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.

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.

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.

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.

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.

An integrated-molecular-beacon based multiple exponential strand displacement amplification strategy for ultrasensitive detection of DNA methyltransferase activity.

DNA methylation is a significant epigenetic mechanism involving processes of transferring a methyl group onto cytosine or adenine. Such DNA modification catalyzed by methyltransferase (MTase) plays important roles in the modulation of gene expression and other cellular activities. Herein, we develop a simple and sensitive biosensing platform for the detection of DNA MTase activity by using only two oligonucleotides. The fluorophore labeled molecular beacon (MB) can be methylated by MTase and subsequently cleaved by endonuclease DpnI at the stem, giving a shortened MB. The shortened MB can then hybridize with a primer DNA, initiating a cycle of strand displacement amplification (SDA) reactions. The obtained SDA products can unfold new MB and initiate another cycle of SDA reaction. Therefore, continuous enlargement of SDA and exponential amplification of the fluorescence signal are achieved. Because the triple functions of substrate, template and probe are elegantly integrated in one oligonucleotide, only two oligonucleotides are necessary for multiple amplification cycles, which not only reduces the complexity of the system, but also overcomes the laborious and cumbersome operation that is always a challenge in conventional methods. This platform exhibits an extremely low limit of detection of 3.3 × 10-6 U mL-1, which is the lowest to our knowledge. The proposed MTase-sensing platform was also demonstrated to perform well in a real-time monitoring mode, which can achieve a further simplified and high-throughput detection. The sensing strategy might be extended to the activity detection of other enzymes, thus showing great application potential in bioanalysis and clinical diagnosis.