Effects of A:T base pairs on the B-Z conformational transitions of DNA

Effects of A:T base pairs on the propensity of B to Z conformational transitions have been investigated by the CD salt titrations on d(CG)5' d(GC)5' terminal or central A:T replaced decamers, and terminal A:T appended dodecamers. The presence of A:T at the center greatly inhibits the B to Z transition of both G:C decamers. Moderate Z inhibitions are shown by terminal A:T replacements and additions to d(CG)5' with the former exhibiting a stronger effect. In contrast, the addition and replacement with A:T at the terminals of d(GC)5 facilitate the B to Z conversion, with the replacement exhibiting a somewhat more pronounced effect. These results may be rationalized in terms of the number of contigous CG sequences present in an oligomer and the relative inhibitory effects of other dinucleotide sequences. Our results also suggest that some short oligomers with purine at the 5'-end, such as d[A(CG)nT] with n greater than or equal to 2, may likely crystallize as Z conformations.     

NMR structure of a parallel-stranded DNA duplex at atomic resolution

DNA dodecamers have been designed with two cytosines on each end and intervening A and T stretches, such that the oligomers have fully complementary A:T base pairs when aligned in the parallel orientation. Spectroscopic (UV, CD and IR), NMR and molecular dynamics studies have shown that oligomers having the sequences d(CCATAATTTACC) and d(CCTATTAAATCC) form a parallel-stranded duplex when dissolved at 1:1 stoichiometry in aqueous solution. This is due to the C:C⁺ clamps on either end and extensive mismatches in the antiparallel orientation. The structure is stable at neutral and acidic pH. At higher temperatures, the duplex melts into single strands in a highly cooperative fashion. All adenine, cytosine and thymine nucleotides adopt the anti conformation with respect to the glycosidic bond. The A:T base pairs form reverse Watson–Crick base pairs. The duplex shows base stacking and NOEs between the base protons T(H6)/A(H8) and the sugar protons (H1′/H2′/H2″) of the preceding nucleotide, as has been observed in antiparallel duplexes. However, no NOEs are observed between base protons H2/H6/H8 of sequential nucleotides, though such NOEs are observed between T(CH₃) and A(H8). A three-dimensional structure of the parallel-stranded duplex at atomic resolution has been obtained using molecular dynamics simulations under NMR constraints. The simulated structures have torsional angles very similar to those found in B-DNA duplexes, but the base stacking and helicoid parameters are significantly different.     

Sequence dependent electrophoretic mobilities and melting temperatures for A-T containing oligodeoxyribonucleotides.

The electrophoretic mobilities and thermal melting properties of self complementary A-T containing dodecamer oligodeoxyribonucleotides have been investigated as a function of solution conditions. The oligomers contained tracts of nonalternating A-T base pairs of 2 (d(A2T2)3), 3 (d(A3T3)2), and 6 (d(A6T6] as well as the fully alternating (d(A-T)6) sequence. The melting temperature increased with the length of the nonalternating sequence and was approximately 12 degrees C higher in the d(A6T6) sequence than in the alternating oligomer. Under denaturing conditions all oligomers had the same electrophoretic mobility on acrylamide gels. Under conditions which favor duplex formation, the oligomers exhibited significant sequence dependent mobility differences. The mobilities of two oligomers, d(A-T)6 and d(A6-T6), were approximately equal and were less than those of the other oligonucleotides. The greatest mobility was observed for d(A2T2)3. These results are best explained by a model which requires bending at a junction of two or more continuous A or T bases with another sequence.     

Circular dichroism spectra of DNA oligomers show that short interior stretches of C.C+ base pairs do not form in duplexes with A.T base pairs

