New G-Triplex DNA Dramatically Activates the Fluorescence of Thioflavin T and Acts as a Turn-On Fluorescent Sensor for Uracil-DNA Glycosylase Activity Detection.

G-triplexes are G-rich oligonucleotides composed of three G-tracts and have absorbed much attention due to their potential biological functions and attractive performance in biosensing. Through the optimization of loop compositions, DNA lengths, and 5'-flanking bases of G-rich sequences, a new stable G-triplex sequence with 14 bases (G3-F15) was discovered to dramatically activate the fluorescence of Thioflavin T (ThT), a water-soluble fluorogenic dye. The fluorescence enhancement of ThT after binding with G3-F15 reached 3200 times, which was the strongest one by far among all of the G-rich sequences. The conformations of G3-F15 and G3-F15/ThT were studied by circular dichroism. The thermal stability measurements indicated that G3-F15 was a highly stable G-triplex structure. The conformations of G3-F15 and G3-F15/ThT in the presence of different metal cations were studied thoroughly by fluorescent spectroscopy, circular dichroism, and nuclear magnetic resonance. Furthermore, using the G3-F15/ThT complex as a fluorescent probe, a robust and simple turn-on fluorescent sensor for uracil-DNA glycosylase activity was developed. This study proposes a new systematic strategy to explore new functional G-rich sequences and their ligands, which will promote their applications in diagnosis, therapy, and biosensing.

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. The improvement of the detection sensitivity and specificity of analytical methods based on mass spectrometry makes it possible to study low-abundance modifications and their biological functions. Some typical nucleic acid modifications and their potential as biomarkers are displayed, and efforts to improve diagnostic accuracy are discussed. Future perspectives are raised for this research field.

Identification and Quantification of Locus-Specific 8-Oxo-7,8-dihydroguanine in DNA at Ultrahigh Resolution Based on G-Triplex-Assisted Rolling Circle Amplification.

Damage of reactive oxygen species to various molecules such as DNA has been related to many chronic and degenerative human diseases, aging, and even cancer. 8-Oxo-7,8-dihydroguanine (OG), the most significant oxidation product of guanine (G), has become a biomarker of oxidative stress as well as gene regulation. The positive effect of OG in activating transcription and the negative effect in inducing mutation are a double-edged sword; thus, site-specific quantification is helpful to quickly reveal the functional mechanism of OG at hotspots. Due to the possible biological effects of OG at extremely low abundance in the genome, the monitoring of OG is vulnerable to signal interference from a large amount of G. Herein, based on rolling circle amplification-induced G-triplex formation and Thioflavin T fluorescence enhancement, an ultrasensitive strategy for locus-specific OG quantification was constructed. Owing to the difference in the hydrogen-bonding pattern between OG and G, the nonspecific background signal of G sites was completely suppressed through enzymatic ligation of DNA probes and the triggered specificity of rolling circle amplification. After the signal amplification strategy was optimized, the high detection sensitivity of OG sites with an ultralow detection limit of 0.18 amol was achieved. Under the interference of G sites, as little as 0.05% of OG-containing DNA was first distinguished. This method was further used for qualitative and quantitative monitoring of locus-specific OG in genomic DNA under oxidative stress and identification of key OG sites with biological function.

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. Also, we established a MS1-only platform for nucleoside profiling and successfully identified sixteen nucleosides in the total RNA of MCF-7 cells. Taking ISF into account, we can realize analysis with higher sensitivity and less ambiguity, not only for nucleosides, but for other molecules with similar protonation and fragmentation behaviors.

Ultrasensitive HPLC-MS Quantification of S-(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 S-(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. The three carboxyl groups of 2SC were all esterified to increase hydrophobicity, greatly improving its ionization efficiency. The sensitivity was increased by 112 times; the limit of detection was reduced to 0.885 fmol, and the protein usage was reduced by at least 10 times. The established method was used to detect the overall concentration of 2SC in fumarate accumulation cells quantitatively.

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. Additionally, the contents of 5mdC and canonical nucleosides could be obtained at the same time. Upon derivatization, the detection sensitivities of 5hmdC, 5fdC, 5hmdU and 5fdU were dramatically enhanced by several hundred times. The established method was further applied to the simultaneous detection of nine nucleosides with different abundances in about 2 µg genomic DNA of breast tissues from 20 breast cancer patients. The DNA consumption was less than other overall reported quantification methods, thereby providing an opportunity to monitor rare, modified nucleosides in precious samples and biology processes that could not be investigated before. The contents of 5hmdC, 5hmdU and 5fdU in tumor tissues and normal tissues adjacent to the tumor were significantly changed, indicating that these three modified nucleosides may play certain roles in the formation and development of tumors and be potential cancer biomarkers. While the detection rates of 5hmdC, 5hmdU and 5fdU alone as a biomarker for breast cancer samples were 95%, 75% and 85%, respectively, by detecting these three cancer biomarkers simultaneously, two of the three were 100% consistent with the overall trend. Therefore, simultaneous detection of multiple cancer biomarkers in clinical samples greatly improved the accuracy of cancer diagnosis, indicating that our method has great application potential in clinical multidimensional diagnosis.

