Highly Efficient Gel Electrophoresis for Accurate Quantification of Nucleic Acid Modifications via in-Gel Digestion with UHPLC-MS/MS

Gel electrophoresis is a powerful technique for the characterization of sequences, sizes and conformations of nucleic acids due to its remarkable separation efficiency. In parallel, liquid chromatography–mass spectrometry (LC-MS) has established itself as a staple tool for the meticulous characterization and accurate quantification of a multitude of DNA modifications. In this study, we devised an in-gel digestion method for coupling gel electrophoresis with LC-MS/MS. This process involves the enzymatic digestion of DNA within the gel by nucleases and release single nucleosides, which subsequently serve as a preprocessing step for (LC-MS/MS) analysis. We demonstrated that ethylenediaminetetraacetic acid (EDTA) in the routine gel electrophoresis buffer reduced the enzymatic digestion efficiency, while Mg2+ could mitigate this inhibition. We further showed EDTA-free gel electrophoresis and the process of digestion of genomic DNA and plasmid DNA within a gel was fluorescently imaged, proving the efficient digestion of DNA. By this improvement, the efficiency of an in-gel digestion could reach 60% or more of the control, compared with direct in-solution digestion. The measured abundances of DNA modifications (5-methylcytosine and N6-methyladenine) via in-gel digestion are consistent with that measured by in-solution digestion. Collectively, we showed an in-gel digestion method, which is a very useful pretreatment technique for the precise quantification of epigenetic modifications in diverse DNA molecules

Bisphenol AP Exposure-Induced Inflammatory Response in Zebrafish Embryos Revealed by Transcriptomic Analysis

Bisphenol AP (BPAP) is a bisphenol analogue that is used as a substitute for BPA in industrial production and is now commonly found in the environment. Currently, there is limited information available on its developmental toxicity in aquatic organisms. This study exploited zebrafish embryos to investigate the adverse effects of BPAP exposure during their early life stages. The results revealed that BPAP had a median lethal concentration (LC50) of 36, 22, and 8.5 μmol/L for zebrafish embryos at 24, 48, and 72 h post fertilization (hpf), respectively. Additionally, exposure to BPAP led to abnormal development in zebrafish embryos, including hatching delay, malformation, and inhibition of spontaneous movements. Transcriptomic analysis further revealed that BPAP exposure upregulated the mRNA expression levels of genes related to inflammatory pathways, such as il1b, ptgs2b, and fosab, indicating the induction of an inflammatory response. Overall, our findings provide early and valuable insights into the environmental and health risks associated with BPAP exposure

Organic Osmolyte Mediated Kinetic Capillary Electrophoresis for Study of Protein−DNA Interactions

An organic osmolyte mediated kinetic capillary electrophoresis is developed and applied to study the binding of Escherichia coli RecA protein to single-stranded (ss) DNA. The complex of RecA and ssDNA can be significantly enhanced and stabilized by 20% glycerol over 5.6 times. Kinetic capillary electrophoresis study reveals that glycerol may function in the preservation of RecA native conformation, enhancement of specific binding affinity (20 times), and decrease in dissociation rate constant (1 time). The combined laser-induced fluorescence polarization (LIFP) study further indicates a tightened assembly of RecA on ssDNA induced by glycerol. The binding of RecA to ssDNA was also enhanced by two additional protecting organic osmolytes, sucrose and PEG 400. In contrast, a denaturing organic osmolyte, urea, even at a concentration of 110 mM can prevent the formation of the RecA−ssDNA complexes. The detection of the binding of E. coli single-stranded binding protein (SSBP) to ssDNA was also improved by the same method. By combining the protection of organic osmolytes with low-temperature storage, a method for preservation of protein−DNA complex up to 43 h is further demonstrated. By use of a long capillary and protecting glycerol together an enhanced kinetic CE analysis was also developed, providing a better separation and analysis of the undissociated complex, the dissociated complex, and the unbound probe. The results demonstrate the wide applicability of the method to the study of various protein−DNA binding

Fluorescence Anisotropy Reduction of An Allosteric G‑Rich Oligonucleotide for Specific Silver Ion and Cysteine Detection Based on the G‑Ag⁺‑G Base Pair