Circular dichroism (CD) experiments were carried out on a series of DNA oligomers to determine if short internal stretches of protonated cytosine-cytosine (C.C+) base pairs could coexist with adenine-thymine (A.T) base pairs. (1) C.C+ base pairs did form in the absence of A.T base pairs in the individual oligomers d(AACC)5 and d(CCTT)5, as indicated by the appearance of a long-wavelength CD band centered at 282-284 nm, when the pH was lowered to 6 or 5 at 0.5 M Na+. A comparison of measured with calculated spectra showed that d(CCTT)5 at pH 5, 0.5 M Na+, 20 degrees C, likely adopted a structure with a central core of stacked C.C+ base pairs and looped-out thymines. Under the same conditions, it appeared that C.C+ base pairs also formed in d(AACC)5, but with the adenines remaining intrahelical. Each of these oligomers showed a cooperative transition for formation of C.C+ base pairs as the temperature was lowered, with C.C+ base pairs forming at a higher temperature in d(CCTT)5 than in d(AACC)5. A.T base formed in equimolar mixtures of d(AACC)5 plus d(CCTT)5 as monitored by an increase in the negative magnitude of the 250-nm CD band. However, a large increase did not appear at about 285 nm in CD spectra of the mixtures, showing that there were no stacked C.C+ base pairs in the d(AACC)5.d(CCTT)5 duplex even though they formed under the same conditions in the individual strands. Thus, in this duplex, A.T base pairs prevented the formation of neighboring internal C.C+ base pairs. (2) CD measurements were also made of d(A10C4T10).(ABSTRACT TRUNCATED AT 250 WORDS)     

The formation of adjacent triplex-duplex domainsin DNA.

The ability of single-stranded DNA oligomers to form adjacent triplex and duplex domains with two DNA structural motifs was examined. Helix-coil transition curves and a gel mobility shift assay were used to characterize the interaction of single-stranded oligomers 12-20 nt in length with a DNA hairpin and with a DNA duplex that has a dangling end. The 12 nt on the 5'-ends of the oligomers could form a triplex structure with the 12 bp stem of the hairpin or the duplex portion of the DNA with a dangling end. The 3'-ends of the 17-20 nt strands could form Watson-Crick pairs to the five base loop of the hairpin or the dangling end of the duplex. Complexes of the hairpin DNA with the single-stranded oligomers showed two step transitions consistent with unwinding of the triplex strand followed by hairpin denaturation. Melting curve and gel competition results indicated that the complex of the hairpin and the 12 nt oligomer was more stable than the complexes involving the extended single strands. In contrast, results indicated that the extended single-stranded oligomers formed Watson-Crick base pairs with the dangling end of the duplex DNA and enhanced the stability of the adjacent triplex region.     

A.T and C.C+ base pairs can form simultaneously in a novel multistranded DNA complex

Previous experiments have established that in certain synthetic oligomeric DNA sequences, including mixtures of d(AACC)5 with d(CCTT)5, adenine-thymine (A.T) base pairs form to the exclusion of neighboring protonated cytosine-cytosine (C.C+) base pairs [Edwards, E., Ratliff, R., & Gray, D. (1988) Biochemistry 27, 5166-5174]. In the present work, circular dichroism and other measurements were used to study DNA oligomers that represented two additional classes with respect to the formation of A.T and/or C.C+ base pairs. (1) One class included two sets of repeating pentameric DNA sequences, d(CCAAT)3-6 and d(AATCC)4,5. For both of these sets of oligomers, an increase in the magnitude of the long-wavelength positive CD band centered at about 280 nm occurred as the pH was lowered from 7 to 5 at 0.1 and 0.5 M Na+, indicating that C.C+ base pairs formed. Even though it may have been possible for these oligomers to form duplexes with two antiparallel A.T base pairs per pentamer, no A.T base pairing was detected by monitoring the CD changes at 250 nm. Thus, spectral data showed that as few as 40% C.C+ base pairs were stable in two sets of oligomers in which A.T base pairs did not form adjacent to, or in place of, C.C+ base pairs. (2) Another class of oligomer was represented by d(C4A4T4C4), which was studied by CD, HPLC, and centrifugation experiments. We confirmed previous work that this sequence was able to form both types of base pairs as the pH and temperature were lowered [Gray, D., Cui, T., & Ratliff, R. (1984) Nucleic Acids Res. 12, 7565-7580].(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of melting transitions of the DNA hairpins formed from the oligomer sequences d[GGATAC(X)4GTATCC] (X = A, T, G, C)

