Porphyrin binding to quadrupled T4G4.

We have recently reported the cation-induced self-assembly of DNA oligomers of the general sequence C4T4G4T1-4G4 into high-molecular weight multistranded structures [Marotta, S.P., Tamburri, P.A., and Sheardy, R.D. (1996) Biochemistry 35, 10484-10492]. The architecture of the proposed structure consists of a series of four leafed G4 tetrads tethered together via one or two T1-4 strands and thus resembles a long four-sided hollow tube with periodic "pockets". These pockets possess electrostatic, hydrogen bonding, and hydrophobic contact points and should be ideal candidates for the binding of small molecules. To assess the potential of using porphyrins as probes for these structures, we have investigated the interaction of tetrakis(4-N-methylpyridyl)porphine (H2TMPyP) with the simple quadruplex formed by T4G4 and with the duplex formed by CGCGATATCGCG. Visible absorption, circular dichroism, and fluorescent energy transfer studies indicate that H2TMPyP binds to both the duplex and quadruplex via intercalation at low [porphyrin]/[DNA molecule] ratios, i.e., in the presence of excess potential DNA binding sites. Analyses of Scatchard plots show that H2TMpyP binds with high affinity to both DNA secondary structures but binds to the quadruplex with an affinity 2 times greater than that of the duplex.

Sequence effects on the relative thermodynamic stabilities of B-Z junction-forming DNA oligomeric duplexes.

Circular dichroism (CD) and ultraviolet absorption techniques were employed in characterizing the sequence-dependent thermodynamic stabilities of B-Z junction-forming DNA duplexes. The Watson strand of the duplexes has the general sequence (5meC-G)4-NXYG-ACTG (where N = A or G and XY represents all permutations of pyrimidine bases). Duplexes were generated by mixing stoichiometric amounts of the complementary strands. Circular dichroism studies indicate that each duplex is fully right-handed at low salt (e.g., 115 mM Na+) but undergoes a salt-induced conformational transition to a structure that possesses both left- and right-handed conformations at high salt (4.5 M Na+), and hence a B-Z junction. Optical melting studies of the DNA duplexes at fixed DNA concentration with total Na+ concentration ranging from 15 mM to 5.0 M were determined. A nonlinear dependence of the melting temperature (Tm) on [Na+] was observed. Thermodynamic parameters at Na+ concentrations of 115 mM and 4.5 M with a wide range of DNA concentrations were determined from UV optical melting studies via construction of van't Hoff plots. A change of a single dinucleotide within these duplexes significantly affected the helix stabilities. The experimentally obtained free energies for the duplex to single-strand transitions were in close agreement with predicted values obtained from two different methods.

Assessing the sequence specificity in the binding of Co(III) to DNA via a thermodynamic approach.

The interaction specificities of Co(III) with DNA were investigated via consideration of thermodynamic characteristics of the duplex to single strand transition for DNA oligomers incubated in the presence of [Co(NH3)5(OH2)](ClO4)3. It has previously been demonstrated that incubation of the DNA oligomer [(5medC-dG)4]2 with this cobalt complex leads to coordination of the cobalt center to the DNA, presumably at N7 of guanine bases [D. C. Calderone, E. J. Mantilla, M. Hicks, D. H. Huchital, W. R. Murphy, Jr. and R. D. Sheardy, (1995) Biochemistry 34, 13841]. In this report, DNA oligomers of different sequence were incubated with [Co(NH3)5(OH2)](ClO4)3 via protocols developed previously and the treated oligomers were subjected to thermal denaturation for comparison to the untreated oligomers. The DNA oligomers were designed in order to investigate the sequence specificity, if any, in the reaction of the cobalt complex with DNA. The values of Tm, delta HvH, and delta n (the differential ion binding term) obtained from the thermal denaturations were used to assess the sequence specificity of the interaction. For all oligomers, treated or untreated, Tm and delta HvH vary linearly with log [Na+] and hence the value of delta n is a function of the Na+ concentration. The results indicate no significant reaction between the cobalt complex and oligomers possessing isolated -GA- or -CG- sites; however, the thermodynamic characteristics of DNA oligomers possessing either an isolated -GG- site or an isolated -GC- site were altered by the treatment. Atomic absorption studies of the treated oligomers demonstrate that only the DNA oligomers possessing isolated -GG- or -GC- sites bind cobalt. Hence, the changes in the thermodynamic properties of these oligomers are a result of cobalt binding with a remarkable sequence specificity.

