Changes in C NMR chemical shifts of DNA as a tool for monitoring drug interactions

The antibiotic drug, netropsin, was complexed with the DNA oligonucleotide duplex [d(GGTATACC)]₂ to explore the effects of ligand binding on the ¹³C NMR chemical shifts of the DNA base and sugar carbons. The binding mode of netrospin to TA-rich tracts of DNA has been well documented and served as an attractive model system. For the base carbons, four large changes in resonance chemical shifts were observed upon complex formation: −0.64 ppm for carbon 4 of either Ado4 or Ado6, 1.36 ppm for carbon 2 of Thd5, 1.33 ppm for carbon 5 of Thd5 and 0.94 for carbon 6 of Thd5. AdoC4 is covalently bonded to a heteroatom that is hydrogen bonded to netropsin; this relatively large deshielding is consistent with the known hydrogen bond formed at AdoN3. The three large shielding increases are consistent with hydrogen bonds to water in the minor groove being disrupted upon netropsin binding. For the DNA sugar resonances, large changes in chemical shifts were observed upon netropsin complexation. The 2′, 3′ and 5′ ¹³C resonances of Thd3 and Thd5 were shielded whereas those of Ado4 and Ado6 were deshielded; the ¹³C resonances of 1′ and 4′ could not be assigned. These changes are consistent with alteration of the dynamic pseudorotational states occupied by the DNA sugars. A significant alteration in the pseudorotational states of Ado4 or Ado6 must occur as suggested by the large change in chemical shift of −1.65 ppm of the C3′ carbon. In conclusion, ¹³C NMR may serve as a practical tool for analyzing structural changes in DNA-ligand complexes.     

Molecular structure of the netropsin-d(CGCGATATCGCG) complex: DNA conformation in an alternating AT segment

The molecular structure of the complex between a minor groove binding drug (netropsin) and the DNA dodecamer d(CGCGATATCGCG) has been solved and refined by single-crystal X-ray diffraction analysis to a final R factor of 20.0% to 2.4-A resolution. The crystal is similar to that of the other related dodecamers with unit cell dimensions of a = 25.48 A, b = 41.26 A, and c = 66.88 A in the space group P2(1)2(1)2(1). In the complex, netropsin binds to the central ATAT tetranucleotide segment in the narrow minor groove of the dodecamer B-DNA double helix as expected. However, in the structural refinement the drug is found to fit the electron density in two orientations equally well, suggesting the disordered model. This agrees with the results from solution studies (chemical footprinting and NMR) of the interactions between minor groove binding drugs (e.g., netropsin and distamycin A) and DNA. The stabilizing forces between drug and DNA are provided by a combination of ionic, van der Waals, and hydrogen-bonding interactions. No bifurcated hydrogen bond is found between netropsin and DNA in this complex due to the unique dispositions of the hydrogen-bond acceptors (N3 of adenine and O2 of thymine) on the floor of the DNA minor groove. Two of the four AT base pairs in the ATAT stretch have low propeller twist angles, even though the DNA has a narrow minor groove. Alternating helical twist angles are observed in the ATAT stretch with lower twist in the ApT steps than in the TpA step.     

Molecular recognition in noncovalent antitumor agent-DNA complexes: NMR studies of the base and sequence dependent recognition of the DNA minor groove by netropsin