Optical melting transitions of the short DNA hairpins formed from the self-complementary DNA oligomers d[GGATACX4GTATCC] where X = A, T, G, or C measured in 100 mM NaCl are presented. A significant dependence of the melting transitions on loop sequence is observed and transition temperatures, tm, of the hairpins vary from 58.3 degrees C for the T4 loop hairpin to 55.3 degrees C for the A4 loop. A nearest-neighbor sequence-dependent theoretical algorithm for calculating melting curves of DNA hairpins is presented and employed to analyze the experimental melting transitions. Experimental melting curves were fit by adjustment of a single theoretical parameter, Fend(n), the weighting function for a hairpin loop comprised of n single-strand bases. Empirically determined values of Fend(n) provide an evaluation of the free-energy of hairpin loop formation and stability. Effects of heterogeneous nearest-neighbor sequence interactions in the duplex stem on hairpin loop formation were investigated by evaluating Fend(n) in individual fitting procedures using two of the published sets of nearest-neighbor stacking interactions in DNA evaluated in 100 mM NaCl and given by Wartell and Benight, 1985. In all cases, evaluated values of Fend(n) were obtained that provided exact theoretical predictions of the experimental transitions. Results of the evaluations indicate: (1) Evaluated free-energies of hairpin loop formation are only slightly dependent on loop sequences examined. At the transition temperature, Tm, the free-energy of forming a loop of four bases is approximately equal for T4, G4, or C4 loops and varies from 3.9 to 4.8 kcal/mole depending on the set of nearest-neighbor interactions employed in the evaluations. This result suggests, in light of the observed differences in stability between the T4, G4, and C4 loop hairpins, that sequence-dependent interactions between base residues of the loop are most likely not the source of the enhanced stability of a T4 loop.(ABSTRACT TRUNCATED AT 400 WORDS)     

Oligodeoxynucleotide folding in solution: loop size and stability of B-hairpins

The secondary structures of the synthetic DNA fragments d(CGCGCGTTTTTCGCGCG) (T5), d(CGCGCGAAAAACGCGCG) (A5), d(CGCGCGTACGCGCG) (TA), and d(CGCGCGATCGCGCG) (AT) were investigated in a combined electrophoretic and spectroscopic study. All the oligomers exist, at low temperature and over a wide range of ionic strength (0.5-100 mM salt) and of nucleotide concentration [0.1-2.0 mM (phosphate)], as a mixture of two slowly interconverting species, identified as the dimeric duplex and the monomeric hairpin structure. The thermodynamic parameters for hairpin denaturation of T5, A5, TA, and AT and for duplex denaturation of d(CGCGCG) show that (a) the hairpins are more stable than the reference hexamer duplex at all accessible nucleotide concentrations; (b) the loop contributes favorably to the enthalpy change of hairpin denaturation in the four DNA fragments; (c) the base composition of the loop (A vs T) and the size of the loop (A5/T5 vs TA/AT) do not appreciably influence the enthalpic contents of the hairpins; (d) hairpins TA and AT, with two AT bases intervening in the CG self-complementary part of the molecule, exhibit a markedly higher thermal stability than hairpins T5 and A5, which is entropic in origin. These findings are consistent with the presence of two-residue loops in the tetradecamers TA and AT.     

Evidence for a DNA triplex in a recombination-like motif: I. Recognition of Watson-Crick base pairs by natural bases in a high-stability triplex

Data are presented on a triplex type with two parallel homologous strands for which triplex formation is almost as strong as duplex formation at least for some sequences and even at pH 7 and 0.2 M NaCl. The evidence mainly rests upon comparing thermodynamic properties of similar systems. A paperclip oligonucleotide d(A12C4T12C4A12) with two linkers C4 obviously can form a triplex with parallel back-folded adenine strand regions, because the single melting transition of this complex splits in two transitions by introducing mismatches only in the third strand region. Respectively, a hairpin duplex d(A12C4T12) and a single strand d(A12) form a triplex as a 1:1 complex in which the second adenine strand is parallel oriented to the homologous one in the Watson-Crick paired duplex. In this system the melting temperature T(m) of the triplex is practically the same as that of the duplex d(A12)-d(T12), at least within a complex concentration range of 0.2-4.0 microM. The melting behaviour of complexes between triplex stabilizing ligand BePI and the system hairpin duplex plus single strand supports the triplex model. Non-denaturing gel electrophoresis suggests the existence of a triplex for a system in which five of the twelve A-T*A base triads are substituted by C-G*C base triads. The recognition between any substituted Watson-Crick base pair (X-Y) in the hairpin duplex d(A4XA7C4T7YT4) and the correspondingly replaced base (Z) in the third strand d(A4ZA7) is mutually selective. All triplexes with matching base substitutions (Z = X) have nearly the same stability (T(m) values from 29 to 33.5 degrees C), whereas triplexes with non-matching substitutions (Z not equal X) show a clearly reduced stability (T(m) values from 15 to 22 degrees C) at 2microM equimolar oligonucleotide concentration. Most nucleic acid triple helices hitherto known are limited to homopurine-homopyrimidine sequences in the target duplex. A stable triplex formation is demonstrated for inhomogeneous sequences tolerating at least 50% pyrimidine content in the homologous strands. On the basis of the surprisingly similar thermodynamic parameters for duplex and triplex, and of the fact that this triplex type seems to be more stable than many other natural DNA triplexes known, and on the basis of semiempirical and molecule mechanical calculations, we postulate bridging interactions of the third strand with the two other strands in the triplex according to the recombination motif. This triplex, denoted by us 'recombination-like form', tolerates heterogeneous base sequences.     