Effect of base pair A/C and G/T mismatches on the thermal stabilities of DNA oligomers that form B-Z junctions.

The thermal stabilities and structures of B-Z junction forming DNA duplexes possessing A/C or G/T base pair mismatches were compared to those of corresponding duplexes possessing perfect matched base pairs. The upper strands of the duplexes have a generalized sequence 5'-(5meCG)-LMN-GACTG-3', where L stands for A or G while M and N are permutations of pyrimidines. The lower strands were either complementary or were such as to create an A/C or G/T mismatch at the position corresponding to L, M, or N. Optical melting and circular dichroism studies were used to investigate the thermal stabilities and structures of both the mismatched base pair and the perfect matched base pair duplexes. Incorporating mismatched A/C or G/T base pairs did not noticeably affect the conformations of the duplexes in 115 mM Na+ but resulted in perturbed B-Z conformations at 4.5 M Na+. For any mismatched base pair duplex, the B-DNA domain of the hybrid B-Z structure formed at 4.5 M Na+ is significantly perturbed while the Z-DNA domain is less perturbed by the presence of the mismatched base pairs. The presence of a mismatch destabilizes a duplex relative to the perfect matched base pair duplex by 1.7-10.0 kcal/mol depending upon position of the mismatch, type of mismatch base pair involved, and Na+ concentration. The thermodynamic destabilization of a mismatched base pair duplex relative to the perfect matched base pair duplex arises from perturbations in nearest neighbor interactions and hydrogen bonding. In general, we observed that the incorporation of an A/C or G/T base pair mismatch in place of a perfect matched base pair at or near a B-Z junction results in a relatively large change in enthalpy and entropy to produce a significant change in the free energy of the duplex to single strand transition. At 4.5 M Na+, where the duplexes possess perturbed B-Z junctions, the farther away from the junction that the mismatch is, the greater the extent of the destabilization.

Conformational properties of Z-forming DNA oligomers bearing terminal unpaired bases.

Three sets of semi-self-complementary deoxyribonucleotide decamers with the sequence XX-(5meCG)4, (5meCG)4-XX, or Y-(5meCG)4-Y, where XX = AA, CC, GG, or TT and Y = A, C, G, or T, were synthesized along with the self-complementary octamer (5meCG)4. The 8-mer duplex readily undergoes a B-to-Z conformational conversion upon increasing the NaCl concentration with a transitional midpoint of approximately 1.1 M NaCl. The 10-mers should form 8-bp duplexes a with core sequence of [(5meCG)4]2 with 5'-XX overhangs, 3'-XX overhangs, or 5',3'-Y/Y mismatches. Circular dichroism was employed to determine the conformations of all oligomers. Salt titrations were performed to measure the effect of overhangs and terminal mismatches on the B-to-Z conversion. In general, the presence of 5'-XX overhangs results in a transition midpoint equal to or slightly higher than the control, whereas the presence of 3'-XX overhangs results in a transition midpoint slightly lower than the control. The 3'-CC and 5'-GG overhangs are exceptions, with transition midpoints much higher than the control. These oligomers apparently form duplexes with 5',3'-C/C or 5',3'-G/G mismatches abutting a [(G5meC)4]2 duplex core. The presence of terminal mismatches in the third set of oligomers results in transition midpoints higher than the control. Ultraviolet absorbance methods were used to evaluate the effect of the various stacking motifs of the 10-mers on the thermodynamics of melting relative to the 8-mer for both B and Z conformations. We found that in both the B and Z conformations, the presence of an overhang stabilizes the [(5meCG)4]2 duplex, with the 5' overhangs having a greater stabilizing effect relative to the 3' overhangs. The presence of 5',3'-Y/Y mismatches also imparts a stabilizing effect on the control 8-mer in both the B and Z conformations. These results are discussed in terms of stacking interactions of the terminal unpaired bases.

