Sequencing Deoxyuracil at Single-Base Resolution via a Hydrazine Reagent-Induced U-to-A Transition

Abstract Uracil is a canonical base in RNA, yet its presence in DNA represents the most common non-canonical modification, with broad implications for diverse physiological processes. However, due to the lack of suitable analytical methods, the distribution and biological functions of deoxyuracil (dU) in the genome remain largely unexplored. Herein, a single-base-resolution sequencing method for dU based on chemical labeling is established. By converting dU into abasic (AP) sites, we achieve specific chemical derivatization of the target residues via the aldehyde−hydrazine reaction. Two hydrazine reagents2-hydrazinyl-4,6-dimethoxy-1,3,5-triazine (MeO) and its methylamino analogue (MeNH)are introduced, both of which are base analogues rich in N or O. During subsequent PCR amplification with Vent (exo-) DNA polymerase, a base transition from uracil (sequenced as T) to adenine (A) is induced, serving as the readout for identifying target sites. Our method is simple, cost-effective, and easy to operate. Sample preparation requires only two enzymatic and labeling steps, and target gene analysis can be completed by Sanger sequencing. The practicality of this approach is validated in complex biological samples, providing an easy-to-use tool for investigating the hotspots of dU in the genome.