NMR studies of pH-dependent conformational polymorphism of alternating (C-T)n sequences.

Alternating (C-T)n sequences are involved in the H-DNA structure associated with (GA)n.(CT)n sequences. Low pH values facilitate H-DNA formation. We have undertaken a detailed analysis of the structural consequences of the (C-T)n sequence as a function of pH. The structures of three DNA oligonucleotides, d(CT)4, d(TC)4 and d(TC)15, have been studied by NMR. We found that their conformations are polymorphic and pH dependent. There are at least three major conformational species: an antiparallel-stranded (APS) duplex with entirely C:T base pairs at pH 7, an antiparallel-stranded (APS) duplex with entirely C+:T base pairs at pH 3, and a possible parallel-stranded (PS) duplex with C+:C and T:T base pairs near pH 5. In the intermediate pH range, the APS duplex may have varying numbers of C+:T and C:T base pairs, and there may be a fast exchange going on between APS duplex species involving these two kinds of base pairs. However, the transition between the APS and PS duplexes is slow. Structural refinement of the two octamers, d(TC)4 and d(CT)4, at pH = 6.9 and pH = 3 using 2D-NOE data suggests that the molecules are likely in the duplex form at 5 degrees C. We lack evidence that the structure at pH 3 is a PS structure with T nucleotides residing in the exterior of the helix. Titration of the longer oligonucleotide, d(TC)15, showed a prominent pKa of approximately 6, approaching the value of 7.0 obtained from the titration of poly-(dC).     

Protonation studies and multivariate curve resolution on oligodeoxynucleotides carrying the mutagenic base 2-aminopurine

2-Aminopurine (P) is a mutagen causing A.T to G.C transitions in prokaryotic systems. To study the base-pairing schemes between P and cytosine (C) or thymine (T), two self-complementary dodecamers containing P paired with either C or T were synthesized, and their protonation equilibria were studied by acid-base titrations and melting experiments. The mismatches were incorporated into the self-complementary sequence d(CGCPCCGGXGCG), where X was C or T. Spectroscopic data obtained from molecular absorption, circular dichroism (CD), and molecular fluorescence spectroscopy were analyzed by a factor-analysis-based method, multivariate curve resolution based on the alternating least squares optimization procedure (MCR-ALS). This procedure allows determination of the number of acid-base species or conformations present in an acid-base or melting experiment and the resolution of the concentration profiles and pure spectra for each of them. Acid-base experiments have shown that at pH 7, 150 mM ionic strength, and 37 degrees C, both C and P are deprotonated. At pH near 4, the majority of species shows C protonated and P deprotonated. Finally, at pH values near 3, the majority of species shows both protonated C and P. These results are in agreement with NMR studies showing a wobble geometry for the P x C base pair and a Watson-Crick geometry for the P x T base pair at neutral pH. Melting experiments were carried out to confirm the proposed acid-base distribution profile. For the sequence including the P x T mismatch, only one transition was observed at neutral pH. However, for the sequence including the P x C mismatch, two transitions were detected by CD but only one by molecular absorption. This behavior agrees with that observed by other authors for oligonucleotides of similar sequence and suggests the following sequence of conformational changes during melting: duplex --> hairpin --> random coil.     

