Nucleotide analogue interference mapping of DNA (dNAIM) is here introduced as a new nonenzymatic interference-based approach that enables high-throughput identification of essential nucleobase functional groups in DNA aptamers and in the catalytic core of deoxyribozymes. Nucleobase-modified ribonucleotides are statistically incorporated into DNA by solid-phase synthesis, employing the 2'-OH group as a chemical tag for analysis of interference effects. This method is exemplified on an AMP-binding DNA aptamer and was further used to identify indispensable nudeobase functional groups for DNA-catalyzed RNA-ligation by the Mg2+-dependent deoxyribozymes 7S11 and 9DB1. dNAIM should prove broadly useful for facile structural probing of functional DNA for which active and inactive variants can be separated based on catalytic or ligand-binding activities.