Sequence and environmental effects on the self-assembly of DNA oligomers possessing GxT2Gy segments.

It is well-known that DNA oligomers possessing contiguous guanine bases can assume non-Watson-Crick type structures through the formation of four-stranded species. The architecture of these four-stranded structures is highly dependent upon the sequence of the DNA and the conditions (e.g., buffer, pH, ionic strength, cations present, and temperature) under which the DNA is prepared. This lab has previously reported the self-assembly of DNA oligomers of sequence C4T4G4T1-4G4 into multistranded high molecular weight species [Dai, T.-Y., Marotta, S. P., & Sheardy, R. D. (1995) Biochemistry 34, 3655-3662]. In order to further investigate the sequence and environmental effects on the self-assembly of DNA oligomers possessing GxT2Gy (where x = 1, 3, or 4 and y = 2-5) segments, the synthesis of a number of such oligomers was undertaken. DNA samples were prepared in standard phosphate buffer (10 mM phosphate, pH 7.0) and NaCl, KCl and/or MgCl2 added to different concentrations in order to evaluate the influence of the cations and their concentrations on the self-assembly of the DNA oligomers. The self-assembly of these oligomers was monitored by nondenaturing polyacrylamide gel electrophoresis and circular dichroism studies. Electrophoresis of the oligomers in either 100 mM K+ or 50 mM Na+ with 50 mM K+ indicated the formation of one or two molecular species for these oligomers. In contrast, electrophoresis of these oligomers in the presence of both 100 mM K+ and 20 mM Mg2+ give a ladder of multiple bands of high molecular weight indicative of multistanded DNA structure formation. The results presented here indicate that self-assembly into high molecular weight species is favored by the presence of Mg2+ as well as the presence of four or more bases in the terminal Gy segment. These results also suggest that the structure of telomeric DNA, which possesses similar sequences, may be quite unusual.

Binding of Co(III) to a DNA oligomer via reaction of [Co(NH3)5(OH2)]3+ with (5medC-dG)4.

The interaction specificities of cobalt(III) ammines with the self-complementary eight-base pair DNA oligomer (5medC-dG)4 have been investigated. Standard protocol for preparing DNA samples calls for heat annealing the DNA oligomer in phosphate buffer in the absence or presence of cobalt(III) ammine complex for 2 min at 80 degrees C, followed by slow cooling to 25 degrees C. An alternative method for DNA preparation is incubation of the oligomer in the presence of the cobalt(III) complex at 37 degrees C followed by exhaustive dialysis. The conformational properties of the thus-treated DNA oligomer were determined by inspection of the UV and CD spectra at 25 and 95 degrees C and thermal denaturation studies. With heat annealing in the absence of any cobalt(III) complex, (5medC-dG)4 assumes a double-stranded, right-handed B conformation at 25 degrees C. Upon heat annealing in the presence of 200 microM [Co(NH3)6]3+, (5medC-dG)4 assumes a double-stranded, left-handed Z conformation at 25 degrees C. In contrast, the CD and UV spectra of (5medC-dG)4 heat annealed in the presence of 200 microM [Co(NH3)5(OH2)]3+ are consistent with a distorted B-like conformation at 25 degrees C. Incubation of the oligomer in the presence of [Co(NH3)5(OH2)]3+ results in modification of the conformational properties of the oligomer at both 25 and 95 degrees C relative to the untreated oligomer. The extent of modification depends upon the incubation concentration of [Co(NH3)5-(OH2)]3+ and the reaction time. Atomic absorption (AA) analyses of these treated DNA samples indicate a high degree of cobalt association to the oligomer.(ABSTRACT TRUNCATED AT 250 WORDS)

Self-assembly of DNA oligomers into high molecular weight species.