We have investigated intermolecular interactions and conformational features of the netropsin complexes with d(G1-G2-A3-A4-T5-T6-C7-C8) duplex (AATT 8-mer) and the d(G1-G2-T3-A4-T5-A6-C7-C8) duplex (TATA 8-mer) by one and two-dimensional NMR studies in solution. We have assigned the amide, pyrrole and methylene protons of netropsin and the base and sugar H1' protons of the nucleic acid from an analysis of the nuclear Overhauser effect (NOESY) and correlated (COSY) spectra of the complex at 25 degrees C. The directionality of the observed distance-dependent NOEs demonstrates that the 8-mer helices remain right-handed and that the arrangement of concave and convex face protons of netropsin are retained in the complexes. The observed changes in NOE patterns and chemical shift changes on complex formation suggest small conformational changes in the nucleic acid at the AATT and TATA antibiotic binding sites and possibly the flanking G.C base pairs. We observe intermolecular NOEs between all three amide and both pyrrole protons on the concave face of the antibiotic and the minor groove adenosine H2 proton of the two central A4.T5 base pairs of the AATT 8-mer and TATA 8-mer duplexes. The concave face pyrrole protons of the antibiotic also exhibit NOEs to the sugar H1' protons of residues 5 and 6 in the AATT and TATA 8-mer complexes. We also detect intermolecular NOEs between the guanidino and propioamidino methylene protons at either end of netropsin and the adenosine H2 proton of the two flanking A3.T6 base pairs in the AATT 8-mer and T3.A6 base pairs in the TATA 8-mer duplexes. These studies establish a set of nine contacts between the concave face of the antibiotic and the minor groove AATT segment and TATA segment of the 8-mer duplexes in solution. The observed magnitude of the NOEs require that there be no intervening water molecules sandwiched between the concave face of the antibiotic and the minor groove of the DNA so that release of the minor groove spine of hydration is a prerequisite for netropsin complex formation. The observed differences in the netropsin amide proton chemical shifts in the AATT 8-mer and TATA 8-mer complexes suggest differences in the strength and/or type of intermolecular hydrogen bonds at the AATT and TATA binding sites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structural and dynamic aspects of the sequence specific binding of netropsin and its bis-imidazole analogue on the decadeoxyribonucleotide d-[CGCAATTGCG]2

High field 1H-NMR techniques have been used to examine the sequence dependent binding of a lexitropsin, the bis-imidazole analogue of netropsin 1, to the decadeoxyribonucleotide d-[CGCAATTGCG]2. The non-exchangeable and imino protons of the 1:1 lexitropsin:DNA complex are assigned by 1D-NOE difference and COSY methods. Addition of 1 to the DNA resulted in marked drug induced chemical shift changes of both the non-exchangeable and imino protons of A(4,5) and T(6,7). These results suggest that the lexitropsin is located in the minor groove along A(4) to T(7) of the DNA. Weaker chemical shift changes are observed for C(3) and G(8) which suggest that the bisimidazole moiety of 1 can also accept G.C sites. Specific NOEs seen between the lexitropsin (H2, H14 and H15) and DNA (AH2(4) and AH2(5] confirmed that the N to C-terminii of 1 is, on average, bound centrally to the sequence in the direction 5'-AATT-3'. However, netropsin 2 is shown to bind tightly only to the AATT sequence. Exchange NMR effects permit the estimate of the rate of exchange of the lexitropsin 1 between the two equivalent sites on the DNA to be approximately 160s and 24s for netropsin under comparable conditions. Several factors contributing to the sequence specificity of lexitropsin binding are discussed.     

Sequence specific molecular recognition and binding by a GC recognizing Hoechst 33258 analogue to the decadeoxyribonucleotide d-[CATGGCCATG]2: structural and dynamic aspects deduced from high field 1H-NMR studies.

The non-exchangeable and imino proton NMR resonances have been assigned of the 1:1 complex of an analogue 2 of Hoechst 33258 1 bound to the decadeoxyribonuycleotide d-[CATGGCCATG]2 by a combination of NOE difference, COSY and NOESYPH techniques. In contrast to Hoechst 33258 which recognizes 5'-AATT sequences exclusively, analogue 2 possesses structural features designed to permit the recognition of GC sites. The NOESY and 1D-NOE experiments place the drug in the minor groove and it is located on the 5'-CCAT sequence. The orientation of the drug in the groove is such as to place the N-methylpiperazine terminus at a GC site. Cross-correlation peaks in the NOESY experiment show that the DNA duplex retains its right-handed B form, similar to that in the free decamer. Specific NOEs locate the benzoxazole moiety on the 5'-CCAT and are consistent with the pyridine nitrogen forming a new hydrogen bond to G(4)-2NH2 at 5'-CCAT. The drug appears to undergo rotation around the C9-C10 bond, at a rate comparable with NMR time scale, even after binding. Variable temperature 1H-NMR studies established that the DNA is thermally stabilized as a result of the drug binding. The drug binding is a dynamic process involving exchange between the equivalent 5'-CCAT sites at approximately 60s-1 with delta G degree of 65 kJ mol-1 at 308K. The experimental evidence is in accord with a slide-swing mechanism for this process.     

