<span class="paragraphSection"><div class="boxTitle">Abstract</div>MTR1 is an <span style="font-style: italic;">in vitro</span>-selected ribozyme that catalyses the transfer of an alkyl group from exogenous <span style="font-style: italic;">O</span>⁶-alkylguanine to N1 of a specific adenine in RNA. We show here that the ribozyme can also efficiently alkylate a DNA substrate strand, with almost complete alkylation in 20 min. We have determined crystal structures of the products of methyl and benzyl transfer. The structures are closely similar to that of the all-RNA ribozyme, binding the guanine product in an identical manner, and alkylation occurs at the equivalent location as in the RNA, i.e. N1 of dA63. 2′-O-methylation of C10 and U45, which are hydrogen-bonded to the exogenous guanine, leads to an order-of-magnitude faster rate of alkyl transfer. The results indicate that MTR1 could be a useful tool for the site-specific modification of DNA, including the creation of fluorescent labels or targets for chemical crosslinking.</span>