Thermal denaturation, gel electrophoresis, and circular dichroism methods were used to characterize DNA oligomers possessing one or two segments of four contiguous G bases in order to investigate their environmentally dependent conformational properties. The sequences of the oligomers studied were the following: HP1-T series, C4T4G4T5-8; HP1-TG series, C4T4G4T1-4G4. In NaCl at concentrations up to 200 mM, the melting profiles of these oligomers are characterized by single inflection points whose Tm values are independent of DNA concentration. In addition, these oligomers run as single bands in polyacrylamide gels under those same conditions as well as in 100 mM K+ or 20 mM Mg2+. These data suggest that these oligomers exist as intramolecular hairpins comprised of four G:C base pairs in the stems, loops of four T bases, and 3'-overhangs of T5-8 or T1-4G4. In the presence of 100 mM K+ plus 20 mM Mg2+, however, gel electrophoresis indicates that oligomers of the HP1-T series exist as equilibria between parent hairpins and four-stranded structures (i.e., quadraplexes). Quadraplex formation for any member of the HP1-T series requires unfolding of the hairpin, exposing the G4 segment prior to quadraplexation. Members of the HP1-TG series self-assemble into multistranded species of high molecular weight in the presence of 100 mM K+ plus 20 mM Mg2+. For this series of oligomers, the data suggest that these higher order species arise from successive additions of parent oligomer to an initially formed quadraplex.(ABSTRACT TRUNCATED AT 250 WORDS)

A thermodynamic investigation of the melting of B-Z junction forming DNA oligomers.

Ultraviolet absorbance methods were used to characterize the thermodynamics of melting of a series of 16 bp deoxyoligonucleotides over a wide range of NaCl concentrations (0-4.5 M) and to obtain complete thermodynamic profiles for their melting at 0.115 and 4.5 M NaCl. The sequence of the series (one strand of duplex) was: 5'-CGCGCGCGAMNGACTG-3', where C indicates m5dC and -MN- was varied to include all combinations of Py:Py stacks (CC, TT, CT, TC). The unmethylated deoxyoligonucleotide 5'-CGCGCGCGACTGACTG-3' was used as a control sequence. All of the methylated oligonucleotides studied undergo a NaCl-induced transition to a hybrid form containing a left-handed, Z-DNA, region joined to a right-handed region by a B-Z junction. Our experiments allowed us to quantitatively evaluate the effects of NaCl, sequence, and methylation and the transition to the hybrid BZ structure on DNA thermal stability. We found that alteration of a single dinucleotide step has profound effects on the thermal stabilities of the 16 bp fragments studied. Methylation was found to destabilize the double helix, resulting in a decrease in Tm. Transition to the hybrid BZ structure, somewhat surprisingly, was found to only slightly destabilize DNA, with an observed decrease in free energy of melting of approximately 0.5 kcal/mol relative to the control, right-handed, sequence in high salt. Transition melting temperatures (Tm) were found, in agreement with previous studies on polymeric DNA, to depend upon NaCl concentration in a complicated, nonlinear fashion. m values increase to maximal values at circa 1.0 M NaCl, but decrease thereafter with further addition of salt.(ABSTRACT TRUNCATED AT 250 WORDS)

Sequence dependence of the free energy of B-Z junction formation in deoxyoligonucleotides.

