dynamics help them reassemble into electrostatically stable bundles. However, we also found that kinetic barriers can be encountered in these systems and limit formation of the hierarchical structures at pH values where charge densities are high. Increasing intermolecular cohesion using longer peptide sequences that form stable ß-sheets was found to suppress superstructure formation. Our findings suggest that low internal cohesion in protein systems could facilitate the conformational rearrangements required to create hierarchical structures.

Encoding Reversible Hierarchical Structures with Supramolecular Peptide-DNA Materials.

Creating soft materials with the tunable hierarchical structures observed in nature remains an enormous challenge. Synthetic hierarchical systems have been reported, yet strategies to reversibly modulate their structure and function are scarce. We report on the programmable self-assembly of peptide-DNA brush copolymers into supramolecular architectures that can be tuned with changes in temperature, pH, or addition of a soluble trigger. A fiber to bundle transition occurs upon mixing peptides bearing complementary oligonucleotides. These hierarchical structures can be reversed using the programmable nature of DNA-DNA interactions. The ability to encode the final assemblies in the composition of both amino acid and DNA building blocks provides a strategy for constructing a unique class of reconfigurable supramolecular materials.

Bioactive DNA-peptide nanotubes enhance the differentiation of neural stem cells into neurons.

We report the construction of DNA nanotubes covalently functionalized with the cell adhesion peptide RGDS as a bioactive substrate for neural stem cell differentiation. Alteration of the Watson-Crick base pairing program that builds the nanostructures allowed us to probe independently the effect of nanotube architecture and peptide bioactivity on stem cell differentiation. We found that both factors instruct synergistically the preferential differentiation of the cells into neurons rather than astrocytes.

Biopolymers and supramolecular polymers as biomaterials for biomedical applications.

Protein- and peptide-based structural biopolymers are abundant building blocks of biological systems. Either in their natural forms, such as collagen, silk or fibronectin, or as related synthetic materials they can be used in various technologies. An emerging area is that of biomimetic materials inspired by protein-based biopolymers, which are made up of small molecules rather than macromolecules and can therefore be described as supramolecular polymers. These materials are very useful in biomedical applications because of their ability to imitate the extracellular matrix both in architecture and their capacity to signal cells. This article describes important features of the natural extracellular matrix and highlight how these features are being incorporated into biomaterials composed of biopolymers and supramolecular polymers. We particularly focus on the structures, properties, and functions of collagen, fibronectin, silk, and the supramolecular polymers inspired by them as biomaterials for regenerative medicine.

Designer peptide-DNA cytoskeletons regulate the function of synthetic cells.

The bottom-up engineering of artificial cells requires a reconfigurable cytoskeleton that can organize at distinct locations and dynamically modulate its structural and mechanical properties. Here, inspired by the vast array of actin-binding proteins and their ability to reversibly crosslink or bundle filaments, we have designed a library of peptide-DNA crosslinkers varying in length, valency and geometry. Peptide filaments conjoint through DNA hybridization give rise to tactoid-shaped bundles with tunable aspect ratios and mechanics. When confined in cell-sized water-in-oil droplets, the DNA crosslinker design guides the localization of cytoskeletal structures at the cortex or within the lumen of the synthetic cells. The tunable spatial arrangement regulates the passive diffusion of payloads within the droplets and complementary DNA handles allow for the reversible recruitment and release of payloads on and off the cytoskeleton. Heat-induced reconfiguration of peptide-DNA architectures triggers shape deformations of droplets, regulated by DNA melting temperatures. Altogether, the modular design of peptide-DNA architectures is a powerful strategy towards the bottom-up assembly of synthetic cells.

Uncovering supramolecular chirality codes for the design of tunable biomaterials.