Hairpin Polyamides That use Parallel and Antiparallel Side-by-Side Peptide Motifs in Binding to DNA

Abstract

Pt-bis-netropsin is a synthetic sequence-specific DNA-binding ligand comprizing two netropsin-like fragments which are linked in a tail-to-tail manner via a cis-diammineplat-inum (II) residue. The CD studies and thermodynamic characterization of the DNA-binding properties exhibited by this compound reveal that it forms two types of complexes with poly[d(AT)]?poly[d(AT)] and DNA oligomers containing nucleotide sequences 5′-CC (TA)_nCC-3′, with n = 4, 5 and 6. The first type corresponds to the binding of Pt-bis-netropsin in the extended conformation and is characterized by the saturating ratio of one bound Pt-bis-netropsin molecule per 9 AT-base pairs. The second type of the complex corresponds to the binding of Pt-bis-netropsin to DNA in the folded hairpin form. The binding approaches saturation level when one Pt-bis-netropsin molecule is bound per four or five AT-base pairs. The hairpin form of Pt-bis-netropsin complex is built on the basis of parallel side-by-side peptide motif which is inserted in the minor DNA groove. The CD spectral profiles reflecting the binding of Pt-bis-netropsin in the hairpin form are different from those observed for binding of another bis-netropsin with the sequence Lys-Gly-Py-Py-Gly-Gly-Gly-Py-Py-Dp, where Py is a N-propylpyrrole amino acid residue and Dp is a dimethylaminopropylamino residue. The hairpin form of this bis-netropsin is formed on the basis of antiparallel side- by-side peptide motif. The CD spectra obtained for complexes of this polyamide in the hairpin form with poly[dAT)]?poly[d(AT)] exhibit positive CD band with a peak at 325 nm, whereas the CD spectral profiles for the second complex of Pt-bis-Nt with poly[d(AT)] ?poly[d(AT)] and short DNA oligomers have two intense positive CD bands near 290 nm and 328 nm. This reflects the fact that two bis-netropsins use different structural motifs on binding to DNA in the hairpin form.     

Circular dichroism measurements show that C.C+ base pairs can coexist with A.T base pairs between antiparallel strands of an oligodeoxynucleotide double-helix.

We have studied the coil-to-helix transition of the DNA oligomer d(C4A4T4C4), using circular dichroism measurements to monitor the formation of A.T base pairs within the central self-complementary A4T4 region and the formation of protonated C.C+ base pairs at the ends of the oligomer. We found that both A.T and C.C+ base pairs formed in a coordinated fashion as the temperature and pH were lowered. The CD data of the helix form of the oligomer were consistent with the presence of paired oligomers, but not with hairpin loops. The pKa for formation of C.C+ base pairs between the C4 ends of the oligomer was higher than the pKa for formation of C.C+ base pairs in d(C8), indicating that the formation of C.C+ base pairs in the oligomer was influenced by the presence of a paired A4T4 region. We conclude that A.T and C.C+ base pairs coexist in the self-complex of the oligomer and, therefore, that C.C+ base pairs can form between antiparallel DNA strands.     

Unusual duplex formation in purine rich oligodeoxyribonucleotides

The purine rich oligodeoxyribonucleotides 1C, d(ATGACGGAATA) and 2C, d(ATGAGCGAATA) alone exhibit highly cooperative melting transitions. Analysis of the concentration dependence of melting, and electrophoretic studies indicate that these oligomers can form an unusual purine rich offset double helix. The unusual duplex is predicted to contain four A.T, two G.C, and four G.A mismatch base pairs as well as a single A base stacked on the 3' end of each chain of the helix. Other possible models for the duplex are unlikely because they are predicted to contain many base pairs of low stability. Changing the central sequence to CGG or GGG should destabilize the duplex and this is observed. The unusual duplex of 2C is more stable than the duplex of 1C indicating that the stability of G.A base pairs is quite sensitive to the surrounding sequence. Addition of 1C and 2C to their complementary pyrimidine strands results in normal duplexes of similar stability. We feel that the unusual duplexes are significantly stabilized by the intrinsic stacking tendency of purine bases.     