Silver is a common heavy metal, and the detection of silver ion (Ag+) is of great importance because of its wide application and hazardous effect on the environment and human health. However, it is a great challenge to produce a large fluorescence anisotropy (FA) change for small molecules (e.g, Ag+). Herein, we describe a novel fluorescence anisotropy reduction approach for the sensitive and specific detection of Ag+. The feasibility of this method is demonstrated through screening a number of guanine-rich oligonucleotide probes. By selectively labeling the oligonucleotides with a single fluorophore tetramethylrhodamine (TMR), the reduction in FA response is associated with the conformation change from the unfolded to a hairpin-like folded structure by inducing formation of the intermolecular G-Ag+-G base pair, which diminishes the interaction between guanine and TMR by photoinduced electron transfer (PET). The change in FA allows the selective detection of Ag+ at a concentration as low as 0.5 nM with a dynamic range from 2.0 to 100 nM. The interference from the other 14 metal ions with a 100-fold even to a 1000-fold excess amount is negligible. This simple and cost-effective probe was further explored to determine cysteine (Cys) based on competing with a guanine-rich oligonucelotide for Ag+-binding

The Ubiquitin Superfamily: Members, Features, and Phylogenies

The ubiquitin superfamily is a rich repository of small, conserved, functionally unique, and important proteins. Its member proteins fold simply and similarly, with kinetic and thermodynamic ease (Sorenson, J. M.; Head-Gordon, T. Toward minimalist models of larger proteins:  A ubiquitin-like protein. Proteins 2002, 46, 368−379). They have been implicated in numerous cancers, neurodegenerations, inflammations, and various disorders affecting signal transduction or protein half-life. These proteins serve the cell generally as portable recognition tags with distinct intracellular roles; indeed, tagging with small protein modifiers has become a new hallmark of post-translational modifications and other signal transduction phenomenon (Finley, D. J. Signal transduction. An alternative to destruction. Nature 2001, 412, 283, 285−286). Because many ubiquitin-like proteins bear similarities in sequence, structure, and function, we gathered protein sequences containing the ubiquitin domain from public databases and created a highly granular and defined protein catabolism database to catalog, summarize, reference, and relate them to their targets and specific ligases (to be described elsewhere). In this paper, we reveal a compilation of proteins possessing the ubiquitin domain. This comprises the first and most important part of our database content. We searched available organismal proteomes for sequence-related members of the ubiquitin superfamily and here present over 200 proteins possessing this domain. These proteins were organized phylogenetically and functionally, thereby defining several new families. To our knowledge, this is the most complete assemblage of ubiquitin domains to date. Keywords: ubiquitin • SUMO-1 • NEDD8 • proteolysis • post-translational modification • signal transduction • catabolism • databas

The Ubiquitin Superfamily: Members, Features, and Phylogenies

The ubiquitin superfamily is a rich repository of small, conserved, functionally unique, and important proteins. Its member proteins fold simply and similarly, with kinetic and thermodynamic ease (Sorenson, J. M.; Head-Gordon, T. Toward minimalist models of larger proteins:  A ubiquitin-like protein. Proteins 2002, 46, 368−379). They have been implicated in numerous cancers, neurodegenerations, inflammations, and various disorders affecting signal transduction or protein half-life. These proteins serve the cell generally as portable recognition tags with distinct intracellular roles; indeed, tagging with small protein modifiers has become a new hallmark of post-translational modifications and other signal transduction phenomenon (Finley, D. J. Signal transduction. An alternative to destruction. Nature 2001, 412, 283, 285−286). Because many ubiquitin-like proteins bear similarities in sequence, structure, and function, we gathered protein sequences containing the ubiquitin domain from public databases and created a highly granular and defined protein catabolism database to catalog, summarize, reference, and relate them to their targets and specific ligases (to be described elsewhere). In this paper, we reveal a compilation of proteins possessing the ubiquitin domain. This comprises the first and most important part of our database content. We searched available organismal proteomes for sequence-related members of the ubiquitin superfamily and here present over 200 proteins possessing this domain. These proteins were organized phylogenetically and functionally, thereby defining several new families. To our knowledge, this is the most complete assemblage of ubiquitin domains to date. Keywords: ubiquitin • SUMO-1 • NEDD8 • proteolysis • post-translational modification • signal transduction • catabolism • databas

Fluorescence Anisotropy Analysis for Mapping Aptamer–Protein Interaction at the Single Nucleotide Level

