2'-Fluorinated Hydantoins as Chemical Biology Tools for Base Excision Repair Glycosylases.

The guanine oxidation products, 5-guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp), are mutagenic and toxic base lesions that are removed by Fpg, Nei, and the Nei-like (NEIL) glycosylases as the first step in base excision repair (BER). The hydantoins are excellent substrates for the NEIL glycosylases in a variety of DNA contexts beyond canonical duplex DNA, implicating the potential impact of repair activity on a multitude of cellular processes. In order to prepare stable derivatives as chemical biology tools, oligonucleotides containing fluorine at the 2'-position of the sugar of 8-oxo-7,8-dihydro-2'-deoxyguanosine2'-F-OG) were synthesized in ribo and arabino configuration. Selective oxidation of 2'-F-OG within a DNA oligonucleotide provided the corresponding 2'-F-Gh or 2'-F-Sp containing DNA. The 2'-F-hydantoins in duplex DNA were found to be highly resistant to the glycosylase activity of Fpg and NEIL1 compared to the unmodified lesion substrates. Surprisingly, however, some glycosylase-mediated base removal from both the 2'-F-ribo- and 2'-F-arabinohydantoin duplex DNA was observed. Notably, the associated ß-lyase strand scission reaction of the 2'-F-arabinohydantoins was inhibited such that the glycosylases were "stalled" at the Schiff-base intermediate. Fpg and NEIL1 showed high affinity for the 2'-F-Gh duplexes in both ribo and arabino configurations. However, binding affinity assessed using catalytically inactive variants of Fpg and NEIL1 indicated higher affinity for the 2'-F-riboGh-containing duplexes. The distinct features of glycosylase processing of 2'-F-ribohydantoins and 2'-F-arabinohydantoins illustrate their utility to reveal structural insight into damage recognition and excision by NEIL and related glycosylases and provide opportunities for delineating the impact of lesion formation and repair in cells.

Targeting Base Excision Repair Glycosylases with DNA Containing Transition State Mimics Prepared via Click Chemistry.

DNA glycosylases of the base excision repair (BER) pathway are front-line defenders in removing compromising modifications of the DNA nucleobases. Aberrantly modified nucleobases mediate genomic mutations and inhibit DNA replication leading to adverse health consequences such as cancer, neurological diseases, and aging. In an effort to develop high-affinity transition state (TS) analogues as chemical biology probes for DNA glycosylases, oligonucleotides containing a propargyl-modified pyrrolidine TS mimic nucleotide were synthesized. A small library of TS mimic-containing oligonucleotides was generated using a structurally diverse set of five azides via copper(I)-catalyzed azide-alkyne cycloaddition "click" chemistry. The relative affinity ( Kd) was evaluated for BER glycosylases Escherichia coli MutY, bacterial formamidopyrimidine glycosylase (Fpg), and human OG glycosylase 1 (hOGG1) with the library of TS mimic DNA duplexes. All of the BER glycosylases were found to exhibit extremely high affinities (approximately picomolar Kd values) for the TS mimics. However, binding preferences, distinct for each glycosylase, for the TS mimic library members were observed, suggesting different modes of binding and transition state stabilization among the three glycosylases. Fpg bound all of the TS mimics with exceptionally high affinities, while the MutY binding affinity correlated inversely with the size of the appended moiety. Of note, we identified one member of the small TS mimic library that exhibited a particularly high affinity for hOGG1. These results strongly support the use of the propargyl-TS mimic oligonucleotides and elaboration via click chemistry in screening and identification of high-affinity ligands for BER glycosylases of interest.

Breast-cancer-secreted miR-122 reprograms glucose metabolism in premetastatic niche to promote metastasis.

Reprogrammed glucose metabolism as a result of increased glycolysis and glucose uptake is a hallmark of cancer. Here we show that cancer cells can suppress glucose uptake by non-tumour cells in the premetastatic niche, by secreting vesicles that carry high levels of the miR-122 microRNA. High miR-122 levels in the circulation have been associated with metastasis in breast cancer patients, and we show that cancer-cell-secreted miR-122 facilitates metastasis by increasing nutrient availability in the premetastatic niche. Mechanistically, cancer-cell-derived miR-122 suppresses glucose uptake by niche cells in vitro and in vivo by downregulating the glycolytic enzyme pyruvate kinase. In vivo inhibition of miR-122 restores glucose uptake in distant organs, including brain and lungs, and decreases the incidence of metastasis. These results demonstrate that, by modifying glucose utilization by recipient premetastatic niche cells, cancer-derived extracellular miR-122 is able to reprogram systemic energy metabolism to facilitate disease progression.

