1H NMR has been used to study the interactions of over 70 clinical and experimental antitumor drugs with DNA. Spectra of the low-field (H-bonded imino proton) resonances of DNA were studied as a function of drug per base pair ratio. From the spectral changes observed, it was possible to distinguish three modes of drug binding (intercalation, groove binding, and nonspecific outside binding), to determine the kinetics of drug binding (approximate lifetime of the bound drug), and, in favorable cases, to determine the specificity of the drugs for A X T or G X C base pairs. This method is a useful assay for general drug-binding characteristics. For the intercalating compounds there appears to be a correlation between drug-binding kinetics and useful antitumor activity.

In this study two-dimensional NMR techniques (COSY and NOESY) have been used in conjunction with one-dimensional NMR results to complete the assignment of the proton NMR spectrum of the double-stranded DNA decamer, d(ATATCGATAT)2, and to obtain qualitative information about numerous interproton distances in this molecule and some limited information about conformational dynamics. COSY and NOESY measurements have been combined to systematically assign many of the resonances from the H1' and H2',2" sugar protons to specific nucleotides in the double helix. This method relies on the fact that sugar protons within a specific nucleotide are scalar coupled and that base protons (AH8, GH8, TH6, and CH6) in right-handed helices can interact simultaneously with their own H2',2" sugar protons and those of the adjacent (5'-3') nucleotide attached to its 5' side (i.e., XpA not ApX). A COSY experiment is used to identify sugar resonances within a residue whereas the NOESY experiment allows the neighboring sugar to be connected (linked). The CH5 and CH6 resonances in the spectrum can immediately be identified by the COSY experiment. The methyl protons of thymine residues exhibit strong through-space interbase interactions both with their own TH6 proton and with AH8 proton on the adjacent (5'-3') adenine residue. These interactions are used both to make assignments of the spectra and to establish that the thymine methyl groups are in close proximity to the AH8 protons of adjacent adenine residues [Feigon, J., Wright, J. M., Leupin, W., Denny, W. A., & Kearns, D. R. (1982) J. Am. Chem. Soc. 104, 5540].(ABSTRACT TRUNCATED AT 250 WORDS)

got the present work underway. We should also like to thank Dr. R. D. Small, Jr., for his involvement in some of the preliminary laser flash experiments, Dr. J. K. Thomas for the use of his ruby laser system, and Dr. R. Walsh for communicating to us the results of his calculations and for acting as liaison officer.

The homeodomain encoded by the Antennapedia (Antp) gene of Drosophila was overproduced in a T7 expression vector in Escherichia coli. The corresponding polypeptide of 68 amino acids was purified to homogeneity. The homeodomain was analysed by ultracentrifugation and assayed for DNA binding. The secondary structure of the isolated homeodomain was determined by nuclear magnetic resonance spectroscopy. DNA‐binding studies indicate that the isolated homeodomain binds to DNA in vitro. It selectively binds to the same sites as a longer Antp polypeptide and a full‐length fushi tarazu (ftz) protein. Therefore, the homeodomain represents the DNA‐binding domain of the homeotic proteins.

The in vitro DNA binding properties of a purified 68-amino acid Antennapedia homeodomain (Antp HD) peptide have been analyzed. Equilibrium and kinetic binding studies showed that stable DNA-protein complexes are formed with a Kd of 1.6 x 10(-9) M and 1.8 x 10(-10) M, respectively. Heterodimer analysis led to the conclusion that Antp HD interacts in vitro as a monomer with the DNA target sites used in our study. The results of methylation and ethylation interference studies indicated that the Antp HD closely approaches the target DNA primarily from one side in a region extending across three phosphate backbones. The DNA binding properties of the Antp HD and prokaryotic DNA binding domains that share a helix-turn-helix motif are compared.