C-3'-Acyl-3'-xylothymidine-containing oligonucleotides have been designed and synthesized for their use as radical precursors in the study of oxidative DNA damage initiated by a C-3'-radical. These oligomers were efficiently obtained using automated DNA synthesis techniques based on H-phosphonate chemistry. CD spectra and melting curves of the synthesized oligonucleotides were compared to those of their unmodified and xylomodified counterparts. The conformational analysis and hybridization studies indicate that the combination of the photolabile acyl group and the inversion of configuration at the sugar has no profound effect on the overall conformation of C-3'-acyl-2'-deoxy-3'-xylonucleotides as compared to their natural analogues. These systems should provide excellent tools for the elucidation of DNA damage processes.