In neurodegenerative diseases, polymorphism and supramolecular assembly of ß-sheet amyloids are implicated in many different etiologies and may adopt either a left- or right-handed supramolecular chirality. Yet, the underlying principles of how sequence regulates supramolecular chirality remains unknown. Here, we characterize the sequence specificity of the central core of amyloid-ß 42 and design derivatives which enable chirality inversion at biologically relevant temperatures. We further find that C-terminal modifications can tune the energy barrier of a left-to-right chiral inversion. Leveraging this design principle, we demonstrate how temperature-triggered chiral inversion of peptides hosting therapeutic payloads modulates the dosed release of an anticancer drug. These results suggest a generalizable approach for fine-tuning supramolecular chirality that can be applied in developing treatments to regulate amyloid morphology in neurodegeneration as well as in other disease states.

Hemin/G-quadruplex-catalyzed aerobic oxidation of thiols to disulfides: application of the process for the development of sensors and aptasensors and for probing acetylcholine esterase activity.

This study describes the novel hemin/G-quadruplex DNAzyme-catalyzed aerobic oxidation of thiols to disulfides and the respective mechanism. The mechanism of the reaction involves the DNAzyme-catalyzed oxidation of thiols to disulfides and the thiol-mediated autocatalytic generation of H2O2 from oxygen. The coupling of a concomitant H2O2-mediated hemin/G-quadruplex-catalyzed oxidation of Amplex Red to the fluorescent resorufin as a transduction module provides a fluorescent signal for probing the catalyzed oxidation of the thiol to disulfides and for probing sensing processes that yield the hemin/G-quadruplex as a functional label. Accordingly, a versatile sensing method for analyzing thiols (L-cysteine, glutathione) using the H2O2-mediated DNAzyme-catalyzed oxidation of Amplex Red to the resorufin was developed. Also, the L-cysteine and Amplex Red system was implemented as an auxiliary fluorescent transduction module for probing recognition events that form the catalytic hemin/G-quadruplex structures. This is exemplified with the development of thrombin aptasensor. The thrombin/thrombin binding aptamer recognition complex binds hemin, and the resulting catalytic complex activates the auxiliary transduction module, involving the aerobic oxidation of l-cysteine and the concomitant formation of the fluorescent resorufin. Finally, the hemin/G-quadruplex DNAzyme/Amplex Red system was used to follow the activity of acetylcholine esterase, AChE, and to probe its inhibition. The AChE-catalyzed hydrolysis of acetylthiocholine to the thiol-functionalized thiocholine enabled the probing of the enzymatic activity of AChE through the hemin/G-quadruplex-catalyzed aerobic oxidation of thiocholine to the respective disulfide and the concomitant generation of the fluorescent resorufin product.

Optical molecular sensing with semiconductor quantum dots (QDs).

Semiconductor quantum dots (QDs) exhibit unique optical and photophysical properties. These features are implemented to develop optical molecular sensor systems. The review addresses the methods to functionalize the QDs with chemical capping layers that enable the use of the resulting hybrid structures for sensing, and discusses the photophysical mechanisms being applied in the different sensor systems. Different methods to design the chemically-modified QDs hybrid structures for sensing low-molecular-weight substrates, metal ions, anions and gases are presented. These include the functionalization of the QDs with ligands that bind ions, the modification of the QDs with substrate-specific ligands or receptor units, and the chemical modification of the QDs upon sensing. Specific emphasis is directed to describe the cooperative catalytic functions of the QDs in the sensing processes, and to address the function of sensing with logic-gate operations.

Statistical Methods for Microrheology of Airway Mucus with Extreme Heterogeneity.