Anthracycline antibiotic arugomycin binds in both grooves of the DNA helix simultaneously: an NMR and molecular modelling study.

Perturbations to the 1H and 31P chemical shifts of DNA resonances together with twenty-four intermolecular nuclear Overhauser effects show that the anthracycline antibiotic arugomycin intercalates between the basepairs of the hexamer duplex d(5'-GCATGC)2 at the 5'-CpA and 5'-TpG binding sites. In the complex two drug molecules are bound per duplex with full retention of the dyad symmetry. Arugomycin adopts a threaded binding orientation with chains of sugars positioned in both the major and minor groove of the helix simultaneously. The complex is stabilized by hydrogen bonding, electrostatic and van der Waals interactions principally in the major groove and involving substituents on the rigidly oriented bicycloamino-glucose sugar of the antibiotic. A specific hydrogen bond is identified between the C2'-hydroxyl and the guanine N7 at the intercalation site. Together, interactions in the major groove appear to account for the intercalation specificity of arugomycin that requires both a guanine and thymine at the intercalation site. We are unable to identify any sequence specific interactions between the minor groove and the arugarose sugar (S1) which binds only weakly, through van der Walls contacts, over the d(GCA).d(TGC) trinucleotide sequence. The data indicate that the sugar chains of arugomycin are flexible and play little part in the interaction of the antibiotic with DNA. The intensity of sequential internucleotide NOEs identifies the intercalation site as being assymmetric. A family of conformers computed using restrained energy minimisation and molecular dynamics indicate that basepair buckling is a feature of the anthracycline intercalation site that may serve to maximise intermolecular van der Waals interactions by wrapping the basepairs around the antibiotic chromophore.     

Netropsin . dG-dG-dA-dA-dT-dT-dC-dC complex. Antibiotic binding at adenine . thymine base pairs in the minor groove of the self-complementary octanucleotide duplex.

The structure of the netropsin . dG-dG-dA-dA-dT-dT-dC-dC complex (one antibiotic molecule/self-complementary octanucleodide duplex) and its dynamics as a function of temperature have been monitored by the nuclear magnetic resonances of the Watson-Crick protons, the nonexchangeable base and sugar protons and the backbone phosphates. The antibiotic forms a complex with the nucleic acid duplex at the dA . dT-containing tetranucleotide segment dA-dA-dT-dT, with slow migration amongst potential binding sites at low temperature. The downfield shifts in the exchangeable protons of netropsin on complex formation demonstrate the contributions of hydrogen-bonding interactions between the antibiotic and the nucleic acid to the stability of the complex. Complex formation results in changes in the glycosidic torsion angles of both thymidine residues and one deoxyadenosine residue as monitored by chemical shift changes in the thymine C-6 and adenine C-8 protons. The close proximity of the pyrrole rings of the antibiotic and the base-pair edges in the minor groove is manifested in the downfield shifts (0.3--0.5 ppm) of the pyrrole C-3 protons of netropsin and one adenine C-2 proton and one thymine N-3 base-pair proton on complex formation. The internucleotide phosphates of the octanucleotide undergo 31P chemical shift changes on addition of netropsin and these may reflect, in part, contributions from electrostatic interactions between the charged ends of the antibiotic and the backbone phosphates of the nucleic acid.     

Use of capillary electrophoresis in the study of ligand-DNA interactions.