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. Herein, an ultrasensitive quantification of OG at single-base resolution was established using real-time fluorescence quantitative polymerase chain reaction as an amplification tool. Based on the coding property of Bsu DNA polymerase that incorporates adenine on the opposite site of OG and the selectivity of the ligase for perfectly matched sequences, the difference between OG and G on the sequence could be enlarged. Well-performed Taq DNA ligase was selected out, and as low as 46.2 zmol of target DNA with an OG site and an OG frequency of 5% could be detected. G contents on a specific site were also detectable based on the similar principle, thus the OG frequency of this locus could be accurately determined by a standard addition method. This strategy was successfully applied to the evaluation of locus-specific OG in both model DNA and genomic DNA from human cervical carcinoma cell lines under multiple oxidative stress, showing the potential for functional research and dynamic monitoring of critical OG sites.

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. Because of the trace amounts of exosomes and matrix complexity, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) as a powerful tool for label-free sensitive and simultaneous determinations of six important modified nucleosides on small RNAs inside exosomes. This system performed well using only approximately 107-108 particles of exosomes to obtain modified nucleoside levels between 0.001 and 0.03, and the most striking result was that the content of m6A in exosomal small RNAs was continuously higher than that in the cells being analyzed. We hope that this conclusion helps establish a greater degree of deciphering accuracy on exosomes, which has considerable application potential in the diagnosis and prognosis of diseases.

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 0.1% for the fully methylated target and high sensitivity of 10 pM have been proved by the successful design of binary-probe hybridization. The successful design of the binary probes makes it possible to quantify the average methylation levels of five CpG sites. Thirty-two DNA strands containing 5, 4, 3, 2, 1 and 0 CpG sites were successfully analyzed with the same pair of binary probes. The higher the average methylation level of the target was, the higher the degree of the hybridization reaction. Based on the simple construction of the binary-probe hybridization, the developed biosensor exhibited signals proportional to the average methylation level of the vimentin gene and could evaluate the average methylation level of artificial mixtures. Furthermore, the method has been used to detect vimentin methylation in a genomic context with good specificity, which indicated its potential in the pre-diagnosis of methylation related disease.

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. G3 has strong affinity with MB and it can cause significant decrease of the diffusion current of MB after binding. Melamine was chosen as the model target. Since melamine can interact with nucleobase thymine (T) to form T-melamine-T structure through complementary hydrogen bonds, a single-strand functional DNA hairpin structure with poly T and G3 elaborately blocked via base pairing was designed. The presence of melamine can trigger the conformation switching of the DNA hairpin to release the G3. The released G3 combined with MB could therefore change the diffusion current, leading to a simple and rapid detection of melamine. The combination of functional DNA hairpin as target recognition element, G3/MB as signal generator, and IME as transducer provided a "Mix and Measure" miniaturized platform for the construction of homogeneous label-free electrochemical biosensors.

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. Samples (in 10 µL) were mixed with methanol as protein precipitation solvent and 2,6-dimethylphenol as internal standard. Protein micro-precipitation was achieved with methanol by vortexing and centrifuging for 5 s each, and propofol was extracted to the supernatant. PSI-MS was performed in negative ionization mode, and MS signal lasted for 1 min. The analysis of a single sample was completed within 2 min. The area ratios of propofol to internal standard were calculated for quantification. Limit of detection of 5.5 ng mL-1 and limit of quantification of 18.2 ng mL-1 were achieved for propofol in whole blood. Calibration curve was linear in the range of 0.02-10 µg mL-1. The developed method was used successfully in monitoring the propofol concentration in 3 patients' whole blood during anesthesia, showing its further application in controlling and feeding-back target concentration infusion. Graphical abstract.

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. For a single SNP target, the assay achieves a limit of detection as low as 0.01-0.02% for three different NRAS mutations in the same codon. For multiple SNP targets, as low as 0.05% abundance can be easily realized. Our method is simple, efficient, ultrasensitive, and universal for multiple SNPs detection without complex enzymatic or chemical ligation reaction, which shows great potential in early clinical diagnosis.

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. Herein, we present a novel strategy based on the sheathless capillary electrophoresis-electrospray ionization-mass spectrometry (CESI-MS) system to characterize snake venom proteins. Through the determination of peptides, we found the characteristic peptides of 8 different snakes with high sensitivity (1 µg mL-1) and high selectivity, which provided a reliable method for the species identification and purity detection of snake venom samples.

