Junyi Chen

All Publications

• Supramolecular Host:Guest Arrays Site-Selectively Recognize Peptide Phosphorylation and Kinase Activity JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Chen, J., Fasihianifard, P., Lian, R., Gibson-Elias, L. J., Moreno, J. L., Chang, C. A., Zhong, W., Hooley, R. J. 2024

  Abstract

  A synergistic combination of cationic styrylpyridinium dyes and water-soluble deep cavitand hosts can recognize phosphorylated peptides with both site- and state-selectivity. Two mechanisms of interaction are dominant: either the cationic dye interacts with Trp residues in the peptide or the host:dye pair forms a heteroternary complex with the peptide, driven by both strong dye-peptide and cavitand-peptide binding (Kd values up to 4 μM). The presence of multiple recognition mechanisms results in varying fluorescence responses dependent on the phosphorylation state and position, eliminating the need for covalent modification of the peptide target. Differential sensing aided by machine learning algorithms permits full discrimination between differently positioned serine phosphorylations with a minimal 3-component array. The array is fully functional in the presence of protein kinase A (PKA) and its required cofactors and capable of site-selective monitoring of serine phosphorylation at the privileged PKA motif, in the presence of serine residues that do not undergo reaction, illustrating the potential of the system in kinase-based drug screening.

  View details for DOI 10.1021/jacs.4c13757

  View details for Web of Science ID 001378664800001

  View details for PubMedID 39680592

• Optical discrimination of terpenes in citrus peels with a host:guest sensing array. Chemical communications (Cambridge, England) Chen, J., Moreno, J. L., Zhang, W., Gibson-Elias, L. J., Lian, R., Najafi, S., Zhang, H., Zhong, W., Hooley, R. J. 2024; 60 (43): 5598-5601

  Abstract

  A simple aqueous host:guest sensing array can selectively discriminate between different types of citrus varietal from peel extract samples. It can also distinguish between identical citrus samples at varying stages of ripening. The discrimination effects stem from detection of changes in the terpenoid composition of the peel extracts by the host:guest array, despite the overwhelming excess of a single component, limonene, in each sample. The hosts are insensitive to limonene but bind other monoterpenes strongly, even though they are similar in structure to the major limonene component. This work demonstrates the capability of host:guest arrays in sensing target molecules in environments with the competing agents present at high abundances in the sample matrix.

  View details for DOI 10.1039/d4cc01309c

  View details for PubMedID 38712724

• Selective recognition and discrimination of single isomeric changes in peptide strands with a host : guest sensing array. Chemical science Chen, J., Fasihianifard, P., Raz, A. A., Hickey, B. L., Moreno, J. L., Chang, C. A., Hooley, R. J., Zhong, W. 2024; 15 (5): 1885-1893

  Abstract

  An indirect competitive binding mechanism can be exploited to allow a combination of cationic fluorophores and water-soluble synthetic receptors to selectively recognize and discriminate peptide strands containing a single isomeric residue in the backbone. Peptide isomerization occurs in long-lived proteins and has been linked with diseases such as Alzheimer's, cataracts and cancer, so isomers are valuable yet underexplored targets for selective recognition. Planar cationic fluorophores can selectively bind hydrophobic, Trp-containing peptide strands in solution, and when paired with receptors that provide a competitive host for the fluorophore, can form a differential sensing array that enables selective discrimination of peptide isomers. Residue variations such as D- and L-Asp, D- and L-isoAsp, D-Ser and D-Glu can all be recognized, simply by their effects on the folded structure of the flexible peptide. Molecular dynamics simulations were applied to determine the most favorable conformation of the peptide : fluorophore conjugate, indicating that favorable π-stacking with internal tryptophan residues in a folded binding pocket enables micromolar binding affinity.

