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

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

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Probing and quantifying DNA–protein interactions with asymmetrical flow field-flow fractionation

Tools capable of measuring binding affinities as well as amenable to downstream sequencing analysis are needed for study of DNA-protein interaction, particularly in discovery of new DNA sequences with affinity to diverse targets. Asymmetrical flow field-flow fractionation (AF4) is an open-channel separation technique that eliminates interference from column packing to the non-covalently bound complex and could potentially be applied for study of macromolecular interaction. The recovery and elution behaviors of the poly(dA)n strand and aptamers in AF4 were investigated. Good recovery of ssDNAs was achieved by judicious selection of the channel membrane with consideration of the membrane pore diameter and the radius of gyration (Rg) of the ssDNA, which was obtained with the aid of a Molecular Dynamics tool. The Rg values were also used to assess the folding situation of aptamers based on their migration times in AF4. The interactions between two ssDNA aptamers and their respective protein components were investigated. Using AF4, near-baseline resolution between the free and protein-bound aptamer fractions could be obtained. With this information, dissociation constants of ∼16nM and ∼57nM were obtained for an IgE aptamer and a streptavidin aptamer, respectively. In addition, free and protein-bound IgE aptamer was extracted from the AF4 eluate and amplified, illustrating the potential of AF4 in screening ssDNAs with high affinity to targets. Our results demonstrate that AF4 is an effective tool holding several advantages over the existing techniques and should be useful for study of diverse macromolecular interaction systems

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

Mapping Molecular Structure of Protein Locating on Nanoparticles with Limited Proteolysis

The molecular structure of a protein could be altered when it is attached to nanoparticles (NPs), affecting the performance of NPs present in biological systems. Limited proteolysis coupled with LC-MS/MS could reveal the changes in protein structure when it binds to a variety of entities, including macro-molecules and small drugs, but it has not yet been applied to study protein-NP interaction. Herein, adsorption of proteins, transferrin, and catalase on the polystyrene (PS) or iron oxide (IO) NPs was analyzed with this method. Both increased and decreased proteolytic efficiency in certain regions on the proteins were observed. Identification of the peptides affected by protein-NP interaction led to proper prediction of alterations to protein function as well as to colloidal stability of NPs. Overall, the present work has demonstrated the utility of limited proteolysis in helping to elucidate the potential biological outcomes of the protein-NP conjugate, obtaining knowledge to guide improvement of the rational design of the protein-conjugated NPs for biomedical applications and to understand the biological behaviors of the engineered NPs

High-throughput profiling of nanoparticle-protein interactions by fluorescamine labeling.

A rapid, high throughput fluorescence assay was designed to screen interactions between proteins and nanoparticles. The assay employs fluorescamine, a primary-amine specific fluorogenic dye, to label proteins. Because fluorescamine could specifically target the surface amines on proteins, a conformational change of the protein upon interaction with nanoparticles will result in a change in fluorescence. In the present study, the assay was applied to test the interactions between a selection of proteins and nanoparticles made of polystyrene, silica, or iron oxide. The particles were also different in their hydrodynamic diameter, synthesis procedure, or surface modification. Significant labeling differences were detected when the same protein incubated with different particles. Principal component analysis (PCA) on the collected fluorescence profiles revealed clear grouping effects of the particles based on their properties. The results prove that fluorescamine labeling is capable of detecting protein-nanoparticle interactions, and the resulting fluorescence profile is sensitive to differences in nanoparticle's physical properties. The assay can be carried out in a high-throughput manner, and is rapid with low operation cost. Thus, it is well suited for evaluating interactions between a larger number of proteins and nanoparticles. Such assessment can help to improve our understanding on the molecular basis that governs the biological behaviors of nanomaterials. It will also be useful for initial examination of the bioactivity and reproducibility of nanomaterials employed in biomedical fields

ZrO₂ Nanofiber as a Versatile Tool for Protein Analysis

Phosphorylation is one of the most important post-translational modifications in proteins. Their essential roles in the regulation of cellular processes and alteration of protein–protein interaction networks have been actively studied. However, phosphorylated proteins are present at low abundance in cells, and ionization of the modified peptides is often suppressed by the more abundant species in mass spectrometry. Effective enrichment techniques are needed to remove the unmodified peptides and concentrate the phosphorylated ones before their identification and quantification. Herein, we prepared ZrO₂ nanofibers by electrospinning, a straightforward and easy fabrication technique, and applied them to enrich phosphorylated peptides and proteins. The fibers showed good size homogeneity and porosity and could specifically bind to the phosphorylated peptides and proteins, allowing their separation from the unmodified analogues when present in either simple protein digests or highly complex cell lysates. The enrichment performance was superior to that of the commercially available nanoparticles. Moreover, modifying the solution pH could lead to selective adsorption of proteins with different p<i>I</i> values, suggesting the fibers’ potential applicability in charge-based protein fractionation. Our results support that the electrospun ZrO₂ nanofibers can serve as a versatile tool for protein analysis with great ease in preparation and handling