Multivalent protein–carbohydrate interactions initiate the first contacts between virus/bacteria and target cells, which ultimately lead to infection. Understanding the structures and binding modes involved is vital to the design of specific, potent multivalent inhibitors. However, the lack of structural information on such flexible, complex, and multimeric cell surface membrane proteins has often hampered such endeavors. Herein, we report that quantum dots (QDs) displayed with a dense array of mono-/disaccharides are powerful probes for multivalent protein–glycan interactions. Using a pair of closely related tetrameric lectins, DC-SIGN and DC-SIGNR, which bind to the HIV and Ebola virus glycoproteins (EBOV-GP) to augment viral entry and infect target cells, we show that such QDs efficiently dissect the different DC-SIGN/R-glycan binding modes (tetra-/di-/monovalent) through a combination of multimodal readouts: Förster resonance energy transfer (FRET), hydrodynamic size measurement, and transmission electron microscopy imaging. We also report a new QD-FRET method for quantifying QD-DC-SIGN/R binding affinity, revealing that DC-SIGN binds to the QD >100-fold tighter than does DC-SIGNR. This result is consistent with DC-SIGN’s higher trans-infection efficiency of some HIV strains over DC-SIGNR. Finally, we show that the QDs potently inhibit DC-SIGN-mediated enhancement of EBOV-GP-driven transduction of target cells with IC50 values down to 0.7 nM, matching well to their DC-SIGN binding constant (apparent Kd = 0.6 nM) measured by FRET. These results suggest that the glycan-QDs are powerful multifunctional probes for dissecting multivalent protein–ligand recognition and predicting glyconanoparticle inhibition of virus infection at the cellular level.

A highly efficient cap‐exchange approach for preparing compact, dense polyvalent mannose‐capped quantum dots (QDs) has been developed. The resulting QDs have been successfully used to probe multivalent interactions of HIV/Ebola receptors DC‐SIGN and DC‐SIGNR (collectively termed as DC‐SIGN/R) using a sensitive, ratiometric Förster resonance energy transfer (FRET) assay. The QD probes specifically bind DC‐SIGN, but not its closely related receptor DC‐SIGNR, which is further confirmed by its specific blocking of DC‐SIGN engagement with the Ebola virus glycoprotein. Tuning the QD surface mannose valency reveals that DC‐SIGN binds more efficiently to densely packed mannosides. A FRET‐based thermodynamic study reveals that the binding is enthalpy‐driven. This work establishes QD FRET as a rapid, sensitive technique for probing structure and thermodynamics of multivalent protein–ligand interactions.

Early B cell factor 1 (EBF1) is a transcription factor involved in the differentiation of several stem cell lineages and it is a negative regulator of estrogen receptors. EBF1 is down-regulated in many tumors, and is believed to play suppressive roles in cancer promotion and progression. However, the functional roles of EBF1 in carcinogenesis are unclear. Liver fluke-infection-associated cholangiocarcinoma (CCA) is an oxidative stress-driven cancer of bile duct epithelium. In this study, we investigated EBF1 expression in tissues from CCA patients, CCA cell lines (KKU-213, KKU-214 and KKU-156), cholangiocyte (MMNK1) and its oxidative stress-resistant (ox-MMNK1-L) cell lines. The formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) was used as an oxidative stress marker. Our results revealed that EBF1 expression was suppressed in cancer cells compared with the individual normal bile duct cells at tumor adjacent areas of CCA tissues. CCA patients with low EBF1 expression and high formation of 8-oxodG were shown to correlate with poor survival. Moreover, EBF1 was suppressed in the oxidative stress-resistant cell line and all of CCA cell lines compared to the cholangiocyte cell line. This suggests that prolonged oxidative stress suppressed EBF1 expression and the reduced EBF1 level may facilitate CCA genesis. To elucidate the significance of EBF1 suppression in CCA genesis, EBF1 expression of the MMNK1 cell line was down-regulated by siRNA technique, and its effects on stem cell properties (CD133 and Oct3/4 expressions), tumorigenic properties (cell proliferation, wound healing and cell migration), estrogen responsive gene (TFF1), estrogen-stimulated wound healing, and cell migration were examined. The results showed that CD133, Oct3/4 and TFF1 expression levels, wound healing, and cell migration of EBF1 knockdown-MMNK1 cells were significantly increased. Also, cell migration of EBF1-knockdown cells was significantly enhanced after 17β-estradiol treatment. Our findings suggest that EBF1 down-regulation via oxidative stress induces stem cell properties, tumorigenic properties and estrogen responses of cholangiocytes leading to CCA genesis with aggressive clinical outcomes.

