Probe-labeled electrochemical approach for highly selective detection of 5-carboxycytosine in DNA.

5-carboxycytosine (5caC) plays a critical role as an intermediate form in DNA methylation and demethylation processes. Its distribution and quantity significantly influence the dynamic equilibrium of these processes, thereby impacting the normal physiological activities of organisms. However, the analysis of 5caC presents a significant challenge due to its low abundance in the genome, making it almost undetectable in most tissues. In response to this challenge, we propose a selective method for 5caC detection using differential pulse voltammetry (DPV) at glassy carbon electrode (GCE), hinging on probe labeling. The probe molecule Biotin LC-Hydrazide was introduced into the target base and the labeled DNA was immobilized onto the electrode surface with the help of T4 polynucleotide kinase (T4 PNK). Leveraging the precise and efficient recognition of streptavidin and biotin, streptavidin-horseradish peroxidase (SA-HRP) on the surface of the electrode catalyzed a redox reaction involving hydroquinone and hydrogen peroxide, resulting in an amplified current signal. This procedure allowed us to quantitatively detect 5caC based on variations in current signals. This method demonstrated good linearity ranging from 0.01 to 100 nM with a detection limit as low as 7.9 pM. We have successfully applied it to evaluate the 5caC levels in complex biological samples. The probe labeling contributes to a high selectivity for 5caC detection, while the sulfhydryl modification via T4 PNK efficiently circumvents the limitation of specific sequences. Encouragingly, no reports have been made about electrochemical methods for detecting 5caC in DNA, suggesting that our method offers a promising alternative for 5caC detection in clinical samples.

Oxime formation coordination-directed detection of genome-wide thymine oxides with nanogram-scale sample input.

Natural chemical modifications of nucleic acids play a vital role in life processes. Compared to other epigenetic modifications, there are multiple ways to quantify the methylated derivatives of cytosine. However, simple and convenient methods for detecting and quantifying thymine derivatives are scarce because they are found in tiny quantities in biological systems. Additionally, exploring easy ways to detect these derivatives can also throw light on their biological significance. This manuscript reports a novel strategy to quantify 5-formyluracil (5fU) and 5-hydroxymethyluracil (5hmU). Differences between modified and unmodified bases are accumulated and amplified by arranging phi29 DNA polymerase to repeat through a circular template labeled thymidine. In combination with real-time quantitative rolling circle amplification (RCA), low-abundance thymine oxides can be quantified precisely. The global levels of 5fU and 5hmU were analyzed in different biological samples, using only 40 ng of sample input on a laboratory real-time PCR instrument. The reported strategy was executed hassle-free and, in principle, can be extended to design methods for detecting other epigenetic modifications in nucleotides that are rare in biological systems.

Determination of 5-Formyluracil via Oxime-Based Nucleotide-Metal Coordination.

2-(Aminooxy)-N-(quinolin-8-yl)acetamide was synthesized, and its ability to regulate activities of DNA polymerase was tested. In addition, we used the isothermal amplification technology to detect the content of 5-formyluracil sites in irradiated genomic DNA, which confirmed its capability for the detection of 5-formyluracil content in general samples. This study presents the first example of the determination of 5fU based on coordination chemistry.

Highly Selective Electrochemical Detection of 5-Formyluracil Relying on (2-Benzimidazolyl) Acetonitrile Labeling.

The identification of formylpyrimidines in DNA is crucial for a better understanding of epigenetics. Although many techniques have been explored to detect their content, more accurate methods of formylpyrimidine determination are still required due to the relatively lower sensitivity or lack of selectivity in current methods. Herein, an electrochemical method based on the covalent bonding of the azido derivative of (2-benzimidazolyl) acetonitrile (azi-BIAN) and the aldehyde group of 5-formyluracil (5fU) was proposed for the selective detection of 5fU in the presence of 5-formylcytosine (5fC) and apyrimidinic (AP) sites. Target DNA containing 5fU was first treated with azi-BIAN and then incubated with DBCO-PEG4-Biotin to introduce a biotin group by copper-free click chemistry. Next, the sulfhydryl group was attached to the 5' end of above DNA through T4 polynucleotide kinase-catalyzed reaction. Subsequently, the labeled DNA was assembled onto the AuNPs-modified glassy carbon electrode (AuNPs/GCE) through Au-S bonds, and the streptavidin-horseradish peroxidase conjugate (SA-HRP) was further immobilized onto the surface of the above electrode by specific recognition between biotin and streptavidin. Finally, HRP catalyzed hydroquinone oxidation to benzoquinone to enhance the current signal, which was related to the amount of 5fU in nucleic acids. This method demonstrated a good linear relationship with 5fU concentrations ranging from 0.1 to 10 nM. Moreover, the level of 5fU in γ-irradiated nucleic acids was also successfully detected, indicating that the combination of molecule-depended chemical recognition and electrochemical sensing is a promising method for the selective and sensitive detection of 5fU.

