Tris-benzimidazole derivatives: design, synthesis and DNA sequence recognition.

Two tris-benzimidazole derivatives have been designed and synthesized based on the known structures of the bis-benzimidazole stain Hoechst 33258 complexed to short oligonucleotide duplexes derived from single crystal X-ray studies and from NMR. In both derivatives the phenol group has been replaced by a methoxy-phenyl substituent. Whereas one tris-benzimidazole carries a N-methyl-piperazine at the 6-position, the other one has this group replaced by a 2-amino-pyrrolidine ring. This latter substituent results in stronger DNA binding. The optimized synthesis of the drugs is described. The two tris-benzimidazoles exhibit high AT-base pair (bp) selectivity evident in footprinting experiments which show that five to six base pairs are protected by the tris-benzimidazoles as compared to four to five protected by the bis-benzimidazoles. The tris-benzimidazoles bind well to sequences like 5'-TAAAC, 5'-TTTAC and 5'-TTTAT, but it is also evident that they can bind weakly to sequences such as 5'-TATGTT-3' where the continuity of an AT stretch is interrupted by a single G*C base pair.

Antimalarial activity of the bisquinoline trans-N1,N2-bis (7-chloroquinolin-4-yl)cyclohexane-1,2-diamine: comparison of two stereoisomers and detailed evaluation of the S,S enantiomer, Ro 47-7737.

The S,S enantiomer of the bisquinoline trans-N1,N2-bis(7-chloroquinolin-4-yl)cyclohexane-1,2-diamine, Ro 47-7737, is significantly more potent against chloroquine-resistant Plasmodium falciparum than the R,R enantiomer and the previously described racemate. Both the enantiomers and the racemate are more potent inhibitors of heme polymerization than chloroquine, and their activities are probably mediated by inhibition of this parasite-specific process. The S,S enantiomer, Ro 47-7737, was studied in more detail and proved to be a potent antimalarial in the treatment of P. vivax ex vivo and P. berghei in vivo. Its suppression of P. berghei growth in a mouse model (50% effective dose, 2.3 mg/kg of body weight) was equal to that of chloroquine and mefloquine, and Ro 47-7737 was found to be more potent than these two drugs in the Rane test, in which the curative effect of a single dose is monitored. The dose at which 50% of animals were permanently cured (34 mg/kg) was markedly superior to those of chloroquine (285 mg/kg) and mefloquine (> 250 mg/kg). When administered orally at 50 mg/kg, Ro 47-7737 also showed a faster clearance of parasites than either chloroquine or mefloquine, and unlike the other two compounds, Ro 47-7737 showed no recrudescence. In a study to compare prophylactic efficacies of oral doses of 50 mg/kg, Ro 47-7737 provided protection for 14 days compared to 3 days for mefloquine and 1 day for chloroquine. The good curative and prophylactic properties of the compound can be explained in part by its long terminal half-life. The ability to generate parasite resistance to Ro 47-7737 was also assessed. With a rodent model, resistance could be generated over eight passages. This rate of resistance generation is comparable to that of mefloquine, which has proved to be an effective antimalarial for many years. Toxicity liabilities, however, ruled out this compound as a candidate for drug development.

Designer DNA-binding drugs: the crystal structure of a meta-hydroxy analogue of Hoechst 33258 bound to d(CGCGAATTCGCG)2.

An analogue of the DNA binding compound Hoechst 33258, which has the para hydroxyl group altered to be at the meta position, together with the replacement of one benzimidazole group by pyridylimidazole, has been cocrystallized with the dodecanucleotide sequence d(CGCGAATTCGCG)2. The X-ray structure has been determined at 2.2 A resolution and refined to an R factor of 20.1%. The ligand binds in the minor groove at the sequence 5'-AATTC with the bulky piperazine group extending over the CxG base pair. This binding is stabilised by hydrogen bonding and numerous close van der Waals contacts to the surface of the groove walls. The meta-hydroxyl group was found in two distinct orientations, neither of which participates in direct hydrogen bonds to the exocyclic amino group of a guanine base. The conformation of the drug differs from that found previously in other X-ray structures of Hoechst 33258-DNA complexes. There is significant variation between the minor groove widths in the complexes of Hoechst 33258 and the meta-hydroxyl derivative as a result of these conformational differences. Reasons are discussed for the inability of this derivative to actively recognise guanine.

