A DNA aptamer for binding and inhibition of DNA methyltransferase 1.

DNA methyltransferases (DNMTs) are enzymes responsible for establishing and maintaining DNA methylation in cells. DNMT inhibition is actively pursued in cancer treatment, dominantly through the formation of irreversible covalent complexes between small molecular compounds and DNMTs that suffers from low efficacy and high cytotoxicity, as well as no selectivity towards different DNMTs. Herein, we discover aptamers against the maintenance DNA methyltransferase, DNMT1, by coupling Asymmetrical Flow Field-Flow Fractionation (AF4) with Systematic Evolution of Ligands by EXponential enrichment (SELEX). One of the identified aptamers, Apt. #9, contains a stem-loop structure, and can displace the hemi-methylated DNA duplex, the native substrate of DNMT1, off the protein on sub-micromolar scale, leading for effective enzymatic inhibition. Apt. #9 shows no inhibition nor binding activity towards two de novo DNMTs, DNMT3A and DNMT3B. Intriguingly, it can enter cancer cells with over-expression of DNMT1, colocalize with DNMT1 inside the nuclei, and inhibit the activity of DNMT1 in cells. This study opens the possibility of exploring the aptameric DNMT inhibitors being a new cancer therapeutic approach, by modulating DNMT activity selectively through reversible interaction. The aptamers could also be valuable tools for study of the functions of DNMTs and the related epigenetic mechanisms

Fluorescamine Labeling for Assessment of Protein Conformational Change and Binding Affinity in Protein–Nanoparticle Interaction

Protein adsorption alters the "biological identity" of nanoparticles (NPs) and could affect how biosystems respond to invading NPs. Study of protein-NP interaction can help understand how the physicochemical properties of NPs impact the interaction and thus potentially guide the design of safer and more effective NPs for biomedical or other applications. Binding affinity between proteins and NPs and the occurrence of protein conformational change upon binding to NPs are two important aspects to be learned, but few methods are currently available to assess both simultaneously in a simple way. Herein, we demonstrated that the fluorescamine labeling method developed by our group not only could reveal protein conformational change upon adsorption to NPs, owing to its capability to label the primary amines exposed on protein surface, but also could be applied to measure the binding affinity. By screening the interaction between a large number of proteins and four types of NPs, the present study also revealed that protein adsorption onto NPs could be strongly affected by structure flexibility. The proteins with high structure flexibility experienced high degrees of conformation change when binding to the polystyrene NPs, which could potentially influence protein function. Overall, we demonstrate that our assay is a quick, simple, and high-throughput tool to reveal potential impacts on protein activity and evaluate the strength of protein-NP binding

Rapid Enrichment and Sensitive Detection of Multiple Metal Ions Enabled by Macroporous Graphene Foam

Nanomaterials have shown great promise in advancing biomedical and environmental analysis because of the unique properties originated from their ultrafine dimensions. In general, nanomaterials are separately applied to either enhance detection by producing strong signals upon target recognition or to specifically extract analytes taking advantage of their high specific surface area. Herein, we report a dual-functional nanomaterial-based platform that can simultaneously enrich and enable sensitive detection of multiple metal ions. The macroporous graphene foam (GF) we prepared displays abundant phosphate groups on the surface and can extract divalent metal ions via metal-phosphate coordination. The enriched metal ions then activate the metal-responsive DNAzymes and produce the fluorescently labeled single-stranded DNAs that are adsorbed and quenched by the GF. The resultant fluorescence reduction can be used for metal quantitation. The present work demonstrated duplexed detection of Pb2+ and Cu2+ using the Pb- and Cu-responsive DNAzymes, achieving a low detection limit of 50 pM and 0.6 nM, respectively. Successful quantification of Pb2+ and Cu2+ in human serum and river water were achieved with high metal recovery. Since the phosphate-decorated GF can enrich diverse types of divalent metal cations, this dual-functional GF-DNAzyme platform can serve as a simple and cost-effective tool for rapid and accurate metal quantification in determination of human metal exposure and inspection of environmental contamination

Prediction of protein corona on nanomaterials by machine learning using novel descriptors

Effective in silico methods to predict protein corona compositions on engineered nanomaterials (ENMs) could help elucidate the biological outcomes of ENMs in biosystems without the need for conducting lengthy experiments for corona characterization. However, the physicochemical properties of ENMs, used as the descriptors in current modeling methods, are insufficient to represent the complex interactions between ENMs and proteins. Herein, we utilized the fluorescence change (FC) from fluorescamine labeling on a protein, with or without the presence of the ENM, as a novel descriptor of the ENM to build machine learning models for corona formation. FCs were significantly correlated with the abundance of the corresponding proteins in the corona on diverse classes of ENMs, including metal and metal oxides, nanocellulose, and 2D ENMs. Prediction models established by the random forest algorithm using FCs as the ENM descriptors showed better performance than the conventional descriptors, such as ENM size and surface charge, in the prediction of corona formation. Moreover, they were able to predict protein corona formation on ENMs with very heterogeneous properties. We believe this novel descriptor can improve in silico studies of corona formation, leading to a better understanding on the protein adsorption behaviors of diverse ENMs in different biological matrices. Such information is essential for gaining a comprehensive view of how ENMs interact with biological systems in ENM safety and sustainability assessments

Accelerating attribute-focused cell culture process development through the deployment of an automatic assay preparation platform

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Fluorescence Labeling and Limited Proteolysis for Demystifying Protein Corona

Engineered nanomaterials (ENMs) have great application potentials in biological systems, while the protein corona formed around ENMs after they encounter any biofluids makes their fate difficult to be predicted. Protein corona provides a brand new “biological identify” for ENMs, which will determine the recognition, targeting, and compatibility of ENMs in vivo. One important property of protein corona is its dynamic exchange of components during incubation time, on which lots of endeavors have been made, but there is still knowledge gap between the intrinsic properties of ENMs and what kinds of protein will form the corona. Moreover, the molecular details of protein in corona, including the orientation, conformational change and aggregation, are also important for the function of protein corona. Due to the non-specific forces behind protein-ENMs interactions, desired and controlled arrangement of protein on ENMs surface has always been a significant but difficult task. Despite of various success made on those two topics, rapid and routine methods for composition analysis and molecular details exploration of protein corona are still in stark deficient. This research will focus on new methods development for those two problems. Firstly, a fluorescamine labeling based high throughput screening method is applied to screen and discriminate interactions between single protein and ENMs, which could indicate either protein binding or unfolding induced by ENMs. With those results as descriptors, correlations between them and protein corona composition have been found, which suggests that a structure activity quantification model using those descriptors could be built for rapid corona prediction. Secondly, limited proteolysis coupled with LC-MS/MS capable to identify binding sites of protein on another molecule has been developed. By applying it to those positive protein-ENMs pairs identified in previous screening, the molecular details including orientation and unfolding of proteins in corona could be unveiled. Despite of the limitation on precision, information obtained from this method could be helpful for further rational design of ENMs on biological application