Sequencing 5-Formyluracil in Genomic DNA at Single-Base Resolution.

Albeit with low content, 5-formyluracil has been an important modification in genomic DNA. 5-formyluracil was found to be widely distributed among living bodies. Due to the equilibrium of keto-enol form, 5-formyluracil could be base-paired with guanine, thus inducing mutations in DNA. The highly reactive aldehyde group of 5-formyluracil could also cross-link with proteins nearby, preventing gene replication and expression. In certain cancerous tissues, the content of 5-formyluracil was found to be higher than the normal tissues adjacent to the tumor, and 5-formyluracil might be an important potential epigenetic mark. Nevertheless, the lack of a higher resolution sequencing technique has hampered the studies of 5-formyluracil. We adjusted the base-pairing of 5-formyluracil during the PCR amplification by changing the pH. Hence, we adopted the Alkaline Modulated 5-formyluracil Sequencing (AMfU-Seq), a single-base resolution analysis method, to profile 5-formyluracil at the genome scale. We analyzed the distribution of 5-formyluracil in the human thyroid carcinoma cells using AMfU-Seq. This technique can be used in the future investigations of 5-formyluracil.

Selective Chemical Labeling and Sequencing of 5-Carboxylcytosine in DNA at Single-Base Resolution.

5-Carboxylcytosine (5caC) plays a vital role in the dynamics of DNA demethylation, and sequencing of its sites will help us dig out more biological functions of 5caC. Herein, we present a novel chemical method to efficiently label 5caC distinguished from other bases in DNA. Combined with bisulfite sequencing, 5caC sites can be located at single-base resolution, and the efficiency of 5caC labeling is 92% based on the Sanger sequencing data. Furthermore, dot blot assays have confirmed that 5caC-containing DNA isolated from HeLa cells was successfully labeled using our method. We expect that our strategy can be further applied to selectively tagging other carboxyl-modified bases and mapping their sites in RNA.

Detection and Application of 5-Formylcytosine and 5-Formyluracil in DNA.

Nucleic acids contain a variety of different base modifications, such as decoration at the fifth position of cytosine, which is one of the most important epigenetic modifications. Nucleic acid epigenetics mediate a wide variety of biological processes, including embryonic development and gene regulation, genomic imprinting, differentiation, and X-chromosome inactivation. Furthermore, the modification level can be aberrantly expressed in distinct sets of tissue that can indicate different tumor onsets and canceration. Thus, the analysis of modified nucleobases may contribute to the understanding of epigenetic modification-related biological processes and the correlation of modified nucleobase patterns with disease states for clinical diagnosis and treatment. In addition to 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine are found in organisms at a low content but are nevertheless extremely important chemical modifications, and 5-hydroxyuracil and 5-formyluracil compounds are also present. 5-Formyluracil is found in bacteriophages, prokaryotes, and mammalian cells. The 5-formyluracil content is higher in certain cancer tissues than in the normal tissues adjacent to the tumor. The content of 5-formyluracil in different cell tissues may have cell type specificity. With the continuous use of chemical tools, new detection technologies have greatly advanced the research on natural pyrimidine modifications. These modifications dynamically regulate the gene expression in eukaryotes and prokaryotes and provide mechanistic insights into the occurrence of diseases. Natural pyrimidine modifications act not only as intermediates for DNA demethylation or oxidative damage products but also as modulators of gene expression. Therefore, the development of more effective chemical tools will help us better understand the dynamic changes of natural pyrimidine modifications in vivo. In this Account, we summarize the recent advanced techniques for the detection of 5-formylpyrimidine (5-formylcytosine and 5-formyluracil) and highlight their great potential as biomarkers in biomedical applications. Focusing on the great urgency for the detection of epigenetic modifications, our group developed a series of methods for the qualitative and quantitative analysis of 5-formylpyrimidine in the past few years, aiming at facilitating the accurate detection and mapping of these epigenetic modifications. By the construction of probes, 5-formylpyrimidine can be selectively labeled. Using mass spectrometry, the epigenetic modifications can be quantified. Upon treatment under specific conditions, 5-formylcytosine can be recognized at single-base resolution. With this Account, we anticipate providing chemical and biological researchers with some insight to unlock the complex mechanism involved in 5-formylpyrimidine-related biological processes and stimulate more collaborative research interests from the different fields of materials, biological, medicine, and chemistry to promote the translational research of epigenetics in tumor diagnosis and treatment.

5-Formyluracil as a cornerstone for aluminum detection in vitro and in vivo: a more natural and sustainable strategy.