Isohelicity and phasing in drug--DNA sequence recognition: crystal structure of a tris(benzimidazole)--oligonucleotide complex.

The crystal structure is reported of a tris(benzimidazole) analogue of the minor-groove drug Hoechst 33258 bound to the sequence d(CGCAAATTTGCG)2. The structure has been refined to an R factor of 17.4% at a resolution of 2.2 A. The ligand covers approximately 7 1/2 base pairs, including the 5'-AAATTT central sequence. This has an exceptionally narrow minor-groove width, together with high propeller twists for individual base pairs. The ligand has a highly twisted structure, with an overall twist of 50 degrees between aromatic rings. All three benzimidazole subunits are in register with the DNA, and there is a symmetric group of six hydrogen bonds between ligand and A.T base-pair edges. By contrast, the ligand does not show an optimal isohelical fit to the DNA. The correct phasing of drug and DNA base pairs is ensured by a number of changes to the DNA such that the central 5'-AAATTT region is slightly unwound relative to the structures of other noncovalent minor-groove drug complexes.

Minor groove hydration of DNA in aqueous solution: sequence-dependent next neighbor effect of the hydration lifetimes in d(TTAA)2 segments measured by NMR spectroscopy.

The hydration in the minor groove of double stranded DNA fragments containing the sequences 5'-dTTAAT, 5'-dTTAAC, 5'-dTTAAA and 5'-dTTAAG was investigated by studying the decanucleotide duplex d(GCATTAATGC)2 and the singly cross-linked decameric duplexes 5'-d(GCATTAACGC)-3'-linker-5'-d(GCGTTAATGC)-3' and 5'-d(GCCTTAAAGC)-3'-linker-5'-d(GCTTTAAGGC)-3' by NMR spectroscopy. The linker employed consisted of six ethyleneglycol units. The hydration water was detected by NOEs between water and DNA protons in NOESY and ROESY spectra. NOE-NOESY and ROE-NOESY experiments were used to filter out intense exchange cross-peaks and to observe water-DNA NOEs with sugar 1' protons. Positive NOESY cross-peaks corresponding to residence times longer than approximately 0.5 ns were observed for 2H resonances of the central adenine residues in the duplex containing the sequences 5'-dTTAAT and 5'-dTTAAC, but not in the duplex containing the sequences 5'-dTTAAA and 5'-dTTAAG. In all nucleotide sequences studied here, the hydration water in the minor groove is significantly more mobile at both ends of the AT-rich inner segments, as indicated by very weak or negative water-A 2H NOESY cross-peaks. No positive NOESY cross-peaks were detected with the G 1'H and C 1'H resonances, indicating that the minor groove hydration water near GC base pairs is kinetically less restrained than for AT-rich DNA segments. Kinetically stabilized minor groove hydration water was manifested by positive NOESY cross-peaks with both A 2H and 1'H signals of the 5'-dTTAA segment in d(GCATTAATGC)2. More rigid hydration water was detected near T4 in d(GCATTAATGC)2 as compared with 5'-d(GCATTAACGC)-3'-linker-5'-d(GCGTTAATGC)-3', although the sequences differ only in a single base pair. This illustrates the high sensitivity of water-DNA NOEs towards small conformational differences.

The width of the minor groove affects the binding of the bisquaternary heterocycle SN-6999 to duplex DNA.

A complex between d(GGGAAAAACGG).d(CCGTTTTTCCC) and the minor groove binding drug SN-6999 has been studied by 1H nuclear magnetic resonance spectroscopy. The drug is found to bind in the d(A)5 tract, but with interactions extending one residue in the 3'-direction along each strand. Doubling of resonances in the complex indicates slow to intermediate exchange between two binding modes. An orientational preference (7:3) is found, the first such example in an SN-6999 complex. Furthermore, the upper limit of the lifetime for the major species is longer than was found for SN-6999 with other DNA duplexes. The preferred orientation of SN-6999 has the pyridinium ring near the 5'-end of the (+) strand; the minor binding mode has the reverse orientation. The orientational preference and slower exchange rate relative to other SN-6999 complexes is attributed to increased stabilization from van der Waals interactions due to better shape complementarity between the DNA duplex and ligand. The comparison of these results with studies of SN-6999 complexed to other DNA duplexes reveals the sensitivity of the binding properties to the delicate interplay between the molecular structure of the ligand and the specific characteristics of the DNA minor groove.

