Insight into the G-Quadruplex Topologies: A Rapid Separation and Identification Method of Multiple Coexisting Conformations by Capillary Zone Electrophoresis.

G-quadruplexes (G4s) are noncanonical secondary structures, which have been demonstrated to be prevalent at gene regulatory regions in various genomes. In solution, G4s derived from single-stranded oligonucleotides can adopt different topologies. Although considerable research over the past three decades has led to in-depth insight into these unusual structures, there is still a great demand to develop a rapid, easy, and universal strategy to separate and identify G4 topologies, especially G4s with various coexisting structures.

[Innovation of chromatographic analysis experiment teaching and cultivation of innovative talents].

Chromatography experiments form an important component of an Instrumental Analysis Laboratory Course. In order to meet the demands of cultivating innovative talents with scientific thinking skills for the evolving educational objectives， the Instrumental Analysis Laboratory Course in the College of Chemistry and Molecular Engineering at Peking University has been reformed since 2017， with a focus on chromatography experiment teaching as a pilot project. A series of well-designed reform measures， including strengthening the design of the experimental content， focusing on the analysis of actual samples， increasing student participation， enhancing their freedom of exploration and the division of labor and cooperation， and adopting a teaching form with both virtual and real components， have been introduced.

LC-MS-based untargeted metabolomics reveals benzoic acid as a predictive biomarker for embryo implantation potential.

Evaluating the quality of embryos and implantation potential is a critical determinant of fertilization and embryo transfer, and it is also one of the main challenges of assisted reproductive technology. A reliable non-invasive method to choose the best candidate with real implantation potential for transfer from two day-3 embryos with equal morphological quality is still lacking clinically. In this article, a sensitive LC-MS method was developed and metabolomic profiling analysis in a 3-day embryo culture medium was performed.

The fabrication of 1,2-dicarbonyl compound-caging isothermal exponential amplification strategy and its application in the highly sensitive detection of tumor exosomal miRNA.

Exponential amplification reaction (EXPAR) possesses the advantage of high amplification efficiency, producing large amount of short nucleic acids in a short time. However, primer-independent DNA autosynthesis and nonspecific amplification caused by DNA self-synthesis increase the risk of high background signals, limiting its application in the fabrication of ultrasensitive sensors. In this study, we developed a new strategy called 1,2-dicarbonyl compound-caging EXPAR (caging-EXPAR) by innovatively modifying EXPAR templates with 1,2-dicarbonyl compounds.

Glycosylase Pretreatment with Chemical Labeling-Assisted HPLC-MS/MS: An Ultrasensitive and Reliable Strategy for Quantification of 8-Oxo-7,8-dihydro-2'-deoxyguanosine in Genomic DNA.

8-Oxo-7,8-dihydro-2'-deoxyguanosine (dOG), the dominant oxidative product of 2'-deoxyguanosine (dG) under high levels of reactive oxygen species, usually serves as a biomarker for oxidative stress and a risk assessment factor for various diseases. Due to the extremely low abundance of dOG and the susceptibility of dOG detection to the interference of spurious oxidation, research on related biological processes is limited by insufficient sensitivity and specificity. In this work, an ultrasensitive and reliable approach for genome-wide dOG quantification was developed through chemical labeling-assisted high-performance liquid chromatography-tandem mass spectrometry with the introduction of glycosylase pretreatment.

Ultrasensitive mass spectrometric quantitation of apurinic/apyrimidinic sites in genomic DNA of mammalian cell lines exposed to genotoxic reagents.

The apurinic/apyrimidinic (AP) site is an important intermediate in the DNA base excision repair (BER) pathway, having the potential of being a biomarker for DNA damage. AP sites could lead to the stalling of polymerases, the misincorporation of bases and DNA strand breaks, which might affect physiological function of cells. However, the abundance of AP sites in genomic DNA is very low (less than 2 AP sites/10 nts), which requires a sensitive and accurate method to meet its detection requirements.

Qualitative and Quantitative Analytical Techniques of Nucleic Acid Modification Based on Mass Spectrometry for Biomarker Discovery.

Nucleic acid modifications play important roles in biological activities and disease occurrences, and have been considered as cancer biomarkers. Due to the relatively low amount of nucleic acid modifications in biological samples, it is necessary to develop sensitive and reliable qualitative and quantitative methods to reveal the content of any modifications. In this review, the key processes affecting the qualitative and quantitative analyses are discussed, such as sample digestion, nucleoside extraction, chemical labeling, chromatographic separation, mass spectrometry detection, and data processing.