The mucus lining of the human airway epithelium contains two gel-forming mucins, MUC5B and MUC5AC. During progression of cystic fibrosis (CF), mucus hyper-concentrates as its mucin ratio changes, coinciding with formation of insoluble, dense mucus flakes. We explore rheological heterogeneity of this pathology with reconstituted mucus matching three stages of CF progression and particle-tracking of 200 nm and 1 micron diameter beads. We introduce statistical data analysis methods specific to low signal-to-noise data within flakes. Each bead time series is decomposed into: (i) a fractional Brownian motion (fBm) classifier of the pure time-series signal; (ii) high-frequency static and dynamic noise; and (iii) low-frequency deterministic drift. Subsequent analysis focuses on the denoised fBm classifier ensemble from each mucus sample and bead diameter. Every ensemble fails a homogeneity test, compelling clustering methods to assess levels of heterogeneity. The first binary level detects beads within vs. outside flakes. A second binary level detects within-flake bead signals that can vs. cannot be disentangled from the experimental noise floor. We show all denoised ensembles, within- and outside-flakes, fail a homogeneity test, compelling additional clustering; next, all clusters with sufficient data fail a homogeneity test. These levels of heterogeneity are consistent with outcomes from a stochastic phase-separation process, and dictate applying the generalized Stokes-Einstein relation to each bead per cluster per sample, then frequency-domain averaging to assess rheological heterogeneity. Flakes exhibit a spectrum of gel-like and sol-like domains, outside-flake solutions a spectrum of sol-like domains, painting a rheological signature of the phase-separation process underlying flake-burdened mucus.

SARS-CoV-2 evolved variants optimize binding to cellular glycocalyx.

Viral variants of concern continue to arise for SARS-CoV-2, potentially impacting both methods for detection and mechanisms of action. Here, we investigate the effect of an evolving spike positive charge in SARS-CoV-2 variants and subsequent interactions with heparan sulfate and the angiotensin converting enzyme 2 (ACE2) in the glycocalyx. We show that the positively charged Omicron variant evolved enhanced binding rates to the negatively charged glycocalyx. Moreover, we discover that while the Omicron spike-ACE2 affinity is comparable to that of the Delta variant, the Omicron spike interactions with heparan sulfate are significantly enhanced, giving rise to a ternary complex of spike-heparan sulfate-ACE2 with a large proportion of double-bound and triple-bound ACE2. Our findings suggest that SARS-CoV-2 variants evolve to be more dependent on heparan sulfate in viral attachment and infection. This discovery enables us to engineer a second-generation lateral-flow test strip that harnesses both heparin and ACE2 to reliably detect all variants of concern, including Omicron.

From the lab to the field: handheld surface enhanced Raman spectroscopy (SERS) detection of viral proteins.

Translating sensors from the lab benchtop to a readily available point-of-need setting is desirable for many fields, including medicine, agriculture, and industry. However, this transition generally suffers from loss of sensitivity, high background signals, and other issues which can impair reproducibility. Here we adapt a label-free surface-enhanced Raman spectroscopy (SERS) sensor for SARS-CoV-2 antigens from a lab-based assay to a handheld device. Utilizing a peptide capture molecule, which we previously employed for a surface-based assay, we optimize a simpler and more cost-efficient nanoparticle-based assay. This new assay allows for the direct detection of these viral antigens by SERS, now with the advantages of robustness and portability. We highlight considerations for nanoparticle modification conditions and warn against methods which can interfere with accurate detection. The comparison of these two assays will help guide further development of SERS-based sensors into devices that can be easily used in point-of-care settings, such as by emergency room nurses, farmers, or quality control technicians.

Fluorescence detection of DNA, adenosine-5'-triphosphate (ATP), and telomerase activity by zinc(II)-protoporphyrin IX/G-quadruplex labels.

The zinc(II)-protoporphyrin IX (ZnPPIX) fluorophore binds to G-quadruplexes, and this results in the enhanced fluorescence of the fluorophore. This property enabled the development of DNA sensors, aptasensors, and a sensor following telomerase activity. The DNA sensor is based on the design of a hairpin structure that includes a "caged" inactive G-quadruplex sequence. Upon opening the hairpin by the analyte DNA, the resulting fluorescence of the ZnPPIX/G-quadruplex provides the readout signal for the sensing event (detection limit 5 nM). Addition of Exonuclease III to the system allows the recycling of the analyte and its amplified analysis (detection limit, 200 pM). The association of the ZnPPIX to G-quadruplex aptamer-substrate complexes allowed the detection of adenosine-5'-triphosphate (ATP, detection limit 10 µM). Finally, the association of ZnPPIX to the G-quadruplex repeat units of telomers allowed the detection of telomerase activity originating from 380 ± 20 cancer 293T cell extract.