Free solution capillary electrophoresis (FSCE) has been used to separate two non-self-complementary 12mer oligonucleotide duplexes: d(AAATTATATTAT).d(ATAA-TATAATTT) and d(GGGCCGCGCCGC).d(GCGGCGCGGCCC). Titration of mixtures of the two oligonucleotides with model intercalators (ethidium bromide andactinomycin D) and minor groove binders (netropsin, Hoechst 33258 and distamycin) has shown the suitability of FSCE as a method to study the sequence selectivity of DNA binding agents. Binding data have shown cooperativity of binding for netropsin and Hoechst 33258 and have provided ligand:DNA binding ratios for all five compounds. Cooperativity of netropsin binding to a 12mer with two potential sites has been demonstrated for the first time. Ligands binding in the minor groove caused changes in migration time and peak shape which were significantly different from those caused by intercalators.     

Solution structure and stability of the DNA undecamer duplexes containing oxanine mismatch

Solution structures of DNA duplexes containing oxanine (Oxa, O) opposite a cytosine (O:C duplex) and opposite a thymine (O:T duplex) have been solved by the combined use of (1)H NMR and restrained molecular dynamics calculation. One mismatch pair was introduced into the center of the 11-mer duplex of [d(GTGACO(6)CACTG)/d(CAGTGX(17)GTCAC), X = C or T]. (1)H NMR chemical shifts and nuclear Overhauser enhancement (NOE) intensities indicate that both the duplexes adopt an overall right-handed B-type conformation. Exchangeable resonances of C(17) 4-amino proton of the O:C duplex and of T(17) imino proton of O:T duplex showed unusual chemical shifts, and disappeared with temperature increasing up to 30 °C, although the melting temperatures were >50 °C. The O:C mismatch takes a wobble geometry with positive shear parameter where the Oxa ring shifted toward the major groove and the paired C(17) toward the minor groove, while, in the O:T mismatch pair with the negative shear, the Oxa ring slightly shifted toward the minor groove and the paired T(17) toward the major groove. The Oxa mismatch pairs can be wobbled largely because of no hydrogen bond to the O1 position of the Oxa base, and may occupy positions in the strands that optimize the stacking with adjacent bases.     

Binding properties and DNA sequence-specific recognition of two bithiazole-linked netropsin hybrid molecules.

We report the DNA binding properties of two hybrid molecules which result from the combination of the DNA sequence-specific minor groove ligand netropsin with the bithiazole moiety of the antitumor drug bleomycin. The drug-DNA interaction has been investigated by means of electric linear dichroism (ELD) spectroscopy and DNase I footprinting. In compound 1 the two moieties are linked by a flexible aliphatic tether while in compound 2 the two aromatic ring systems are directly coupled by a rigid peptide bond. The results are consistent with a model in which the netropsin moiety of compound 1 resides in the minor groove of DNA and where the appended bithiazole moiety is projected away from the DNA groove. This monocationic hybrid compound has a weak affinity for DNA and shows a strict preference for A and T stretches. ELD measurements indicate that in the presence of DNA compound 2 has an orientation typical of a minor groove binder. Similar orientation angles were measured for netropsin and compound 2. This ligand which has a biscationic nature tightly binds to DNA (Ka = 6.3 x 10(5) M-1) and is mainly an AT-specific groove binder. But, depending on the nature of the sequence flanking the AT site first targeted by its netropsin moiety, the bithiazole moiety of 2 can accommodate various types of nucleotide motifs with the exception of homooligomeric sequences. As evidenced by footprinting data, the bithiazole group of bleomycin acts as a DNA recognition element, offering opportunities to recognize GC bp-containing DNA sequences with apparently a preference (although not absolute) for a pyrimidine-G-pyrimidine motif. Thus, the bithiazole unit of bleomycin provides an additional anchor for DNA binding and is also capable of specifically recognizing particular DNA sequences when it is appended to a strongly sequence selective groove binding entity. Finally, a model which schematizes the binding of compound 2 to the sequence 5'-TATGC is proposed. This model readily explains the experimentally observed specificity of this netropsin-bithiazole conjugate.     