Sequence and length dependent thermodynamic differences in heterocyclic diamidine interactions at AT base pairs in the DNA minor groove

With the goal of developing a better understanding of the antiparasitic biological action of DB75, we have evaluated its interaction with duplex alternating and nonalternating sequence AT polymers and oligomers. These DNAs provide an important pair of sequences in a detailed thermodynamic analysis of variations in interaction of DB75 with AT sites. The results for DB75 binding to the alternating and nonalternating AT sequences are quite different at the fundamental thermodynamic level. Although the Gibbs energies are similar, the enthalpies for DB75 binding with poly(dA).poly(dT) and poly(dA-dT).poly(dA-dT) are +3.1 and -4.5 kcal/mol, respectively, while the binding entropies are 41.7 and 15.2 cal/mol.K, respectively. The underlying thermodynamics of binding to AT sites in the minor groove plays a key role in the recognition process. It was also observed that DB75 binding with poly(dA).poly(dT) can induce T.A.T triplet formation and the compound binds strongly to the dT.dA.dT triplex.     

The duplex-hairpin conformational transition of d(CGCGCGATCGCGCG) and d(CGCGCGTACGCGCG): a thermodynamic and kinetic study

We have studied the duplex-hairpin conformational transition in two perfectly palindromic sequences, d(CGCGCGATCGCGCG)(I) and d(CGCGCGTACGCGCG)(II), by means of UV-melting, electrophoretic and T-jump experiments. Both tetradecamers exhibit biphasic thermal profiles. The lower temperature transition is concentration dependent whereas the higher temperature transition is not. The former transition has been characterized by gel electrophoresis and shows two distinct bands, whose intensity depends on temperature. This behavior is due to the occurrence of a slow premelting interconversion between the duplex and hairpin forms in both tetradecamers. The kinetics of hairpin formation from the duplex is studied by T-jump experiments. Relaxation spectra are well reproduced by a single relaxation time with rate constants characterized by a high temperature coefficient. In 10 mM NaCl, the duplex-hairpin conversion of I is characterized by an apparent activation energy of 96 +/- 6 kcal/mol, a value rather close to the expected denaturation enthalpy. In 1 mM NaCl a value slightly lower has been obtained. The rate of duplex-hairpin interconversion has been found to decrease as the salt concentration is raised. These data suggest that the transformation from the duplex to the hairpin form should imply a transition state with a simultaneous breaking of most base pairs, if not total strand separation.     

The Duplex-Harpin Conformational Transition of d(CGCGCGATCGCGCG) and d(CGCGCGTACGCGCG): A Thermodynamic and Kinetic Study

Abstract

We have studied the duplex-hairpin conformational transition in two perfectly palindromic sequences, d(CGCGCGATCGCGCG)(I) and d(CGCGCGTACGCGCG)(II), by means of UV-melting, electrophoretic and T-jump experiments. Both tetradecamers exhibit biphasic thermal profiles. The lower temperature transition is concentration dependent whereas the higher temperature transition is not. The former transition has been characterized by gel electrophoresis and shows two distinct bands, whose intensity depends on temperature. This behavior is due to the occurrence of a slow premelting interconversion between the duplex and hairpin forms in both tetradecamers. The kinetics of hairpin formation from the duplex is studied by T-jump experiments. Relaxation spectra are well reproduced by a single relaxation time with rate constants characterized by a high temperature coefficient. In 10 mM NaCl, the duplex-hairpin conversion of I is characterized by an apparent activation energy of 96 ± 6 kcal/mol, a value rather close to the expected denaturation enthalpy. In 1 mM NaCl a value slightly lower has been obtained. The rate of duplex-hairpin interconversion has been found to decrease as the salt concentration is raised. These data suggest that the transformation from the duplex to the hairpin form should imply a transition state with a simultaneous breaking of most base pairs, if not total strand separation.     