Structural characterization of aptamer–protein interactions is challenging and limited despite the tremendous applications of aptamers. Here we for the first time report a fluorescence anisotropy (FA) approach for mapping the interaction of an aptamer and its protein target at the single nucleotide level. Nine fluorescently labeled aptamers, each conjugated to a single tetramethylrhodamine at a specified nucleotide in the aptamer, were used to study their interactions with thrombin. Simultaneous monitoring of both fluorescence anisotropy changes and electrophoretic mobility shifts upon binding of the fluorescently modified aptamer to the protein provides unique information on the specific nucleotide site of binding. T25, T20, T7 and the 3′-end were identified as the close contact sites, and T3, C15T, and the 5′-end were identified as the sites distant from the binding. This approach is highly sensitive and does not require cross-linking reactions. Studies of aptamer–protein interactions using this technique are potentially useful for design, evolution, and modification of functional aptamers for a range of bioanalytical, diagnostic, and therapeutic applications

Identification of Allosteric Nucleotide Sites of Tetramethylrhodamine-Labeled Aptamer for Noncompetitive Aptamer-Based Fluorescence Anisotropy Detection of a Small Molecule, Ochratoxin A

Aptamer-based fluorescence anisotropy (FA) assay combines the advantages of affinity aptamers in good stability, easy generation, and facile labeling and the benefits of FA in homogeneous analysis, such as robustness, simplicity, and high reproducibility. By using a fluorophore-labeled aptamer, FA detection of a small molecule is not as easy as detection of protein because the binding of a small molecule cannot cause significant increase of molecular weight of the dye-labeled aptamer. The intramolecular interaction between labeled tetramethylrhodamine (TMR) and DNA aptamer bases dramatically affects the local rotation and FA of TMR. This intramolecular interaction can be altered by aptamer conformation change upon target binding, leading to a significant change of FA of TMR. Taking this unique feature of a TMR-labeled aptamer, we described a noncompetitive aptamer-based fluorescence anisotropy assay for detection of small molecules by using ochratoxin A (OTA) as a model. We successfully identified the specific TMR-labeling sites of aptamers with sensitive FA response to OTA from the 5′-end, 3′-end and the internal thymine (T) bases. The aptamer with a TMR labeled on the 10th T base exhibited a remarkable FA reduction response to OTA (Δr = 0.078), without requiring any proteins or nanomaterials as FA signal enhancers. This FA approach for OTA showed high sensitivity with a detection limit of 3 nM, a dynamic range from 3 nM to 3 μM, and good selectivity over the tested compounds with similar structures to OTA. The new strategy allowed the detection of OTA in diluted red wine and urine samples

Quantification of Epigenetic DNA Modifications in the Subchromatin Structure Matrix Attachment Regions by Stable Isotope Dilution UHPLC-MS/MS Analysis

To date, subchromatin structure-based quantification of epigenetic DNA modifications is limited. Matrix attachment regions (MARs), an important subchromatin structure, contain DNA elements that specifically bind chromatin to the nuclear matrix in eukaryotes and are involved in a number of diseases. Here, we exploited a high-salt extraction-based subchromatin fractionation approach for the isolation of MAR DNA and other fractions and further developed heavy stable isotope-diluted ultrahigh-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) for the specific quantification of epigenetic DNA modifications in the subchromatin structures. By this approach, we showed for the first time that the content of a DNA demethylation intermediate, 5-hydroxymethylcytosine (5hmdC), in MARs decreased significantly in four tested cell lines compared to the contents in genomic DNA. In particular, the content of DNA 5hmdC in the MARs of 293T cell lines decreased the most at approximately 41.09%. Together, our findings implicate that MAR DNA is less sensitive than genomic DNA to DNA demethylation

Artificially Evolved Superbinder for Specific Recognition of N⁶‑Methyladenine Base Modification in DNA and RNA

N6-Methyladenine (m6A/6mA) is a functional epigenetic base modification found in RNA and DNA. By selecting one RNA m6A reader as a template, we created a series of libraries of 3 × 108 RNA m6A reader mutants and developed a yeast surface-display-based evolution approach. Using high-throughput fluorescence-activated cell sorting, we ultimately obtained three evolved 6mA-binding proteins (e6mABPs), which displayed increased affinity for 6mA-containing DNA and reduced affinity for 6mA-free DNA. These e6mABPs are applicable for m6A/6mA enrichment and are potentially applied for modulating cell behavior