NMR studies of DNA duplexes singly cross-linked by different synthetic linkers.

Molecular modelling studies resulted in the design of a variety of non-nucleotidic covalent linkers to bridge the 3'-end of the (+)-strand and the 5'-end of the (-)-strand in DNA duplexes. Three of these linkers were synthesized and used to prepare singly cross-linked duplexes d(GTGGAATTC)-linker-d(GAATTCCAC). Linker I is an assembly of a propylene-, a phosphate- and a second propylene-group and is thought to mimic the backbone of two nucleotides. Linkers II and III consist of five and six ethyleneglycol units, respectively. The melting temperatures of the cross-linked duplexes are 65 degrees C for I and 73 degrees C for II and III, as compared with 36 degrees C for the corresponding non-linked nonadeoxynucleotide duplex. The three cross-linked duplexes were structurally characterized by nuclear magnetic resonance spectroscopy. The 1H and 31P resonance assignments in the DNA stem were obtained using standard methods. For the resonance assignment of the linker protons, two-dimensional 1H-31P heteronuclear COSY and two-quantum-experiments were used. Distance geometry calculations with NOE-derived distance constraints were performed and the resulting structures were energy-minimized. In duplex I, the nucleotides flanking the propylene-phosphate-propylene-linker do not form a Watson-Crick base pair, whereas in duplexes II and III the entire DNA stem is in a B-type double helix conformation.

Hydration of DNA in aqueous solution: NMR evidence for a kinetic destabilization of the minor groove hydration of d-(TTAA)2 versus d-(AATT)2 segments.

The residence times of the hydration water molecules near the base protons of d-(GTGGAATTCCAC)2 and d-(GTGGTTAACCAC)2 were investigated by nuclear magnetic resonance (NMR) spectroscopy. Nuclear Overhauser effects (NOE) were observed between base protons of the DNA and hydration water in NOESY and ROESY experiments. Large positive NOESY cross peaks observed between the resonances of the water and the adenine 2H protons of the central d-(AATT)2 segment in the duplex d-(GTGGAATTCCAC)2 indicate the presence of a 'spine of hydration' with water molecules exhibiting residence times on the DNA longer than 1 nanosecond. In contrast, no positive intermolecular NOESY cross peaks were detected in the d-(TTAA)2 segment of the duplex d-(GTGGTTAACCAC)2, indicating that no water molecules bound with similarly long residence times occur in the minor groove of this fragment. These results can be correlated with the larger width of the minor groove in d-(TTAA)2 segments as compared to that in d-(AATT)2 segments, as observed previously in single crystal structures of related oligonucleotide duplexes in B type conformation. The present experiments confirm earlier experimental results from single crystal studies and theoretical predictions that a 5'-dTA-3' step in the nucleotide sequence interrupts the spine of hydration in the minor groove.

Homeodomain proteins in development and therapy.

Homeobox genes encode transcriptional regulators found in all organisms ranging from yeast to humans. In Drosophila, a specific class of homeobox genes, the homeotic genes, specifies the identity of certain spatial units of development. Their genomic organization, in Drosophila, as well as in vertebrates, is uniquely connected with their expression which follows a 5'-posterior-3'-anterior rule along the longitudinal body axis. The 180-bp homeobox is part of the coding sequence of these genes, and the sequence of 60 amino acids it encodes is referred to as the homeodomain. Structural analyses have shown that homeodomains consist of a helix-turn-helix motif that binds the DNA by inserting the recognition helix into the major groove of the DNA and its amino-terminal arm into the adjacent minor groove. Developmental as well as gene regulatory functions of homeobox genes are discussed, with special emphasis on one group, the Antennapedia (Antp) class homeobox genes and a representative 60-amino acid Antennapedia peptide (pAntp). In cultured neuronal cells, pAntp translocates through the membrane specifically and efficiently and accumulates in the nucleus. The internalization process is followed by a strong induction of neuronal morphological differentiation, which raises the possibility that motoneuron growth is controlled by homeodomain proteins. It has been demonstrated that chimeric peptide molecules encompassing pAntp are also captured by cultured neurons and conveyed to their nuclei. This may be of enormous interest for the internalization of drugs.