Binding of meso-tetrakis(N-methylpyridinium-4-yl)porphyrin to AT oligomers: effect of chain length and the location of the porphyrin stacking

Circular dichroism (CD) spectra of meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (TMPyP) that are associated with various duplex and triplex AT oligomers were investigated in this study. A strong positive CD was apparent for both the TMPyP complexed with duplex d[(A-T)(12)](2), d(A)(12).d(T)(12) and triplex d(A)(12).d[(T)(12)](2) at a low mixing ratio. As the mixing ratio increased, bisignate excitonic CD was produced for TMPyP complexed with duplexes, whereas the positive CD signal remained the same for the TMPyP-d(A)(12).d[(T)(12)](2) complex. This difference in the CD spectrum in the presence of duplex and triplex oligomers indicates that the moderate stacking of TMPyP occurs at the major groove of the duplex and the monomeric binding occurs in (or near) the minor groove. When TMPyP forms a complex with duplex d[(A-T)(6)](2) only excitonic CD was observed, even at a very low mixing ratio. Therefore, at least seven or more basepairs are required for TMPyP to exhibit a monomeric CD spectrum. After close analysis of the CD spectrum, the TMPyP-poly[d(A-T)(2)] complex could be explained by a combination of the CD spectrum of the monomeric, moderately stacked, and extensively stacked TMPyP.     

Interaction between the Z-type DNA duplex and 1,3-propanediamine: crystal structure of d(CACGTG)2 at 1.2 A resolution

The crystal structure of a hexamer duplex d(CACGTG)(2) has been determined and refined to an R-factor of 18.3% using X-ray data up to 1.2 A resolution. The sequence crystallizes as a left-handed Z-form double helix with Watson-Crick base pairing. There is one hexamer duplex, a spermine molecule, 71 water molecules, and an unexpected diamine (Z-5, 1,3-propanediamine, C(3)H(10)N(2)) in the asymmetric unit. This is the high-resolution non-disordered structure of a Z-DNA hexamer containing two AT base pairs in the interior of a duplex with no modifications such as bromination or methylation on cytosine bases. This structure does not possess multivalent cations such as cobalt hexaammine that are known to stabilize Z-DNA. The overall duplex structure and its crystal interactions are similar to those of the pure-spermine form of the d(CGCGCG)(2) structure. The spine of hydration in the minor groove is intact except in the vicinity of the T5A8 base pair. The binding of the Z-5 molecule in the minor grove of the d(CACGTG)(2) duplex appears to have a profound effect in conferring stability to a Z-DNA conformation via electrostatic complementarity and hydrogen bonding interactions. The successive base stacking geometry in d(CACGTG)(2) is similar to the corresponding steps in d(CG)(3). These results suggest that specific polyamines such as Z-5 could serve as powerful inducers of Z-type conformation in unmodified DNA sequences with AT base pairs. This structure provides a molecular basis for stabilizing AT base pairs incorporated into an alternating d(CG) sequence.     

Conformations of duplex structures formed by oligodeoxynucleotides covalently linked to the intercalator 2-methoxy-6-chloro-9-aminoacridine

A family of covalent complexes between oligonucleotides and derivatives of the intercalating agent 9-amino acridine has been synthesized (Asseline, U., Thuong, N.T. and Helene, C. (1983) C.R.Acad. Sci. (Paris) 297 (III), 369-372) and studied (Lancelot, G., Asseline, U., Thuong, N.T., and Helene, C. (1985) Biochemistry 24, 2521-2529; Lancelot, G., Asseline, U., Thuong, N.T., and Helene, C. (1985) J. Biomol. Str. Dyn. 3, 913-921) with a view to understand nucleic acid-nucleic acid recognition. In order to understand the nature of interactions between the intercalator and the oligonucleotides in such complexes and the sensitivity of such interactions to the polymorphic form of the DNA, we have carried out molecular mechanics simulations on duplex deoxyoligonucleotides d(A)6.d(T)6 (A and B forms) and d(TATC).d(GATA) (B form) covalently bound to 2-methoxy-6-chloro-9-aminoacridine through a pentamethylene linker chain. Structures in which the acridine derivative is end stacked (at the 3' and 5' ends) and in which the dye is intercalated between the terminal base pairs (at both the ends) and between second and third base pairs from the 3' end are all of reasonably low energy in both A and B forms of DNA. Our studies on 3' end complexes find that in the B form, intercalation of the dye between the second and third base pairs is preferred over the other two modes of binding, while in the A form, intercalation between the terminal base pairs is preferred. In the 5' end A and B form complexes, outside stacking and intercalation between the terminal base pairs are preferred, respectively. Our calculations suggest the possibility that the presence of the dye attached covalently to the DNA can induce conformational transitions in the DNA. For example, intercalation of the dye two base pairs from the end could induce an A----B transition.