The sequence specificity and time course of covalent DNA adduct formation of the novel platinum-acridine conjugate [PtCl(en)(ACRAMTU)](NO(3))(2) [PT-ACRAMTU, 2; en = ethane-1,2-diamine, ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea] have been investigated using restriction enzyme cleavage and transcription footprinting assays and compared to the damage produced by the clinical agent cis-diamminedichloroplatinum(II) (cisplatin, 1). The rate of DNA binding of 1 and 2 was also monitored by atomic emission spectrometry. Restriction enzymes were chosen that cleave the phosphodiester linkage at, or adjacent to, the predicted damage sites. While conjugate 2 selectively protected supercoiled plasmid from cleavage by EcoRI and DraI enzymes at their respective restriction sites, G downward arrow AATTC and TTT downward arrow AAA, 1 inhibited DNA hydrolysis by HindIII and PspOMI at A downward arrow AGCTT and G downward arrow GGCCC (arrows mark cleavage sites) more efficiently. Transcription footprinting using T7 RNA polymerase revealed major single-base damage sites for 2 at adenine in 5'-TA and 5'-GA sequences. In addition, the enzyme is efficiently stalled at guanine bases, primarily in the sequence 5'-CGA where the damaged nucleobase is flanked by two high-affinity intercalation sites of ACRAMTU. While 1 targets poly(G) sequences, the binding of 2 appears to be dominated by the groove and sequence recognition of the intercalator. The biochemical assays used confirm previous structural information extracted from mass spectra of DNA fragments modified by 2 isolated from enzymatic digests [Barry, C. G., et al. (2003) J. Am. Chem. Soc. 125, 9629-9637]. Possible DNA-binding mechanisms and biological consequences of the unprecedented modification of alternating TA sequences by 2, which occurred at a faster rate than binding to G, are discussed.