Determination of the NMR solution structure of the Hoechst 33258-d(GTGGAATTCCAC)2 complex and comparison with the X-ray crystal structure.

BACKGROUND: The chromosomal stain, Hoechst 33258, binds to the minor groove of the DNA double helix and specifically recognizes a run of four A-T base pairs. Extensive biochemical and biophysical studies have been aimed at understanding the binding of the dye to DNA at the atomic level. Among these studies there have been several crystal structure determinations and some preliminary structural studies by NMR. RESULTS: On the basis of our own previously reported NMR data, we have now determined the three-dimensional solution structure of the 1:1 complex between Hoechst 33258 and the self-complementary DNA duplex d(GTGGAATTCCAC)2. Two coexisting families of con formers, which exhibit differences in their intermolecular hydrogen bonding pattern, were found and the two terminal rings of the dye displayed greater internal mobility than the rest of the molecule. CONCLUSIONS: The observed multiple ligand-binding modes in the complex between Hoechst 33258 and DNA and differential internal mobility along the bound ligand provide a novel, dynamic picture of the specific inter actions between ligands that bind in the minor groove and DNA. The dynamic state revealed by these studies may account for some of the significant differences previously observed between different crystal structures of Hoechst 33258 complexed with a different DNA duplex, d(CGCGAATTCGCG)2.

Two-dimensional NMR studies of d(GGTTAATGCGGT).d(ACCGCATTAACC) complexed with the minor groove binding drug SN-6999.

The dodecadeoxynucleotide duplex d(GGTTAATGCGGT).d(ACCGCATTAACC) and its 1:1 complex with the minor groove binding drug SN-6999 have been prepared and studied by two-dimensional 1H nuclear magnetic resonance spectroscopy. Complete sequence-specific assignments have been obtained for the free duplex by standard methods. The line widths of the resonances in the complex are greater than those observed for the free duplex, which complicates the assignment process. Extensive use of two-quantum spectroscopy was required to determine the scalar correlations for identifying all of the base proton and most of the 1'H-2'H-2''H spin subsystems for the complex. This permitted unambiguous sequence-specific resonance assignments for the complex, which provides the necessary background for a detailed comparison of the structure of the duplex, with and without bound drug. A series of intermolecular NOEs between drug and DNA were identified, providing sufficient structural constraints to position the drug in the minor groove of the duplex. However, the combination of NOEs observed can only be rationalized by a model wherein the drug binds in the minor groove of the DNA in both orientations relative to the long helix axis and exchanges rapidly between the two orientations. The drug binds primarily in the segment of five consecutive dA-dT base pairs d(T3T4A5A6T7).d(A18T19T20A21A22), but surprisingly strong interactions are found to extend one residue in the 3' direction along each strand to G8 and C23. The observation of intermolecular contacts to residues neighboring the AT-rich region demonstrates that the stabilization of the bis(quaternary ammonium) heterocycle family of AT-specific, minor groove binding drugs is not based exclusively on interactions with dA-dT base pairs.

Conformational studies of the duplex d-(CCAAAAATTTCC).d-(GGAAATTTTTGG) containing a (dA)5 tract using two-dimensional 1H-n.m.r. spectroscopy.