In-source fragmentation of nucleosides in electrospray ionization towards more sensitive and accurate nucleoside analysis.

Nucleosides have been found to suffer in-source fragmentation (ISF) in electrospray ionization mass spectrometry, which leads to reduced sensitivity and ambiguous identification. In this work, a combination of theoretical calculations and nuclear magnetic resonance analysis revealed the key role of protonation at N3 near the glycosidic bond during ISF. Therefore, an ultrasensitive liquid chromatography-tandem mass spectrometry system for 5-formylcytosine detection was developed with 300 fold signal enhancement.

Ultrasensitive HPLC-MS Quantification of -(2-Succino) Cysteine Based on Ethanol/Acetyl Chloride Derivatization in Fumarate Accumulation Cells.

Succination is a nonenzymatic and irreversible post-translational modification (PTM) with important biological significance, yielding -(2-succino) cysteine (2SC) residue. This PTM is low in abundance and often requires a large amount of protein samples for 2SC quantification. In this work, an efficient quantification method based on ethanol/acetyl chloride chemical derivatization was developed.

Ultrasensitive Simultaneous Detection of Multiple Rare Modified Nucleosides as Promising Biomarkers in Low-Put Breast Cancer DNA Samples for Clinical Multi-Dimensional Diagnosis.

Early cancer diagnosis is essential for successful treatment and prognosis, and modified nucleosides have attracted widespread attention as a promising group of cancer biomarkers. However, analyzing these modified nucleosides with an extremely low abundance is a great challenge, especially analyzing multiple modified nucleosides with a different abundance simultaneously. In this work, an ultrasensitive quantification method based on chemical labeling, coupled with LC-MS/MS analysis, was established for the simultaneous quantification of 5hmdC, 5fdC, 5hmdU and 5fdU.

Ultrasensitive and Single-Base Resolution Quantification of 8-Oxo-7,8-dihydroguanine in DNA by Extension and Ligation-Based qPCR.

Oxidative DNA damage is tightly linked to the development of multiple age-related diseases. The prominent oxidation product is 8-oxo-7,8-dihydroguanine (OG), which has been proved to be an important epigenetic-like biomarker. Quantification of the locus-specific OG frequency includes quantitative and locating information, which is of great significance for exploring the functional roles of OG in disease induction and gene regulation.

Systematic Profiling of Exosomal Small RNA Epigenetic Modifications by High-Performance Liquid Chromatography-Mass Spectrometry.

Exosomes are nanosized extracellular vesicles that have a critical role in intercellular communication and tumor microenvironment regulation. Extensive research has shown that exosomal small RNAs contribute to metastasis in multiple tumor types and that abnormal epigenetic modifications in nucleic acids also have an association with diverse diseases. However, the content of modified nucleosides on exosomal small RNAs has not been quantitatively reported.

Detection of average methylation level of specific genes by binary-probe hybridization.

We developed a simple and highly-selective method for 5-methylcytosine detection of specific gene sequence based on binary-probe DNA hybridization. The sequence complementary to the target was designed into two probes, and each fragment of binary probes bound to a relatively short sequence of the target, which made it sensitive to the base mismatches introduced by bisulfite treatment. The advantages of a low detection limit of methylation abundance of 0.

A novel miniaturized homogeneous label-free electrochemical biosensing platform combining integrated microelectrode and functional nucleic acids.

A miniaturized platform combining integrated microelectrode (IME) and functional nucleic acids was developed for homogeneous label-free electrochemical biosensing. IME was constructed with a carbon fiber microelectrode and a platinum wire in a θ type glass tube as a two-electrode system for electrochemical monitoring at microliter level. A newly reported G-triplex/methylene blue (G3/MB) complex was used as the signal generator in the homogeneous label-free electrochemical biosensor.

Rapid determination and continuous monitoring of propofol in microliter whole blood sample during anesthesia by paper spray ionization-mass spectrometry.

Propofol is a widely used intravenous anesthetic agent in sedation and general anesthesia. To improve the safety and maintain the depth of anesthesia, it is important to develop a rapid, sensitive, and reliable method to monitor the concentration of propofol in blood during anesthesia continuously. Here, we present a novel strategy based on paper spray ionization-mass spectrometry (PSI-MS) to detect propofol.

Ultrasensitive Multiplex Detection of Single Nucleotide Polymorphisms Based on Short-Chain Hybridization Combined with Online Preconcentration of Capillary Electrophoresis.

