Abstract: Genomic DNA contains embedded ribonucleotides (rNMPs) that are incorporated during DNA replication and repair, or formed during DNA damage. The modifications have been linked to genome instability and disease, but no method currently exists to profile their locations genome-wide. rNMP incorporation has been extensively studied in recent years; however, locating sites of rNMP incorporation in genomic DNA has not yet been possible. Disclosed herein is a unique method for mapping rNMPs in genomic DNA that exploits the unique ligation mechanism of Arabidopsis thaliana tRNA ligase (AtRNL), normally involved in tRNA maturation. As disclosed herein AtRNL captures 2?,3?-cyclic phosphate or 2?-phosphate termini of DNA derived from alkaline cleavage of a DNA oligonucleotide (oligo) at an embedded rNMP, ligating the 2?-phosphate end to the 5?-phosphate terminus of the same DNA molecule and producing a ssDNA circle containing an embedded rNMP.