  View details for DOI 10.1039/d3sc06087j

  View details for PubMedID 38303931

  View details for PubMedCentralID PMC10829040

• Selective Molecular Recognition and Indicator Displacement Sensing of Neurotransmitters in Cellular Environments. ACS sensors Chen, J., Tabaie, E. Z., Hickey, B. L., Gao, Z., Raz, A. A., Li, Z., Wilson, E. H., Hooley, R. J., Zhong, W. 2023; 8 (8): 3195-3204

  Abstract

  Flexible, water-soluble hosts are capable of selective molecular recognition in cellular environments and can detect neurotransmitters such as choline in cells. Both cationic and anionic water-soluble self-folded deep cavitands can recognize suitable styrylpyridinium dyes in cellular interiors. The dyes selectively accumulate in nucleotide-rich regions of the cell nucleus and cytoplasm. The hosts bind the dyes and promote their relocation to the outer cell membrane: the lipophilic cavitands predominantly reside in membrane environments but are still capable of binding suitable targets in other cellular organelles. Incubating the cells with structurally similar biomarkers such as choline, cholamine, betaine, or butyrylcholine illustrates the selective recognition. Choline and butyrylcholine can be bound by the hosts, but minimal binding is seen with betaine or cholamine. Varying the dye allows control of the optical detection method, and both "turn-on" sensing and "turn-off" sensing are possible.

  View details for DOI 10.1021/acssensors.3c00886

  View details for PubMedID 37477362

• Applications of Synthetic Receptors in Bioanalysis and Drug Transport. Bioconjugate chemistry Chen, J., Hooley, R. J., Zhong, W. 2022; 33 (12): 2245-2253

  Abstract

  Synthetic receptors are powerful tools for molecular recognition. They can bind to guests with high selectivity and affinity, and their structures are tunable and diversified. These features, plus the relatively low cost and high simplicity in synthesis and modification, support the feasibility of array-based molecular analysis with synthetic receptors for improved selectivity in the recognition of a wide range of targets. More attractively, host-guest interaction is reversible and guest displacement allows biocompatible and gentle release of the host-bound molecules, simplifying the stimulation designs needed to control analyte sensing, enrichment, and transportation. Here, we highlight a few recent advancements in using synthetic receptors for molecular analysis and manipulation, with the focus on macrocyclic receptors and their applications in displacement sensing, separation, imaging, and drug transport.

  View details for DOI 10.1021/acs.bioconjchem.2c00096

  View details for PubMedID 35362963

• Sensing Base Modifications in Non-Canonically Folded DNA with an Optimized Host:Guest Sensing Array. ACS sensors Chen, J., Hickey, B. L., Gao, Z., Raz, A. A., Hooley, R. J., Zhong, W. 2022; 7 (8): 2164-2169

  Abstract

  An arrayed host:guest fluorescence sensor system can discriminate DNA G-quadruplex structures that differ only in the presence of single oxidation or methylation modification in the guanine base. These small modifications make subtle changes to G4 folding that are often not detectable by CD but induce differential fluorescence responses in the array. The sensing is functional in diluted serum and is capable of distinguishing individual modifications in DNA mixtures, providing a powerful method of detecting folding changes caused by DNA damage.

  View details for DOI 10.1021/acssensors.2c00839

  View details for PubMedID 35917160

• Machine Learning Aids Classification and Discrimination of Noncanonical DNA Folding Motifs by an Arrayed Host:Guest Sensing System. Journal of the American Chemical Society Chen, J., Gill, A. D., Hickey, B. L., Gao, Z., Cui, X., Hooley, R. J., Zhong, W. 2021; 143 (32): 12791-12799

  Abstract

  An arrayed host:guest fluorescence sensor system can discriminate among and classify multiple different noncanonical DNA structures by exploiting selective molecular recognition. The sensor is highly selective and can discriminate between folds as similar as native G-quadruplexes and those with bulges or vacancies. The host and guest can form heteroternary complexes with DNA strands, with the host acting as mediator between the DNA and dye, modulating the emission. By applying machine learning algorithms to the sensing data, prediction of the folding state of unknown DNA strands is possible with high fidelity.

