Chemical Synthesis of Native ADP-Ribosylated Oligonucleotides Enables Analysis of DNA ADP-Ribosylation Hydrolase Specificity.

ADP-ribosylation (ADPr) is a modification by which an ADP-ribose moiety is conjugated to different molecules by ADP-ribosyltransferases (ARTs) to control various cellular processes in organisms from all kingdoms of life. While traditionally considered primarily as a post-translational modification of proteins, recent evidence has demonstrated that ADPr of nucleic acids (DNA and RNA) has emerged as a widespread type of ADPr. However, the precise biological roles and underlying mechanisms remain elusive, largely due to the lack of ADP-ribosylated nucleic acid molecular tools. Inspired by the native thymidine modification sites of ADP-ribosylated DNA, we developed a synthetic method for the chemo- and stereoselective assembly of both anomers of ADP-ribosylated thymidine (T-ADPr). We further tailored our method to successfully synthesize ADP-ribosylated oligonucleotides with desired sequences via an automatic solid-phase synthesis. We used the synthetic ADPr substrates, including the ADP-ribosylated DNA sequence motif TCTC that is targeted by bacterial ARTs to control DNA replication or antiphage response, and showed the utility of newly developed tools to characterize the specificity and mechanism of action of different hydrolases in ADPr reversal.