Base-resolution analysis of deoxyuridine at genome scale based on artificial incorporation modified nucleobase

Deoxyuridine (dU) in DNA can result from deamination of cytosine and dUMP misincorporation. The easy single-nucleotide resolution assay for dU are crucial to understanding its role in genome. Herein, we present a new concept of “base substitution” for accurate single-nucleotide resolution profiling of dU. The method termed AI-Seq (Artificial Incorporation of a modified nucleobase for sequencing) was developed using artificial cytosine (N3-C) to replace dU. After the artificial base construction, dU can read as cytosine during polymerase chain reaction assay. AI-seq was validated on synthetic DNA and then applied to the genome of HEK293T cell line. Collectively, the “base substitution” provides a novel approach for generating comprehensive information about the distribution of dU and can be potentially adapted to detect other epigenetic modifications. N3-C is an artificially synthesized modified cytosine, which is mainly composed of three parts. Hydroxylamine group can label the UNG-treated dU. Cytosine can be incorporated to replace the dU, leading to U-to-C signals in the sequencing reads. Such base substitution enabled U-to-C transition was used from the sequencing reads to obtain dU at single-base resolution. Moreover, the azide group of N3-C can react with DBCO-biotin reagents through a copper-free click reaction for enriching the dU-containing fragment.