  View details for DOI 10.1021/jacs.1c06031

  View details for PubMedID 34346209

• Selective discrimination and classification of G-quadruplex structures with a host-guest sensing array. Nature chemistry Chen, J., Hickey, B. L., Wang, L., Lee, J., Gill, A. D., Favero, A., Pinalli, R., Dalcanale, E., Hooley, R. J., Zhong, W. 2021; 13 (5): 488-495

  Abstract

  The secondary structures of nucleic acids have an important influence on their cellular functions but can be difficult to identify and classify quickly. Here, we show that an arrayed suite of synthetic hosts and dyes is capable of fluorescence detection of oligonucleotide secondary structures. Multivariate analysis of different fluorescence enhancements-generated using cationic dyes that show affinity for both DNA G-quadruplexes and the synthetic hosts-enables discrimination between G-quadruplex structures of identical length and highly similar topological types. Different G-quadruplexes that display the same folding topology can also be easily differentiated by the number of G-quartets and sequence differences at the 3' or 5' ends. The array is capable of both differentiation and classification of the G-quadruplex structures at the same time. This simple non-invasive sensing method does not require the discovery and synthesis of specific G-quadruplex binding ligands, but employs a simple multicomponent approach to ensure wide applicability.

  View details for DOI 10.1038/s41557-021-00647-9

  View details for PubMedID 33795843

  View details for PubMedCentralID 3334823

• Solvent Effects and Internal Functions Control Molecular Recognition of Neutral Substrates in Functionalized Self-Assembled Cages JOURNAL OF ORGANIC CHEMISTRY Woods, C. Z., Sharma, K., Chen, C., Yang, L., Chen, J., Wu, Y., Farooqi, N. S., Zhang, J., Julian, R. R., Hooley, R. J. 2024

  Abstract

  A suite of internally functionalized Fe4L6 cage complexes has been synthesized with lipophilic end groups to allow dissolution in varied solvent mixtures, and the scope of their molecular recognition of a series of neutral, nonpolar guests has been analyzed. The lipophilic end groups confer cage solubility in solvents with a wide range of polarities, from hexafluoroisopropanol (HFIP) to tetrahydrofuran, and the hosts show micromolar affinities for neutral guests, despite having no flat panels enclosing the cavity. These hosts allow interrogation of the effects of an internal functional group on guest binding properties, as well as solvent-based driving forces for recognition. Introducing polar effects to the interior of the cavity enhances guest binding affinity in nonpolar solvents; adding space-filling aliphatic groups reduces affinity in all cases. While high dielectric solvents such as acetonitrile strongly favor guest binding, "low dielectric, high polarity" solvents such as HFIP strongly occupy the cavity and prevent guest recognition. Analysis of the cage optical transitions shows that the guests interact with the central ligand cores and reside in close proximity to the internal functions. These results have implications for supramolecular catalysis: balancing directed host:guest interactions (e.g., H-bonds) with entropic effects from solvent displacement is essential for reactions in these (and related) biomimetic hosts.

  View details for DOI 10.1021/acs.joc.4c02190

  View details for Web of Science ID 001378662500001

  View details for PubMedID 39680645

• Colocalization of protein and microRNA markers reveals unique extracellular vesicle subpopulations for early cancer detection. Science advances Li, Z., Guo, K., Gao, Z., Chen, J., Ye, Z., Cao, M., Wang, S. E., Yin, Y., Zhong, W. 2024; 10 (9): eadh8689

  Abstract

  Extracellular vesicles (EVs) play important roles in cell-cell communication but are highly heterogeneous, and each vesicle has dimensions smaller than 200 nm with very limited amounts of cargos encapsulated. The technique of NanOstirBar (NOB)-EnabLed Single Particle Analysis (NOBEL-SPA) reported in the present work permits rapid inspection of single EV with high confidence by confocal fluorescence microscopy, thus enables colocalization assessment for selected protein and microRNA (miRNA) markers in the EVs produced by various cell lines, or present in clinical sera samples. EV subpopulations marked by the colocalization of unique protein and miRNA combinations were discovered to be able to detect early-stage (stage I or II) breast cancer (BC). NOBEL-SPA can be adapted to analyze other types of cargo molecules or other small submicron biological particles. Study of the sorting of specific cargos to heterogeneous vesicles under different physiological conditions can help discover distinct vesicle subpopulations valuable in clinical examination and therapeutics development and gain better understanding of their biogenesis.

