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.

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 5carboxycytosine 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...

Simultaneous fluorogenic switch-on detection and single-base resolution analysis of 5fC through yielding an intramolecular cyclization nucleobase has been presented.

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.

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Biotinylated o-phenylenediamine directly tethered to naphthalimide can both enrich and fluorogenically label 5-formyluracil in DNA under physiological conditions.

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.

5-Formyluracil (5fU) is a vital DNA marker that is widely distributed in the cells of organisms. A unique feature of 5fU is the possession of a potentially reactive aldehyde group in its structure that could realize addition and condensation reactions. However, the biological functional details of 5fU remain mostly elusive, especially, regarding its relatedness with proteins. In this current study, we show that 5fU bases have a strong affinity toward nucleosome core particles, and that could yield regulable DNA-protein conjugates (DPCs) via chemical interactions between amino and aldehyde groups, and reductants could be applied to stabilize or dissociate the interactions. Besides, we developed a photocaged method to exploit the relationship between 5fU and nucleosomes. Finally, by applying a combination of the existence of 5fU-histone interactions in vivo by ChIP analysis of histone H4 with liquid chromatography-mass spectrometry (LC-MS), we probed further, the DPCs' influence on nucleosome and enzyme. Collectively, our results showed that the 5fU-protein interactions increase the occupancy and stability of nucleosomes, affect enzyme recognition, and block DNA replication. These might imply that, in vivo, the DPCs between 5fU and nucleosome core particles might play a key role in 5fU-associated pathways such as DNA repair, transcriptional regulation, or development.