A variety of two-dimensional nuclear magnetic resonance (n.m.r.) techniques were utilized for structural studies of the dodecadeoxy-nucleotide duplex d-(CCAAAAATTTCC).d-(GGAAATTTTTGG). Overall, the duplex was found to adopt a conformation of the B-DNA type. Direct measurement of 1H-1H coupling constants of the deoxyribose rings and comparison of nuclear Overhauser effects (NOEs) involving characteristic intra- and inter-nucleotide proton-proton connectivities indicate that the sugar rings are all in predominantly C2'-endo/S-type conformation. The appearance of NOEs between adenine 2H and sugar l'H resonances indicates a narrowing of the minor groove at the 3' end of the (dA)n tracts. Several lines of evidence indicate that a conformational change in the DNA duplex is introduced by the (dA)5 tract, in support of the junction model for curved DNA. The results are compared with previous structural studies of the evidence for curvature in duplex DNA containing (dA)n tracts.

Distamycin-induced inhibition of homeodomain-DNA complexes.

The mobility shift assay was used to study the competition of the minor groove binder distamycin A with either an Antennapedia homeodomain (Antp HD) peptide or derivatives of a fushi tarazu homeodomain (ftz HD) peptide for their AT-rich DNA binding site. The results show that distamycin and the homeodomain peptides compete under the conditions: (i) preincubation of DNA with distamycin and subsequent addition of HD peptide; (ii) simultaneous incubation of DNA with distamycin and HD peptide; and (iii) preincubation of DNA with HD peptide and subsequent addition of distamycin. There is also competition when using a peptide which lacks the N-terminal arm of ftz HD that is involved in contacts in the minor groove. It is proposed that the protein's binding affinity is diminished by distamycin-induced conformational changes of the DNA. The feasibility of the propagation of conformational changes upon binding in the minor groove is also shown for the inhibition of restriction endonucleases differing in the AT content of their recognition site and of their flanking DNA sequences. Thus, it is demonstrated that minor groove binders can compete with the binding of proteins in the major groove, providing an experimental indication for the influence of biological activities exerted by DNA ligands binding in the minor groove.

Dynamics and binding mode of Hoechst 33258 to d(GTGGAATTCCAC)2 in the 1:1 solution complex as determined by two-dimensional 1H NMR.

We have investigated the interaction of the bisbenzimidazole derivative Hoechst 33258 with the self-complementary dodecadeoxynucleotide duplex d(GTGGAATTCCAC)2 using one-dimensional (1D) and two-dimensional (2D) proton nuclear magnetic resonance (1H NMR) spectroscopy. To monitor the extent of complex formation, we used the imino proton region of the 1D 1H NMR spectra acquired in H2O solution. These spectra show that the DNA duplex loses its inherent C2v symmetry upon addition of the drug, indicating that the two molecules form a kinetically stable complex on the NMR time scale (the lifetime of the complex has been measured to be around 450 ms). We obtained sequence-specific assignments for all protons of the ligand and most protons of each separate strand of the oligonucleotide duplex using a variety of homonuclear 2D 1H NMR experiments. The aromatic protons of the DNA strands, which are symmetrically related in the free duplex, exhibit exchange cross peaks in the complex. This indicates that the drug binds in two equivalent sites on the 12-mer, with an exchange rate constant of 2.2 +/- 0.2 s-1. Twenty-five intermolecular NOEs were identified, all involving adenine 2 and sugar 1' protons of the DNA and protons in all four residues of the ligand, indicating that Hoechst 33258 is located in the minor groove at the AATT site. Only protons along the same edge of the two benzimidazole moieties of the drug show NOEs to DNA protons at the bottom of the minor groove. Using molecular mechanics, we have generated a unique model of the complex using distance constraints derived from the intermolecular NOEs. We present, however, evidence that the piperazine group may adopt at least two locally different conformations when the drug is bound to this dodecanucleotide.

Comparative NMR analysis of the decadeoxynucleotide d-(GCATTAATGC)2 and an analogue containing 2-aminoadenine.

The dodecadeoxynucleotide duplex d-(GCATTAATGC)2 has been prepared with all adenine bases replaced by 2-NH2-adenine. This modified duplex has been characterized by nuclear magnetic resonance (NMR) spectroscopy. Complete sequence-specific 1H resonance assignments have been obtained by using a variety of 2D NMR methods. Multiple quantum-filtered and multiple quantum experiments have been used to completely assign all sugar ring protons, including 5'H and 5'H resonances. The assignments form the basis for a detailed comparative analysis of the 1H NMR parameters of the modified and parent duplex. The structural features of both decamer duplexes in solution are characteristic of the B-DNA family. The spin-spin coupling constants in the sugar rings and the relative spatial proximities of protons in the bases and sugars (as determined from the comparison of corresponding nuclear Overhauser effects) are virtually identical in the parent and modified duplexes. Thus, substitution by this adenine analogue in oligonucleotides appears not to disturb the global or local conformation of the DNA duplex.