  View details for DOI 10.1126/sciadv.adh8689

  View details for PubMedID 38416840

  View details for PubMedCentralID PMC10901469

• Low-Dose Exposure of WS2 Nanosheets Induces Differential Apoptosis in Lung Epithelial Cells. Environmental science & technology Coreas, R., Li, Z., Chen, J., Zhong, W. 2023; 57 (39): 14493-14501

  Abstract

  Escalating the production and application of tungsten disulfide (WS2) nanosheets inevitably increases environmental human exposure and warrants the necessity of studies to elucidate their biological impacts. Herein, we assessed the toxicity of WS2 nanosheets and focused on the impacts of low doses (≤10 μg/mL) on normal (BEAS-2B) and tumorigenic (A549) lung epithelial cells. The low doses, which approximate real-world exposures, were found to induce cell apoptosis, while doses ≥ 50 μg/mL cause necrosis. Focused studies on low-dose exposure to WS2 nanosheets revealed more details of the impacts on both cell lines, including reduction of cell metabolic activity, induction of lipid peroxidation in cell membranes, and uncoupling of mitochondrial oxidative phosphorylation that led to the loss of ATP production. These phenomena, along with the expression situations of a few key proteins involved in apoptosis, point toward the occurrence of mitochondria-dependent apoptotic signaling in exposed cells. Substantial differences in responses to WS2 exposure between normal and tumorigenic lung epithelial cells were noticed as well. Specifically, BEAS-2B cells experienced more adverse effects and took up more nanosheets than A549 cells. Our results highlight the importance of dose and cell model selection in the assessment of nanotoxicity. By using doses consistent with real-world exposures and comparing normal and diseased cells, we can gain knowledge to guide the development of safety precautions for mitigating the adverse impacts of nanomaterial exposure on human health.

  View details for DOI 10.1021/acs.est.3c01843

  View details for PubMedID 37726893

• Selective anion sensing in high salt water via a remote indicator displacement assay. Chemical communications (Cambridge, England) Hickey, B. L., Raz, A. A., Chen, J., Moreno, J. L., Hartman, J. D., Zhong, W., Hooley, R. J. 2023; 59 (50): 7819-7822

  Abstract

  Water-soluble deep cavitands with cationic functions at the lower rim can selectively bind iodide anions in purely aqueous solution. By pairing this lower rim recognition with an indicator dye that is bound in the host cavity, optical sensing of anions is possible. The selectivity for iodide is high enough that micromolar concentrations of iodide can be detected in the presence of molar chloride. Iodide binding at the "remote" lower rim causes a conformational change in the host, displacing the bound dye from the cavity and effecting a fluorescence response. The sensing is sensitive, selective, and works in complex environments, so will be important for optical anion detection in biorelevant media.

  View details for DOI 10.1039/d3cc01974h

  View details for PubMedID 37272374

• Biological Impacts of Reduced Graphene Oxide Affected by Protein Corona Formation. Chemical research in toxicology Coreas, R., Castillo, C., Li, Z., Yan, D., Gao, Z., Chen, J., Bitounis, D., Parviz, D., Strano, M. S., Demokritou, P., Zhong, W. 2022; 35 (7): 1244-1256