NMR chemical shift mapping of the binding site of a protein proteinase inhibitor: changes in the (1)H, (13)C and (15)N NMR chemical shifts of turkey ovomucoid third domain upon binding to bovine chymotrypsin A(alpha)

The substrate-like inhibition of serine proteinases by avian ovomucoid domains has provided an excellent model for protein inhibitor-proteinase interactions of the standard type. 1H,15N and 13C NMR studies have been undertaken on complexes formed between turkey ovomucoid third domain (OMTKY3)2 and chymotrypsin A(alpha) (Ctr) in order to characterize structural changes occurring in the Ctr binding site of OMTKY3. 15N and 13C were incorporated uniformly into OMTKY3, allowing backbone resonances to be assigned for OMTKY3 in both its free and complex states. Chemical shift perturbation mapping indicates that the two regions, K13-P22 and N33-A40, are the primary sites in OMTKY3 involved in Ctr binding, in full agreement with the 12 consensus proteinase-contact residues of OMTKY3 defined previously on the basis of X-ray crystallographic and mutational analysis. Smaller chemical shift perturbations in selected other regions may result from minor structural changes on binding. Through-bond 15N-13C correlations between P1-13C' and P1'-15N in two-dimensional H(N)CO and HN(CO) NMR spectra of selectively labeled OMTKY3 complexed with Ctr indicate that the scissile peptide bond between L18 and E19 of the inhibitor is intact in the complex. The chemical shifts of the reactive site peptide bond indicate that it is predominantly trigonal, although the data are not inconsistent with a slight perturbation of the hybridization of the peptide bond toward the first tetrahedral state along the reaction coordinate.     

Structural and dynamic aspects of binding of a prototype lexitropsin to the decadeoxyribonucleotide d(CGCAATTGCG)2 deduced from high-resolution 1H NMR Studies

Structural and dynamic properties of the self-complementary decadeoxyribonucleotide d(CGCAATTGCG)2 and the interaction between a prototype lexitropsin, or information-reading oligopeptide, and the decadeoxyribonucleotide are deduced by using high-resolution 1H NMR techniques. The nonexchangeable and imino proton resonances of d(CGCAATTGCG)2 have been completely assigned by two-dimensional NMR studies. The decadeoxyribonucleotide exists as a right-handed B-DNA. In the 1H NMR spectrum of the 1:1 complex, the selective chemical shifts and removal of degeneracy of AH2(4), AH2(5), T-CH3(6), and T-CH3(7) due to the anisotropy effects of the heterocyclic moieties of the ligand, and with lesser effects at the flanking base sites C(3) and G(8), locate the drug centrally in the decadeoxyribonucleotide. This conclusion is supported by plots of individual chemical shift changes across the decadeoxyribonucleotide. Similarly, imino protons IV and V experience larger shifts and II and III smaller shifts in accord with this conclusion while drug complexation permits the detection of imino proton I. Strong nuclear Overhauser effects (NOEs) between pyrrole H5 and AH2(5), and weaker NOEs to AH1'(5), TH3'(6), and AH2'(5), firmly locate the ligand in the minor groove. Intraligand NOEs between the adjacent heterocyclic moieties indicate that the lexitropsin is subject to propeller twisting about the N6-C9 bond in both the bound and free forms. Nuclear Overhauser effect spectroscopy (NOESY) and correlated spectroscopy (COSY) experiments also indicate that the removal of degeneracy of the C16 methylene protons upon complexation may arise from restricted rotation about the C15-N9, C15-C16, and C16-C17 bonds. Specific hydrogen bonds between amide NH groups on the concave face of the ligand (N4H, N6H, N9H) and adenine N3 or thymine O2 on the floor of the minor groove are in accord with displacement of the hydration shell by the drug. NOE measurements on the decadeoxyribonucleotide in the 1:1 complex confirm it exists as a right-handed helix and belongs to the B family. Exchange NMR effects permit an estimate of a rate of approximately equal to 44 s-1 for the two-site exchange of the lexitropsin between two equivalent sites on the decamer with delta G++ approximately equal to 70 +/- 5 kJ mol-1 at 294 K. Alternative mechanisms for this exchange process are considered.     

Spectroscopic probe of distribution of the non-intercalating drug netropsin between DNA and heparin.