Two-dimensional 1H NMR studies of synthetic immobile Holliday junctions.

Two 32 base pair, four-arm immobile Holliday junctions have been prepared and studied by two-dimensional 1H nuclear magnetic resonance (NMR) spectroscopy. Two-quantum spectroscopy provides scalar (through bond) correlations for the 1'H, 2'H, and 2''H resonances of the deoxyribose sugar rings and the nonlabile cytosine and thymine base protons. Assignments in the deoxyribose sugars are extended to the 3'H resonances principally from relayed connectivities in total correlation spectra. Severe overlap of resonances in the standard regions of two-dimensional nuclear Overhauser enhancement (NOE) spectra necessitated the use of a unique approach for obtaining sequence-specific assignments of duplex DNA, wherein all possible NOE connectivities in the spectra are analyzed. These studies of 64-residue structures represent a substantial step forward with respect to the size of oligonucleotide for which virtually complete assignments are obtained. The assignments form the critical background for the detailed analysis of Holliday junction structure and dynamics that is required to address key issues in understanding the role of Holliday junctions in genetic recombination and repair.

DNA binding properties of the purified Antennapedia homeodomain.

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.

Application of 13C(omega 1)-half-filtered [1H,1H]-NOESY for studies of a complex formed between DNA and a 13C-labeled minor-groove-binding drug.

The complex formed between the anticancer drug 4-[p-[p-(4-quinolylamino)benzamido]anilino]pyridine (SN 6999) and the decadeoxyribonucleoside nonaphosphate d-(GCATTAATGC)2 was investigated using two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) with a 13C(omega 1)-half-filter. The two quaternary methyl groups in SN 6999 had been labeled with 13C for these experiments. The simplified subspectra of [1H,1H]-NOESY obtained with this procedure greatly facilitate the identification and assignment of intermolecular NOEs. Quite generally, the combined use of isotope labeling and heteronuclear filters in [1H,1H]-NOESY provides an improved experimental basis for structural studies of drug/DNA complexes.

Conformational studies of d-(AAAAATTTTT)2 using constraints from nuclear overhauser effects and from quantitative analysis of the cross-peak fine structures in two-dimensional 1H nuclear magnetic resonance spectra.

The conformation at the dA-dT junction in d-(AAAAATTTTT)2 was investigated by using a variety of phase-sensitive two-dimensional nuclear magnetic resonance experiments at 500 MHz for detailed studies of the deoxyribose ring puckers. Conformational constraints were collected from two-dimensional nuclear Overhauser enhancement spectra recorded with short mixing times and from quantitative simulations of the cross-peaks in two-dimensional correlated spectra. Overall, the decamer duplex adopts a conformation of the B-DNA type, and for dA4 and dA5 the pseudorotation phase angle P is in the standard range 150-180 degrees. The deoxyribose puckers for the other nucleotides deviate significantly from the standard B-DNA structure. Spectrum simulations assuming either static deviations from standard B-DNA or a simple two-state dynamic equilibrium between the C2'-endo and C3'-endo forms of the deoxyribose were used to analyze the experimental data. It was thus found that the ring pucker for dT6 deviates from the regular C2'-endo form of B-DNA by a static distortion, with the pseudorotation phase angle P in the range 100-130 degrees, and a similar value of P is indicated for dT7. For the peripheral base pairs dynamic distortions of the C2'-endo form of the deoxyribose were found. In agreement with recent papers on related duplexes containing (dA)n tracts, we observed prominent nuclear Overhauser effects between adenine-2H and deoxyribose-1'H, which could be largely due to pronounced propeller twisting as observed in the crystal structures of (dA)n-containing compounds.

Isolation and sequence-specific DNA binding of the Antennapedia homeodomain.

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.