5-Formyluracil (5fU) based probes were designed and synthesized to detect Al3+ ions in vitro and in biological systems. These probes were synthesised using furan-2-carbohydrazide (fr) and 5fU derivatives. We also selectively labelled 5-formyluracil in DNA with the help of Al3+.

Selective Labeling Aldehydes in DNA.

A naphthalimide hydroxylamine probe has been designed and synthesized to selectively label the whole natural aldehydes present in DNAs including 5-formylcytosine, 5-formyluracil, and abasic sites. The fluorescence characteristics of the generated nucleosides have been examined in detail, and the reaction activities of hydroxylamine, amine groups toward aldehydes in DNA have been discussed with others, which will be a vital reference for designing chemicals for selective labeling of DNAs.

A novel nucleic acid aptamer tag: a rapid fluorescence strategy using a self-constructing G-quadruplex from AGG trinucleotide repeats.

Herein, we have developed a novel fluorescence labeling strategy for nucleic acid aptamers based on self-assembling between AGG tri-nucleotide repeats and a pyrene-modified oligonucleotide. This strategy could be an effective tool for developing targeting-imaging systems and biosensor systems to detect target molecules.

5-Formyluracil as a Multifunctional Building Block in Biosensor Designs.

In organisms 5-formyluracil (5fU), which is known as a vital natural nucleobase, is widely present. Despite the recent development of sensor designs for organic fluorescent molecules for selective targeting applications, biocompatible and easily operated probe designs that are based on natural nucleobase modifications have rarely been reported. Here, we introduce the idea of 5fU as a multifunctional building block to facilitate the design and synthetic development of biosensors. The azide group was derived from the sugar of a nucleoside, which can be further used in the selective binding of cells or organelles through click chemistry with alkynyl-modified targeting groups. The aldehyde group of 5fU can react with different chemicals to generate environmentally sensitive nucleobases that have obvious characteristics, which precious reactants cannot achieve for selective fluorogenic switch-on detection of a specific target. We first synthesized 5fU analogues that had aggregation-induced emission properties, and then we used triphenylphosphonium as a mitochondria-targeting group to selectively image mitochondria in cancer cells and mouse embryonic stem cells. Additionally, the reagents exhibit a high selectivity for reaction with 5fU, which means that the method can also be used for the detection of 5fU. Combining the two characteristics, the idea of 5fU as a multifunctional building block in biosensor designs may potentially be applicable in 5fU site-specific microenvironment detection in future research.

Gene specific-loci quantitative and single-base resolution analysis of 5-formylcytosine by compound-mediated polymerase chain reaction.

5-Formylcytosine (5fC) is known as one of the key players in the process of active DNA demethylation and displays essential epigenetic functions in mammals. In spite of the blooming development of whole genome sequencing methods for this modified cytosine base, the easily operated gene specific-loci detection of 5fC has rarely been reported. Herein, we present a compound-mediated analysis of the content and site of 5fC by the polymerase chain reaction (PCR) assay. The molecule, namely azi-BP, which can selectively label 5fC and form a huge group through a click chemistry reaction, hindering the amplification activity of Taq DNA polymerase, acts as a "roadblock" and enables the quantitative analysis of 5fC by quantitative polymerase chain reaction (qPCR). The existence of 5fC in several fragment-specific genomic DNAs of mouse embryonic stem cells (mESCs) was successfully demonstrated using this method. In addition, the gene fragment containing 5fC can be easily biotinylated and enriched after labeling with azi-BP. Moreover, after azi-BP incorporation, the loss of the exocyclic 4-amino group of 5fC leads to C-to-T conversion and subsequent pairing with adenine (A) in the PCR, which can accurately identify 5fC sites at single-base resolution by site-specific mutation. Azi-BP shows high selectivity to 5fC among all modified pyrimidine bases, revealing that this compound-mediated assay can be applied for content and single-base resolution analysis for gene specific-loci of 5fC.

Naphthalimide derivatives as multifunctional molecules for detecting 5-formylpyrimidine by both PAGE analysis and dot-blot assays.

An azide and hydrazine tethered to a naphthalimide analogue was created to selectively react with 5-formyluracil in one system and fluorogenically label 5-formylcytosine in another system. A biotin group was also introduced by copper-free click chemistry through selective labelling of the aldehyde present in 5-formylpyrimidine.

Fluorogenic labeling and single-base resolution analysis of 5-formylcytosine in DNA.

5-Formylcytosine (5fC), which plays an important role in epigenetic functions, has received widespread attention in many related fields. Here, we demonstrate a new design for both the fluorogenic switch-on detection and single-base resolution analysis of 5fC through selectively reacting a reagent with 5fC to yield an intramolecular cyclization nucleobase. The generated product, bearing a similar benzothiazole-iminocoumarin scaffold, is highly fluorescent and enables us to qualitatively and quantitatively detect 5fC moieties in γ-irradiated calf thymus DNA. Additionally, losing the exocyclic 4-amino group in 5fC causes the incorporation of dATP through base pairing with the generated nucleobase during polymerase extension, which helped us to analyze the 5fC sites in both single- and double-stranded oligonucleotides. Our Sanger and Illumina sequencing results show great potential in single-base resolution analysis of 5fC. It is hopeful that a similar design may be used for more detection targets.