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.

Distribution profiling of circulating microRNAs in serum.

Circulating microRNAs (miRNAs) are potential biomarkers useful in cancer diagnosis. They have been found to be bound to various carriers like proteins, lipoprotein particles, and exosomes. It is likely that only miRNAs in particular carriers, but not the overall quantity, are directly related to cancer development. Herein, we developed a method for rapid separation of different miRNA carriers in serum using asymmetrical flow field flow fractionation (AF4). Sera from two healthy individuals (control) or from two cancer patients (case) were fractionated. Six fractions enriching different types of miRNA carriers, such as the lipoprotein particles and exosomes, were collected. The quantities of eight selected miRNAs in each fraction were obtained by RT-qPCR to yield their distribution profiles among the carriers. Larger changes in miRNA quantity between the control and the case were detected in the fractionated results compared to the sum values. Statistical analysis on the distribution profiles also proved that, the quantities of 4 miRNAs within particular fractions showed significant difference between the controls and the cases. On the contrary, if the overall quantity of the miRNA was subject to the same statistical analysis, only 2 miRNAs exhibited significant difference. Moreover, principle component analysis revealed good separation between the controls and the cases with the fractionated miRNA amounts. All in all, we have demonstrated that, our method enables comprehensive screening of the distribution of circulating miRNAs in the carriers. The obtained distribution profile enlarges the miRNA expression difference between healthy individuals and cancer patients, facilitating the discovery of specific miRNA biomarkers for cancer diagnosis.

Size and surface functionalization of iron oxide nanoparticles influence the composition and dynamic nature of their protein corona.

Nanoparticles (NPs) adsorb proteins when in the biological matrix, and the resulted protein corona could affect NP-cell interactions. The corona has a dynamic nature with the adsorbed proteins constantly exchanging with the free proteins in the matrix at various rates. The rapidly exchanging proteins compose the soft corona, which responds more dynamically to environment changes than the hard corona established by the ones with slow exchange rates. In the present study, the corona formed on the superparamagnetic iron oxide NPs (SPIONs) in human serum was studied by flow field-flow fractionation and ultracentrifugation, which rapidly differentiated the corona proteins based on their exchange rates. By varying the surface hydrophobicity of the SPIONs with a core size around 10 nm, we found out that, the more hydrophobic surface ligand attracted proteins with higher surface hydrophobicity and formed a more dynamic corona with a larger portion of the involved proteins with fast exchange rates. Increasing the core diameter of the SPIONs but keeping the surface ligand the same could also result in a more dynamic corona. A brief investigation of the effect on the cellular uptake of SPIONs using one selected corona protein, transferrin, was conducted. The result showed that, only the stably bound transferrin could significantly enhance cellular uptake, while transferrin bound in a dynamic nature had negligible impact. Our study has led to a better understanding of the relationship between the particle properties and the dynamic nature of the corona, which can help with design of nanomaterials with higher biocompatibility and higher efficacy in biosystems for biomedical applications.

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.

Dissociation-based screening of nanoparticle-protein interaction via flow field-flow fractionation.

A protein corona will be formed on nanoparticles (NPs) entering a biological matrix, which can influence particles' subsequent behaviors inside the biological systems. For proteins bound stably to the NPs, they can exhibit different association/dissociation rates. The binding kinetics could affect interaction of the NPs with cell surface receptors and possibly contribute to the outcomes of NPs uptake. In the present study, a method to differentiate the corona proteins based on their relative dissociation rates from the NPs was developed, employing flow field-flow fraction (F4) in combination with centrifugation. The proteins bound to the superparamagnetic iron oxide NPs (SPION) present in an IgG/albumin depleted serum were isolated via collection of the SPIONs by either F4 or centrifugation. They were subsequently analyzed by LC-MS/MS and identified. Because the SPION-protein complexes injected to F4 dissociated continuously under the nonequilibrium separation condition, only the proteins with slow enough dissociation rates would be collected with the NPs in the eluent of F4. However, in centrifugation, proteins with good affinity to the SPIONs were collected regardless of the dissociation rates of the complexes. In both cases, the nonbinding ones were washed off. Capillary electrophoresis and circular dichroism were employed to verify the binding situations of a few SPION-protein interactions, confirming the effectiveness of our method. Our results support that our method can screen for proteins binding to NPs with fast on-and-off rates, which should be the ones quickly exchanging with the free matrix proteins when the NPs are exposed to a new biological media. Thus, our method will be useful for investigation of the temporal profile of protein corona and its evolution in biological matrices as well as for high-throughput analysis of the dynamic feature of protein corona related to particle properties.