Circular dichroism has been used as a monitoring tool to probe the distribution of the non-intercalating drug netropsin (NTPS) between the two biomolecules DNA and heparin. The stoichiometry of the interaction of the individual biomolecules and the drug is determined from conductometric titrations; the titration in each case shows two breaks corresponding to two stoichiometries of interaction. Though netropsin is non-intercalating, DNA wins over heparin in binding the drug due to strong hydrogen bonding capability of NTPS in the minor grooves of DNA through its greater than NH donor groups. Potential hydrogen bond breakers like KF and urea reduce the induced dichroism of NTPS-DNA system, probably dislodging some drug from DNA through hydrogen bond breaking.     

Cooperative stabilization of Zn2+:DNA complexes through netropsin binding in the minor groove of FdU-substituted DNA

The simultaneous binding of netropsin in the minor groove and Zn²⁺ in the major groove of a DNA hairpin that includes 10 consecutive FdU nucleotides at the 3′-terminus (3′FdU) was demonstrated based upon NMR spectroscopy, circular dichroism (CD), and computational modeling studies. The resulting Zn²⁺/netropsin: 3′FdU complex had very high thermal stability with aspects of the complex intact at 85?°C, conditions that result in complete dissociation of Mg²⁺ complexes. CD and ¹⁹F NMR spectroscopy were consistent with Zn²⁺ binding in the major groove of the DNA duplex and utilizing F5 and O4 of consecutive FdU nucleotides as ligands with FdU nucleotides hemi-deprotonated in the complex. Netropsin is bound in the minor groove of the DNA duplex based upon 2D NOESY data demonstrating contacts between AH2 ¹H and netropsin ¹H resonances. The Zn²⁺/netropsin: 3′FdU complex displayed increased cytotoxicity towards PC3 prostate cancer (PCa) cells relative to the constituent components or separate complexes (e.g. Zn²⁺:3′FdU) indicating that this new structural motif may be therapeutically useful for PCa treatment.

An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:32     

Structure and Dynamics of Netropsin-Poly(dA-dT)· Poly(dA-dT) Complex: 500 MHz 1H NMR Studies

Abstract

Antibiotic netropsin is known to bind specifically to A and T regions in DNA; the mode of binding being non-intercalative. Obviously, H-bonding between the proton donors of netropsin and acceptors N3 of A and 02 of T comes as a strong possibility which might render this specificity. In netropsin there could be 8 proton donors: four terminal amino groups and four internal imino groups. However, methylation of the terminal amino groups does not alter the binding affinity of netropsin to DNA—but the modification of the internal imino groups significantly lowers the binding affinity. Hence, the logical conclusion is that netropsin may specifically interact with A and T through H-bonding and in order to do so, it should approach the helix from the minor groove. The present paper provides experimental data which verify the conclusion mentioned above.

Using poly(dA-dT)? poly(dA-dT) as a model system it was observed following a thorough theoretical stereochemical analysis that netropsin could bind to -(T-A-T) sequence of the polymer in the B-form through the minor groove by forming specific B-bonding. Models could be either right or left-handed B-DNA with a mono or dinucleotide repeat.

By monitoring the ³¹P signals of free poly(dA-dT) ? poly(dA-dT) and netropsin-poly(dA-dT)? poly(dA-dT) complex we show that the drug changes the DNA structure from essentially a mononucleotide repeat to that of very dominant dinucleotide repeat; however the base- pairing in the DNA-drug complex remain to be Watson-Crick. Whether H-bonding is the specific mode of interaction was judged by monitoring the imino protons of netropsin in the presence of poly(dA-dT) ? poly(dA-dT). This experiment was conducted in 90% H₂O + 10% D₂O Using the time-shared long pulse. It was found that exchangeable imino protons of netropsin appear in the drug-DNA complex and disappear upon increasing the D₂O content; thus confirming that H-bonding is indeed the specific mode of interaction. From these and several NOE measurements, we propose a structure for poly(dA-dT)? poly(dA-dT(-netropsin complex.