The NaCl-induced transition from the right-handed B form to a hybrid form containing both left and right-handed DNA, joined by a B-Z junction, was investigated. Transition curves were constructed from circular dichroism spectra collected as a function of NaCl concentration for a series of 16 bp deoxyoligonucleotides. The sequence of the series (one strand of the duplex) was: 5'CGCGCGCGAMNGACTG, where C indicates m5dC, and -MN- was varied to include the possible Py:Py stacks: -CC-, -CT-, -TC- and -TT-. Transition curves for the conversion of all deoxyoligonucleotides were found to be biphasic. Singular value decomposition was used to analyze the experimental circular dichroism spectra obtained as a function of NaCl, and showed that the transition was not a simple two-state process, but rather required at least three species to account for the experimental data. A sequential three-state model, B<-->I<-->BZ, was derived and applied to analyze experimental transition curves. Non-linear least-squares analysis was used to evaluate the salt-dependent equilibrium constants for each step in the sequential reaction model. The results indicate that the free energy change for B-Z junction formation (delta Gj) depends on the dinucleotide sequence near the junction. At a Na activity of 5, delta Gj values ranging from +1.2 to +1.7 kcal mol-1 were determined, depending on the sequence near the junction. delta Gj was found to be strongly dependent on salt concentration, with its magnitude decreasing with increasing Na activity. In addition to studies on linear duplex molecules, the B to BZ transition was also investigated in "dumbbell" forms of selected sequences. In these molecules, the ends were covalently linked by a single-stranded T4 segment. These studies show that junction formation is energetically more costly in dumbbells than in their linear counterparts. A striking correlation was found between delta Gj and two independent conformational properties of the dinucleotide steps that were introduced into the linear duplex molecules. These are the free energy of unstacking and the estimated free energy change for the conversion of the dinucleotide from the B to the A conformation. The more stable against unstacking, or the more resistant to the B to A conversion, the sequence near the junction site is, the more costly is B-Z junction formation. These correlations reveal that the DNA sequence near the junction apparently must be pliable in order to accommodate the unusual structure of the junction.(ABSTRACT TRUNCATED AT 400 WORDS)

The interface between an alternating CG motif and a random sequence motif displays altered nuclease activity.

Previously we described the B-Z junctions produced in oligomers containing (5meCG)4 segments in the presence of 5.0 M NaCl or 50 uM Co(NH3)6+3 [Sheardy, R.D. & Winkle, S.A., Biochemistry 28, 720-725 (1989); Winkle, S.A., Aloyo, M.C., Morales, N., Zambrano, T.Y. & Sheardy, R.D., Biochemistry 30, 10601-10606 (1991)]. The circular dichroism spectra of an analogous unmethylated oligomer containing (CG)4, termed BZ-IV, in 5.0 M NaCl and in 50 uM Co(NH3)+3 suggest, however, that this oligomer does not form a B-Z hybrid. BZ-IV possesses Hha I sites (CGCG) in the (CG)4 segment and an Mbo I site (GATC) at the terminus of the (CG)4 segment. BZ-IV is equally digestible in the presence and absence of cobalt hexamine by Hha I, further indicating that the structure of BZ-IV is fully B-like under these conditions. The Mbo I cleavage site at the juncture between the (CG)4 segment and the adjacent random segment displays enhanced cleavage by both Mbo I and its isoschizomer Sau3AI in the presence of cobalt hexamine. In addition, exonuclease III digestion of BZ-IV is inhibited at this juncture. Actinomycin inhibits Mbo I activity in the presence of cobalt hexamine but not in the absence. Together, these results suggest that enzymes recognize the interfaces of (CG)n and adjacent random sequences as altered substrates even in the absence of a B-Z junction formation.

Conformational properties of B-Z junctions in DNA.

The structural consequences of specific base sequences in DNA can exert a strong influence on the function of DNA. It has previously been reported that the presence of multiple B-Z conformational junctions in constructed DNA oligomers results in unusually enhanced electrophoretic gel mobilities of these oligomers [Winkle, S. A., & Sheardy, R. D. (1990) Biochemistry 29, 6514-6521]. In order to investigate this phenomenon further, we designed and synthesized several DNA oligomers capable of pure Z or B-Z junction formation for polyacrylamide gel electrophoresis studies. The results indicate that both pure Z-DNA and polymorphic B-Z-DNA oligomers exhibit unusual gel migratory properties. The results of gel mobility studies in the absence and presence of cobalt hexamine indicate that a B-Z junction corresponds to a stiff bend of the helix axis, with two or more conformers accessible at the junction site. This is a different bend and mechanism than that in oligo(A) tracts.