Spin state modulation of iron spin crossover complexes via hydrogen-bonding self-assembly.

Iron complexes derived from 6-diaminotriazyl-2,2'-bipyridines display spin crossover behaviour, and hydrogen bonding-controlled self-assembly with a suitable barbiturate partner can modulate the crossover from mixed low and high spin to high spin. This system is the first to use solution-phase self-assembly of complementary hydrogen-bonding organic species to modulate spin state.

Aptamer-protein binding detected by asymmetric flow field flow fractionation.

Asymmetric flow field flow fractionation (AF4) should be suitable for the study of aptamer-target binding, because its gentle separation would impose little disturbance to the complex structure, and it can use carrier solutions with high salt concentrations to provide the most optimal interaction environment to the complex. However, no report has been found for such applications. Herein, we investigated the utility of AF4 as an effective tool for detection of the aptamer-protein complex. With the model system of human immunoglobulin E (IgE) and its aptamer, impacts on aptamer binding from the incubation and AF4 carrier solutions, as well as the flow conditions used during the sample focusing step, were studied. We found that the composition of the carrier solution, in particular, the presence of Mg(2+), strongly influenced the complex's integrity in AF4. Also, the focusing conditions during sample injection in AF4 affected the binding equilibrium. Our findings highlight the necessity of maintaining the optimal binding environment during the time course of complex measurement; and demonstrated the good compatibility of AF4 with salty buffers and its high simplicity in conducting on-channel incubation. With its capability to carry out size-based separation of analytes with a wide range of dimensions, AF4 can be employed for detection of large proteins and even biological particles using aptamers. AF4 is also valuable for study of aptamer-target binding under different buffer environments for better understanding of the structure-function relationship of aptamers.

Impact of carrier fluid composition on recovery of nanoparticles and proteins in flow field flow fractionation.

Flow field flow fractionation (F4) is an invaluable separation tool for large analytes, including nanoparticles and biomolecule complexes. However, sample loss due to analyte-channel membrane interaction limits extensive usage of F4 at present, which could be strongly affected by the carrier fluid composition. This work studied the impacts of carrier fluid (CF) composition on nanoparticle (NP) recovery in F4, with focus on high ionic strength conditions. Successful analysis of NPs in a biomolecules-friendly environment could expand the applicability of F4 to the developing field of nanobiotechnology. Recovery of the unfunctionalized polystyrene NPs of 199, 102, and 45 nm in CFs with various pH (6.2, 7.4 and 8.2), increasing ionic strength (0-0.1M), and different types of co- and counter-ions, were investigated. Additionally, elution of the 85 nm carboxylate NPs and two proteins, human serum albumin (HSA) and immunoglobulin (IgG), at high ionic strengths (0-0.15M) was investigated. Our results suggested that (1) electrostatic repulsion between the negatively charged NPs and the regenerated cellulose membrane was the main force to avoid particle adsorption on the membrane; (2) larger particles experienced higher attractive force and thus were influenced more by variation in CF composition; and (3) buffers containing weak anions or NPs with weak anion as the surface functional groups provided higher tolerance to the increase in ionic strength, owing to more anions being trapped inside the NP porous structure. Protein adsorption onto the membrane was also briefly investigated in salted CFs, using HSA and IgG. We believe our findings could help to identify the basic carrier fluid composition for higher sample recovery in F4 analysis of nanoparticles in a protein-friendly environment, which will be useful for applying F4 in bioassays and in nanotoxicology studies.

Advances in field-flow fractionation for the analysis of biomolecules: instrument design and hyphenation.

Field-flow fractionation (FFF) separates analytes by use of an axial channel-flow and a cross-field. Its soft separation capability makes it an ideal tool for initial fractionation of complex mixtures, but large elution volumes and high flow rates have limited its applicability without significant user handling. Recent advances in instrumentation and miniaturization have successfully reduced channel size and elution speed, and thus the volume of each fraction, making it possible to conveniently couple FFF with orthogonal separation techniques for improved resolution. More detailed analysis can also be performed on the fractions generated by FFF by use of diverse analytical techniques, including MS, NMR, and even X-ray scattering. These developmental trends have given FFF more power in the analysis of different types of molecule, and will be the direction of choice for further advances in FFF technology.