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. By using miRNAs as a driving force, a two-stage isothermal exponential amplification reaction was proposed to generate G3 through miRNAs. The generated G3 can combine with MB and produce observable current changes, which depend on the concentration of miRNAs. Therefore, a novel homogeneous label-free electrochemical biosensor for miRNA detection was successfully constructed. By choosing let-7a, the down-regulation of which is possibly associated with the over-expression of RAS and HMGA2 oncogenes, as a model, we discovered that this biosensor demonstrated excellent analytical performance in detecting let-7a, with an ultralow limit of detection (0.45 fM) and high specificity (discriminating one nucleotide variation). Moreover, the proposed biosensor was successfully applied in monitoring the expression levels of the low-abundant miRNAs in the human lung adenocarcinoma cell lines. This assay successfully verified the feasibility of G-triplex/MB as an efficient and sensitive probe for immobilization-free and label-free electrochemical detection of nucleic acids, which would greatly promote the rapid development of homogeneous label-free electrochemical biosensors.

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. Here, this work reports an approach to fabricate dissolvable polyacrylamide beads, by crosslinking acrylamide with disulfide bridges that can be cleaved with dithiothreitol. The beads can be rapidly dissolved in drops and release DNA barcode primers. The dissolvable beads are easy to synthesize, and the primer cost for the beads is significantly lower than that for the previous barcoding beads. Furthermore, the dissolvable beads can be loaded into drops with >95% loading efficiency of a single bead per drop and the dissolution of beads does not influence reverse transcription or the polymerase chain reaction (PCR) in drops. Based on this approach, the dissolvable beads are used for single cell RNA and protein analysis.

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 MnO2 oxidation and hydrazine-s-triazine reagent (i-Pr2N) 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. This strategy improved the detection sensitivity of 5hmC by 178 times, and the limit of detection was as low as 14 amol. With simple preparation steps, 5hmC-MIQuant could quantify the 5hmC level in as little as 340 pg genomic DNA (equivalent to 57 copies of diploid genome). cfDNA samples from human plasma were successfully analyzed using 5hmC-MIQuant. This method is promising for the identification of 5hmC function in precious samples and the 5hmC-based noninvasive disease diagnosis.

Synthesis of a pH-Responsive Functional Covalent Organic Framework via Facile and Rapid One-Step Postsynthetic Modification and Its Application in Highly Efficient N1-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 N1-methyladenosine (m1A). Glutathione (GSH) was judiciously designed as the functional group for extracting and releasing m1A 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. Owing to the several-to-one immobilization of GSH on Au NPs and the large surface area of TpPa-1, this functional COF was constructed with abundant m1A binding sites. TpPa-1@Au@GSH showed excellent selectivity for m1A extraction by capturing m1A from a mixture of 14 nucleoside analogues followed by mass spectrometry analysis. It was proved to have ultrafast adsorption ability (only 1 min incubation time), high binding capacity (5 mg g-1, m1A/TpPa-1@Au@GSH), good reusability (at least 5 times), and good storage stability (at least 8 months at room temperature). Great performance was also achieved in extracting m1A from both animal and plant biological samples. The adsorption mechanism was demonstrated to be based on the electrostatic interaction. This work proposed a new approach for m1A extraction, demonstrated the high potential of COFs in biological sample pretreatment, and offered an effective and versatile route for functionalization of COFs.

DNAzyme-powered nucleic acid release from solid supports.

Here, we demonstrate use of a Mg2+-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.

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. Since the small difference in duplex stability caused by a single-nucleotide mismatch was amplified by the steric effects of these reactive groups for the ligation reaction of an unstable duplex, CuAAC-LA exhibited an ultra-sensitive discrimination ability for a mutant type target in the presence of large amounts of wild type targets. As low as 0.05% SNP could be clearly detected, which was better than most previously reported methods by various DNA ligases, indicating that a simple and rapid synthetic method i.e., the DNA template-directed click reaction held the potential to replace the ligase for SNP detection.

Ultrasensitive Determination of Rare Modified Cytosines Based on Novel Hydrazine Labeling Reagents.

Modified cytosines are important epigenetic marks that exert critical influences in a variety of cellular processes in living organisms. However, biological functions of rare modified cytosines, especially certain functions of 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), are still unclear due to the extremely low abundance in biological samples. In this work, a series of novel hydrazine-based reagents, which held a hydrazine group as the reaction group, a hydrophobic triazine group, and two easily charged tertiary amine groups with different alkyl chains for adjusting the hydrophobicity of the labeling reagents, were first explored to label rare modified cytosines such as 5fC and 5caC. The derivatization reaction between 5fC and the labeling reagents was extremely fast, and more than 99% derivatization efficiency could be achieved only by vortexing without additional reaction time. The detection sensitivity of 5fC increased with the increase of the hydrophobicity of the labeling reagents, the best of which was dramatically enhanced by 125-fold. The limit of detection was as low as 10 amol, realizing the most sensitive genome-wide overall quantification for 5fC. Moreover, the labeling reagents were also successfully applied for the detection of 5caC with 100-fold improvement of sensitivity. With this method, we achieved the simultaneous detection of 5fC and 5caC in different mammalian tissues using only about 600 ng of genomic DNA, which was less than one-tenth of the sample consumption for other reported methods, providing an opportunity to monitor 5fC and 5caC in precious samples and biology processes that could not be investigated before.