Enrichment and fluorogenic labelling of 5-formyluracil in DNA.

Recently, the detection of natural thymine modified 5-formyluracil has attracted widespread attention. Herein, we introduce a new insight into designing reagents for both the selective biotin enrichment and fluorogenic labelling of 5-formyluracil in DNA. Biotinylated o-phenylenediamine directly tethered to naphthalimide can switch on 5-formyluracil, under physiological conditions, which can then be used in cell imaging after exposure to γ-irradiation. In addition, its labelling property caused the polymerase extension to stop in the 5-formyluracil site, which gave us more information than the fluorescence did itself. The idea of detecting 5-formyluracil might be used in the synthesis of other modified diaminofluoresceins.

Upregulation of TRPC1/6 may be involved in arterial remodeling in rat.

BACKGROUND: Hypertension and its complications are associated with arterial remodeling. Transient receptor potential cationic channels (TRPCs) are important nonselective cationic channels that regulate calcium homeostasis in mammalian cell membranes. We aimed to study the expression of various TRPC isoforms in spontaneously hypertensive rat (SHR) carotid arterial remodeling and explore the relationship between SHR carotid arterial remodeling and TRPC expression. MATERIALS AND METHODS: Thirty male SHRs were randomly divided into three groups and sacrificed at ages 4, 8, and 18 wk, respectively, with matching control male Wistar-Kyoto rats (n = 10). Caudal artery systolic blood pressure (SBP) was measured every 2 wk. Carotid artery remodeling parameters including carotid artery wall thickness (MT), lumen diameter (LD), medial area, collagen area rate, and average nuclear area in media cells were determined after histologic staining. Real-time polymerase chain reaction and immunoblot assays were performed to assess TRPC expression. Carotid artery remodeling and TRPC expression were reevaluated after ginsenoside Rb1 treatment from eighth to eighteenth week. RESULTS: Carotid artery remodeling of SHRs was aggravated gradually with age and SBP, as well as MT, LD, MT/LD, medial area, average nuclear area in media cells, and collagen deposition, most obvious at 18 wk. Interestingly, expression of TRPC1, 3, and 6 increased with age and SBP, with TRPC1/6 showing significant differences between the Wistar-Kyoto and 18 wk groups; TRPC4/5 expression was unchanged and TRPC7 was barely detected. Importantly, after ginsenoside Rb1 treatment, TRPC1/6 expressions were significantly inhibited, SBP decreased, and the carotid artery remodeling in SHRs relieved. CONCLUSIONS: Upregulation of TRPC1 and TRPC6 may be involved in carotid arterial remodeling in SHRs.

TRPC1, CaN and NFATC3 signaling pathway in the pathogenesis and progression of left ventricular hypertrophy in spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHR) was used to study left ventricular hypertrophy (LVH) and its dynamic change after the interventions with Telmisartan and Amlodipine. The results showed that the expression of TRPC1, CaN and NFATC3 increased gradually with the pathogenesis and progression of LVH. Telmisartan reduced blood pressure and LVH, and down-regulated the expression of TRPC1, CaN and NFATC3 in left ventricle of SHR. Amlodipine reduced the blood pressure in SHR but had no impact on the hypertrophy and expression of above factors. Our data suggest that the pathogenesis and progression of LVH in SHR are related to upregulation of TRPC1, CaN and NFATC3 signaling pathway.

1,4-Benzoxazine-3(4H)-ones as potent inhibitors of platelet aggregation: design, synthesis and structure-activity relations.

A series of novel potentially platelet aggregation-inhibiting 1,4-benzoxazine-3(4H)-one derivatives was designed and synthesized through Smiles rearrangement, reduction and acetylation reactions. The antiaggregatory activities of the target molecules on arterial blood samples from rabbits, expressed by IC50 values (µM), were then evaluated in vitro against ADP induced platelet aggregation. The favorable IC50 values of compound 8c (IC50=8.99 µM) and 8d (IC50=8.94 µM) indicated that these two compounds were the most potent molecules among all the synthesized compounds. A detailed molecular docking study to explore the interaction of compounds 8c and 8d with GP IIb/IIIa receptor showed that they these two compounds were docked into the active site of GPIIb/IIIa receptor. These results suggest that the 1,4-benzoxazine-3(4H)-one derivatives are promising lead compounds to develop new platelet aggregation inhibitors.