DNA lesions such as 1,N6-ethenoadenine (εA) and 3,N4-ethenocytosine (εC) are ubiquitously present in genomes of different organisms and show increasing levels upon exposure to mutagenic substances or under conditions of chronic inflammations and infections. To facilitate investigations of the mutagenic properties and repair mechanisms of etheno-base adducts, access to oligonucleotides bearing these lesions at defined positions is of great advantage. In this study, we report a new synthetic strategy to sequence-specifically generate etheno-adducts in a single-stranded unmodified DNA sequence making use of a DNA-templated approach that positions the alkylating agent close in space to the respective target base. In contrast to solid-phase synthesis of modified oligonucleotides such DNA-templated methods can be applied to single-stranded nucleic acids of unrestricted lengths. The modular nature of the system allows straightforward adaptation to different sequences.