  Abstract

  Applications of reduced graphene oxide (rGO) in many different areas have been gradually increasing owing to its unique physicochemical characteristics, demanding more understanding of their biological impacts. Herein, we assessed the toxicological effects of rGO in mammary epithelial cells. Because the as-synthesized rGO was dissolved in sodium cholate to maintain a stable aqueous dispersion, we hypothesize that changing the cholate concentration in the dispersion may alter the surface property of rGO and subsequently affect its cellular toxicity. Thus, four types of rGO were prepared and compared: rGO dispersed in 4 and 2 mg/mL sodium cholate, labeled as rGO and concentrated-rGO (c-rGO), respectively, and rGO and c-rGO coated with a protein corona through 1 h incubation in culture media, correspondingly named pro-rGO and pro-c-rGO. Notably, c-rGO and pro-c-rGO exhibited higher toxicity than rGO and pro-rGO and also caused higher reactive oxygen species production, more lipid membrane peroxidation, and more significant disruption of mitochondrial-based ATP synthesis. In all toxicological assessments, pro-c-rGO induced more severe adverse impacts than c-rGO. Further examination of the material surface, protein adsorption, and cellular uptake showed that the surface of c-rGO was coated with a lower content of surfactant and adsorbed more proteins, which may result in the higher cellular uptake observed with pro-c-rGO than pro-rGO. Several proteins involved in cellular redox mediation were also more enriched in pro-c-rGO. These results support the strong correlation between dispersant coating and corona formation and their subsequent cellular impacts. Future studies in this direction could reveal a deeper understanding of the correlation and the specific cellular pathways involved and help gain knowledge on how the toxicity of rGO could be modulated through surface modification, guiding the sustainable applications of rGO.

  View details for DOI 10.1021/acs.chemrestox.2c00042

  View details for PubMedID 35706338

  View details for PubMedCentralID PMC9842398

• Enantioselective sensing of insect pheromones in water. Chemical communications (Cambridge, England) Hickey, B. L., Chen, J., Zou, Y., Gill, A. D., Zhong, W., Millar, J. G., Hooley, R. J. 2021; 57 (98): 13341-13344

  Abstract

  An arrayed combination of water-soluble deep cavitands and cationic dyes has been shown to optically sense insect pheromones at micromolar concentration in water. Machine learning approaches were used to optimize the most effective array components, which allows differentiation between small structural differences in targets, including between different diastereomers, even though the pheromones have no innate chromophore. When combined with chiral additives, enantiodiscrimination is possible, dependent on the size and shape of the pheromone.

  View details for DOI 10.1039/d1cc05540b

  View details for PubMedID 34817473

• Monitoring the crosstalk between methylation and phosphorylation on histone peptides with host-assisted capillary electrophoresis. Analytical and bioanalytical chemistry Lee, J., Chen, J., Sarkar, P., Xue, M., Hooley, R. J., Zhong, W. 2020; 412 (24): 6189-6198

  Abstract

  Post-translational modifications (PTMs) greatly increase protein diversity and regulate their functions by changing the structures, properties, and molecular interactions of proteins. In peptide regions with high density of PTMs, PTMs can influence modification on residues in proximity or even at distal positions, adding another layer of regulation. Methods that can monitor the activities of PTM enzymes on peptides carrying multiple modifications are valuable tools for better understanding of PTM crosstalk. Herein, we developed a host-assisted capillary electrophoresis (CE) method to separate histone peptides with methylation and phosphorylation and applied it to monitor the crosstalk between serine phosphorylation and lysine methylation when they were added by Aurora B kinase and G9a lysine methyltransferase, respectively. A synthetic receptor molecule, 4-hexasulfonatocalix[6]arene (CX6), was included in the CE buffer to improve the resolution of the corresponding substrates and products. A linear polyacrylamide-coated capillary was employed to effectively reduce wall adsorption of the cationic histone peptides. The peptide substrates were labeled with fluorescein to enhance their detectability during CE separation. Our method successfully revealed that the activity of G9a methyltransferase was completely inhibited by the adjacent phosphorylation, while 25% reduction in the activity of Aurora B kinase was observed with the presence of dimethylation on the nearby residue. The PTM crosstalk was examined not only using a pure peptide substrate, but also in a competitive reaction environment, in which the modified and unmodified peptides were mixed and the enzyme actions on both peptides were monitored simultaneously. Our work demonstrates that host-assisted CE is an effective method for study of PTM crosstalk, which could offer the advantages of fast separation, high resolution, and low sample consumption. Graphical abstract.

  View details for DOI 10.1007/s00216-020-02486-y

  View details for PubMedID 32064571