The natural chemical modifications of messenger RNA (mRNA) in living organisms have shown essential roles in both physiology and pathology. The mapping of mRNA modifications is critical for interpreting their biological functions. In another dimension, the synthesized nucleoside analogs can enable chemical labeling of cellular mRNA through a metabolic pathway, which facilitates the study of RNA dynamics in a pulse-chase manner. In this regard, the sequencing tools for mapping both natural modifications and nucleoside tags on mRNA at single base resolution are highly necessary. In this work, we review the progress of chemical sequencing technology for determining both a variety of naturally occurring base modifications mainly on mRNA and a few on transfer RNA and metabolically incorporated artificial base analogs on mRNA, and further discuss the problems and prospects in the field.

The development of a simple and cost-effective method to map the distribution of RNA polymerase II (RNPII) genome-wide at a high resolution is highly beneficial to study cellular transcriptional activity. Here we report a mutation-based and enrichment-free global chromatin run-on sequencing (mGROseq) technique to locate active RNPII sites genome-wide at nearbase resolution. An adenosine triphosphate (ATP) analog named N 6 -allyladenosine triphosphate (a 6 ATP) was designed and could be incorporated into nascent RNAs at RNPII-located positions during a chromatin run-on reaction. By treatment of the run-on RNAs with a mild iodination reaction and subjection of the products to reverse transcription into complementary DNA (cDNA), base mismatch occurs at the original a 6 A incorporation sites, thus making the RNPII locations detected in the high-throughput cDNA sequencing. The mGRO-seq yields both the map of RNPII sites and the chromatin RNA abundance and holds great promise for the study of single-cell transcriptional activity.

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DNA tagging with base analogues has found numerous applications. To precisely record the DNA labelling information, it would be highly beneficial to develop chemical sequencing tags that can be encoded into DNA as regular bases and decoded as mutant bases following a mild, efficient and bioorthogonal chemical treatment. Here we reported such a DNA tag, N 4 -allyldeoxycytidine (a 4 dC), for labeling and identifying DNA by in vitro assays. The iodination of a 4 dC led to fast and complete formation of 3, N 4 -cyclized deoxycytidine, which induced base misincorporation during DNA replication and thus could be located at single base resolution. We explored the applications of a 4 dC in pinpointing DNA labelling sites at single base resolution, mapping epigenetic marker N 4 -methyldeoxycytidine, and imaging nucleic acids in situ. In addition, mammalian cellular DNA could be metabolically labelled with a 4 dC. Our study sheds light on the design of next generation DNA tags with chemical sequencing power.