Optical aptasensors for the analysis of the vascular endothelial growth factor (VEGF).

The vascular endothelial growth factor, VEGF, is an important biomarker for different diseases and clinical disorders. We present a series of optical aptasensor-based sensing platforms for VEGF that include the following: (i) A FRET-based sensor that involves the VEGF-induced separation of aptamer-functionalized quantum dots blocked by a quencher nucleic acid (detection limit 1 nM). (ii) A FRET-based sensor based on the VEGF-induced assembly of the aptamer subunits functionalized with QDs and a dye acceptor (Cy5), respectively (detection limit 12 nM). (iii) A chemiluminescence aptasensor based on VEGF-induced assembly of a hemin/G-quadruplex catalyst (detection limit 18 nM). (iv) A chemiluminescence aptasensor based on the VEGF-stimulated assembly of two aptamer subunits into the hemin/G-quadruplex catalyst (detection limit 2.6 nM). (v) A chemiluminescence resonance energy transfer (CRET) aptasensor based on the VEGF-induced assembly of a semiconductor QDs-hemin/G-quadruplex supramolecular structure (detection limit 875 pM). Furthermore, an amplified optical aptasensor system based on the Exonuclease III (Exo III) recycling of the VEGF analyte was developed. In this system, one aptamer subunit is modified at its 5' and 3' ends with QDs and a black hole quencher, respectively. The VEGF-induced self-assembly of the aptamer subunits result in the digestion of the quencher units and the autonomous recycling of the analyte, while triggering-on the luminescence of the QDs (detection limit 5 pM). The system was implemented to analyze VEGF in human sera samples.

Amplified fluorescence aptamer-based sensors using exonuclease†III for the regeneration of the analyte.

Quick and easy detection: The Exo III-stimulated regeneration of the analyte by the digestion of supramolecular aptamer-analyte complexes provides a means to develop amplified optical aptasensors (see figure).

Amplified multiplexed analysis of DNA by the exonuclease III-catalyzed regeneration of the target DNA in the presence of functionalized semiconductor quantum dots.

Quantum dots (QDs) functionalized with a black-hole quencher are used as optical tracer for the detection of DNA using exonuclease as a biocatalyst. The binding of the target DNA or of a target/open hairpin complex to the functionalized QDs leads to the exonuclease-stimulated recycling of the target DNA or the target/hairpin complex. This results in the triggering of the luminescence of the QDs that provides a readout signal for the amplified sensing process. By using different-sized QDs, the multiplexed detection of DNAs is demonstrated.

GlycoGrip: Cell Surface-Inspired Universal Sensor for Betacoronaviruses.

Inspired by the role of cell-surface glycoproteins as coreceptors for pathogens, we report the development of GlycoGrip: a glycopolymer-based lateral flow assay for detecting SARS-CoV-2 and its variants. GlycoGrip utilizes glycopolymers for primary capture and antispike antibodies labeled with gold nanoparticles for signal-generating detection. A lock-step integration between experiment and computation has enabled efficient optimization of GlycoGrip test strips which can selectively, sensitively, and rapidly detect SARS-CoV-2 and its variants in biofluids. Employing the power of the glycocalyx in a diagnostic assay has distinct advantages over conventional immunoassays as glycopolymers can bind to antigens in a multivalent capacity and are highly adaptable for mutated strains. As new variants of SARS-CoV-2 are identified, GlycoGrip will serve as a highly reconfigurable biosensor for their detection. Additionally, via extensive ensemble-based docking simulations which incorporate protein and glycan motion, we have elucidated important clues as to how heparan sulfate and other glycocalyx components may bind the spike glycoprotein during SARS-CoV-2 host-cell infection. GlycoGrip is a promising and generalizable alternative to costly, labor-intensive RT-PCR, and we envision it will be broadly useful, including for rural or low-income populations that are historically undertested and under-reported in infection statistics.