In summary, experimental data indicate that netropsin binds to poly(dA-dT)? poly(dA-dT) by forming specific hydrogen bonds and that the binding interaction causes the structure to adopt a Watson-Crick paired dinucleotide repeat motif. The proposed hydrogen bonds can form only if the drug approaches the DNA from the minor groove. Within the NMR time scale the interaction between the ligand and DNA is a fast one. From the NOE experimental data, it appears that poly(dA-dT)? poly(dA-dT) in presence of netropsin exists as an equilibrium mixture of right- and left-handed B-DNA duplexes with a dinucleotide repeat—with a predominance of the left-handed form. The last conclusion is a soft one because it was very difficult to make sure the absence of spin diffusion. In a 400 base pairs long DNA duplex- drug complex (as used in this study), equilibrium between right and left-handed helices can also mean the existence of both helical domains in the same molecule with fast interchange between these domains or/and unhindered motion/propagation of these domains along the helix axis.     

Hydration of the RNA duplex r(CGCAAAUUUGCG)2 determined by NMR.

The so-called spine of hydration in the minor groove of AnTn tracts in DNA is thought to stabilise the structure, and kinetically bound water detected in the minor groove of such DNA species by NMR has been attributed to a narrow minor groove [Liepinsh, E., Leupin, W. and Otting, G. (1994) Nucleic Acids Res. 22, 2249-2254]. We report here an NMR study of hydration of an RNA dodecamer which has a wide, shallow minor groove. Complete assignments of exchangeable protons, and a large number of non-exchangeable protons in r(CGCAAAUUUGCG)2 have been obtained. In addition, ribose C2'-OH resonances have been detected, which are probably involved in hydrogen bonds. Hydration at different sites in the dodecamer has been measured using ROESY and NOESY experiments at 11.75 and 14.1 T. Base protons in both the major and minor grooves are in contact with water, with effective correlation times for the interaction of approximately 0.5 ns, indicating weak hydration, in contrast to the hydration of adenine C2H in the homologous DNA sequence. NOEs to H1' in the minor groove are consistent with hydration water present that is not observed in the analogous DNA sequence. Hydration kinetics in nucleic acids may be determined by chemical factors such as hydrogen-bonding more than by simple conformational factors such as groove width.     

Sequence-dependent recognition of DNA duplexes. Netropsin complexation to the AATT site of the d(G-G-A-A-T-T-C-C) duplex in aqueous solution

We have investigated intermolecular interactions and conformational features of the netropsin X d(G-G-A-A-T-T-C-C) complex by one- and two-dimensional NMR studies in aqueous solution. Netropsin removes the 2-fold symmetry of the d(G-G-A-A-T-T-C-C) duplex at the AATT binding site and to a lesser extent at adjacent dG X dC base pairs resulting in doubling of resonances for specific positions in the spectrum of the complex at 25 degrees C. We have assigned the amide, pyrrole, and CH2 protons of netropsin, and the base and sugar H1' protons of the nucleic acid from an analysis of the nuclear Overhauser effect (NOESY) and correlated (COSY) spectra of the complex at 25 degrees C. We observe intermolecular nuclear Overhauser effects (NOE) between all three amide and both pyrrole protons on the concave face of the antibiotic and the minor groove adenosine H2 proton of the two central A4 X T5 base pairs of the d(G1-G2-A3-A4-T5-T6-C7-C8) duplex. Weaker intermolecular NOEs are also observed between the pyrrole concave face protons and the sugar H1' protons of residues T5 and T6 in the AATT minor groove of the duplex. We also detect intermolecular NOEs between the guanidino CH2 protons at one end of netropsin and adenosine H2 proton of the two flanking A3 X T6 base pairs of the octanucleotide duplex. These studies establish a set of intermolecular contacts between the concave face of the antibiotic and the minor groove AATT segment of the d(G-G-A-A-T-T-C-C) duplex in solution. The magnitude of the NOEs require that there be no intervening water molecules sandwiched between the antibiotic and the DNA so that release of the minor groove spine of hydration is a prerequisite for netropsin complex formation.