Colorimetric aptasensor based on assembly of salt-induced gold nanoparticles (AuNPs) is a promising biosensor. However, the molecular mechanism of the aptasensor is far from being fully understood. Herein, molecular dynamics (MD) simulation was used to investigate molecular interactions in the detection of ochratoxin A (OTA) including the following: (i) the molecular recognition of the anti-OTA aptamer, (ii) OTA-aptamer interactions in monovalent (Na) and divalent (Mg) electrolytes, (iii) the binding mode of citrate on the AuNP surface, (iv) interactions of the aptamer with citrate-capped AuNPs, and (v) a detailed mechanism of the aptasensor. Our MD simulations revealed a specific binding of the OTA-aptamer complex, compared with OTB and warfarin. Compared with Na, Mg ions exerted a more effective attractive force between OTA and anti-OTA aptamer. Three different binding modes of citrate on AuNP surfaces were found. The kinetics of the adsorption of unfolded aptamers onto the citrate-capped AuNP was also elucidated. Most importantly, our MD simulation revealed an insightful analysis of the molecular mechanisms in the AuNP-based aptasensor and paved the way for the design of a novel colorimetric aptasensor for other target molecules, which is not limited to OTA detection.

A visual strategy for 8-oxo-dG monitoring based upon the dispersion of citrate-capped gold nanoparticles has been developed.

The concentration of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) in urine or serum is associated with the degree of oxidative damage of DNA and broadly used as a sensitive biomarker for various diseases. However, determination of a low concentration of 8-oxo-dG in biosamples is not an easy task owing to the complexity of coexisting substances. Herein, we design an aptasensor based on aptamer-mediated aggregation of cysteamine-capped gold nanoparticles (Cyst/AuNPs) for the detection of 8-oxo-dG by molecular dynamics simulation. Our simulations reveal that a positively charged Cyst modified onto the surfaces of AuNP exists in two conformers including gauche and trans. The trans conformer was prevalent on the AuNP surfaces and can stabilize AuNPs in the aqueous solution, even in the presence of 8-oxo-dG. Molecular recognition between 8-oxo-dG and the aptamer was demonstrated and bonding between these biomolecules was thoroughly elucidated. During the complex formation, van der Waals stacking interactions between 8-oxo-dG molecules were observed and found to play a significant role in the binding stability. The sensing mechanism of the colorimetric aptasensor was studied and the feasibility study of the proposed aptasensor was assessed by experimental validation. The experimental results are in good agreement with the computational study. Our in silico design can pave the way for, but is not limited to, a highly sensitive aptasensor for the naked-eye detection of 8-oxo-dG.

A highly efficient cap‐exchange approach for preparing compact, dense polyvalent mannose‐capped quantum dots (QDs) has been developed. The resulting QDs have been successfully used to probe multivalent interactions of HIV/Ebola receptors DC‐SIGN and DC‐SIGNR (collectively termed as DC‐SIGN/R) using a sensitive, ratiometric Förster resonance energy transfer (FRET) assay. The QD probes specifically bind DC‐SIGN, but not its closely related receptor DC‐SIGNR, which is further confirmed by its specific blocking of DC‐SIGN engagement with the Ebola virus glycoprotein. Tuning the QD surface mannose valency reveals that DC‐SIGN binds more efficiently to densely packed mannosides. A FRET‐based thermodynamic study reveals that the binding is enthalpy‐driven. This work establishes QD FRET as a rapid, sensitive technique for probing structure and thermodynamics of multivalent protein–ligand interactions.

We describe single‐chain polymer nanoparticles (SCNPs) possessing intramolecular dynamic covalent crosslinks that can transform into polymer films through a molecular recognition‐mediated crosslinking process. The SCNPs utilise molecular recognition with surface‐immobilised proteins to concentrate upon a substrate, bringing the SCNPs into close spatial proximity with one another and allowing their dynamic covalent crosslinkers to undergo intra‐ to interpolymer chain crosslinking leading to the formation of polymeric film. SCNPs must possess both the capacity for specific molecular recognition and a dynamic nature to their intramolecular crosslinkers to form polymer films, and an investigation of the initial phase of film formation indicates it proceeds from features which form upon the surface then grow predominantly in the xy directions. This approach to polymer film formation presents a potential method to “wrap” surfaces displaying molecular recognition motifs—which could potentially include viral, cellular and bacterial surfaces or artificial surfaces displaying multivalent recognition motifs—within a layer of polymer film.