Reliable multiple single nucleotide polymorphisms (SNPs) detection at low abundance is of great significance for disease diagnosis and biomedical research. Herein, we have developed a novel and simple method for multiple SNPs detection combining solid-phase capture by specific hybridization with online preconcentration of capillary gel electrophoresis-laser-induced fluorescence (CGE-LIF). The method presents an excellent performance due to its favorable traits: the solid-phase short-chain hybridization ensures the high specificity of SNP detection; the effective separation ability of CGE can easily achieve multiplex detection; the simple online preconcentration significantly improves the detection sensitivity of fluorescent probe by nearly 100-fold.

Snake venom characteristic peptides: novel fingerprints for species identification by sheathless capillary electrophoresis-electrospray ionization-mass spectrometry.

Snake venom is a complex mixture mainly consisting of proteins and peptides which varies with different species. These variations lead to different toxic mechanisms and different anti-venom serums for treatment and the determination of their use as drugs. Hence, it is important to develop a sensitive and reliable method to identify the species of snakes from venoms.

Ultrasensitive detection of microRNA based on a homogeneous label-free electrochemical platform using G-triplex/methylene blue as a signal generator.

The electrochemical methods for microRNA (miRNA) detection have received increasing attention because high portability and affordability of electrochemical biosensors may facilitate point-of-care quantitative detection of miRNAs. Among these biosensors, the homogenous label-free electrochemical biosensors for miRNAs are rarely reported due to the lack of a universal and efficient signal read-out-mode. A newly discovered G-triplex, 5'-CTGGGAGGGAGGGA-3' (denoted as G3), can specifically bind with methylene blue (MB), leading to a significant decrease of the diffusion current of MB.

Dissolvable Polyacrylamide Beads for High-Throughput Droplet DNA Barcoding.

Droplet-based single cell sequencing technologies, such as inDrop, Drop-seq, and 10X Genomics, are catalyzing a revolution in the understanding of biology. Barcoding beads are key components for these technologies. What is limiting today are barcoding beads that are easy to fabricate, can efficiently deliver primers into drops, and thus achieve high detection efficiency.

DNAzyme-powered nucleic acid release from solid supports.

Here, we demonstrate use of a Mg-dependent, site-specific DNA enzyme (DNAzyme) to cleave oligos from polyacrylamide gel beads, which is suitable for use in drop-based assays. We show that cleavage efficiency is improved by use of a tandem-repeat cleavage site. We further demonstrate that DNAzyme-released oligos function as primers in reverse transcription of cell-released mRNA.

5hmC-MIQuant: Ultrasensitive Quantitative Detection of 5-Hydroxymethylcytosine in Low-Input Cell-Free DNA Samples.

Cell-free DNA (cfDNA)-based biomarkers such as mutation and methylation offer promising noninvasive strategies for disease diagnosis and prognosis. However, besides high-throughput sequencing, there has been no alternative approach to date to detect the epigenetic marks, such as 5-hydroxymethylcytosine (5hmC), in cfDNA. Here, we described a MnO oxidation and hydrazine--triazine reagent (i-PrN) labeling based method named 5hmC-MIQuant that achieved ultrasensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) quantification of 5hmC in low-input DNA samples.

Synthesis of a pH-Responsive Functional Covalent Organic Framework via Facile and Rapid One-Step Postsynthetic Modification and Its Application in Highly Efficient -Methyladenosine Extraction.

A facile and rapid postsynthetic modification strategy for functionalization of covalent organic framework (COF) was developed to synthesize a tailor-made pH-responsive COF called TpPa-1@Au@GSH for highly efficient extraction of -methyladenosine (mA). Glutathione (GSH) was judiciously designed as the functional group for extracting and releasing mA by pH variations. With the aid of gold nanoparticles (Au NPs) as linkers, GSH was successfully introduced to the robust substrate TpPa-1 in only one step spending only 1 h.

High-throughput ultra-sensitive discrimination of single nucleotide polymorphism via click chemical ligation.

Single nucleotide polymorphisms (SNPs) have been proven to be important biomarkers for disease diagnosis, prognosis and disease pathogenesis. Here, taking the advantages of a self-assembled oligonucleotide sandwich structure and robust chemical reactions, we have developed a simple, high-throughput and effective colorimetric analytical technique termed CuAAC-based ligation-assisted assays (CuAAC-LA) for SNP detection using a DNA-BIND 96-well plate. With the 5'-azide and 3'-alkyne groups labelled on two oligonucleotide probes, the target DNA can direct a Cu(i)-catalyzed alkyne-azide cycloaddition (CuAAC) click reaction.