Drug binding to a DNA BZ molecule: analysis by chemical footprinting.

The polymorphism in a DNA 16-mer (designated BZ-II) has been investigated by means of circular dichroism (CD) spectroscopy and chemical footprinting. CD spectra indicate that, in low salt, the oligomer is fully right-handed whereas, in high salt, it possesses a B-Z conformational junction: half of the duplex is right-handed while the other half is left-handed. Treatment of BZ-II with diethyl pyrocarbonate (DEPC) confirms the existence of a left-handed segment of the duplex in high salt: enhanced DEPC scission occurs at the G residues in the alternating CG sequence. The scission patterns of the upper and lower strands in BZ-II by the reactive chemical probe MPE.Fe(II), and the antitumor antibiotics dynemicin and Fe-(II).bleomycin, are different under low salt conditions. The 3'-terminal region of both upper and lower strands and the middle region of the upper strand of BZ-II are preferential cleavage sites in low salt. This result suggests that the methylated cytosines or the alternating CG domain in the molecule perturbs the DNA structure. Under high salt conditions, the reactivity of the Z-DNA segment of BZ-II for MPE.Fe(II) and Fe(II).bleomycin is dramatically enhanced, while it is reduced in the case of dynemicin. Excess propidium (PI) eliminates preferential cleavage by each of these chemical probes in high salt conditions. This is due in part to conversion of the BZ-DNA molecule into B-DNA, as is seen by a DEPC modification experiment.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced reactivity of a B-Z junction for cleavage by the restriction enzyme MboI.

We have been investigating the structure, dynamics, and ligand-binding properties of the interface that exists between a right-handed conformation and a left-handed conformation (i.e., a B-Z junction) in synthetic DNA oligomers. Since exo- and endonuclease activity is known to be sensitive to the conformation of the template DNA, we have designed and synthesized a DNA oligonucleotide of 20 base pairs (designated as BZ-III) with an MboI recognition site (GATC) at the location of a potential B-Z junction. The activity of the MboI enzyme toward this molecule and DNA oligomers that contain multiple MboI sites located at B-Z junctions was monitored in the absence and presence of the Z-conformation-inducing reagent cobalt hexaammine. In all cases, the activity of the enzyme was enhanced in the presence of cobalt hexaammine. The activity of MboI toward BZ-III, in the presence and absence of cobalt hexaammine, was also examined when the DNA oligomer is also in the presence of the DNA binding drugs actinomycin D, ametantrone, or ethidium bromide. In all cases, the activity of the enzyme was inhibited in the presence of drug. The results suggest that B-Z junctions are structurally unique and that this uniqueness may alter nuclease activity at sites in or near the junction.

Unusual binding of ethidium to a deoxyoligonucleotide containing a B-Z junction.

Equilibrium binding studies are reported that describe the interaction of the simple intercalator ethidium with a hexadecadeoxynucleotide (BZ-1) that contains a stable B-Z junction. The transition of BZ-1 from its low-salt right-handed form to its high-salt hybrid form containing the junction between left- and right-handed DNA was found to greatly facilitate ethidium binding. Ethidium binding to all conformational forms of BZ-1 was found to be complex and highly cooperative, a result that indicates an unusually robust conformational mobility in the molecule. Binding results were quantitatively analyzed by using an allosteric model that provides a description of energetics of the conformational transitions in BZ-1, in addition to estimates of the binding constants for ethidium binding to the different conformations. These results provide a quantitative description of a structurally specific intercalator-DNA interaction.

Anomalous gel migration of DNA oligomers containing multiple conformational junctions.

We have previously shown that a short 16 base pair DNA oligomer can accommodate a B-Z conformational junction [Sheardy, R. D., & Winkle, S. A. (1989) Biochemistry 28, 720-725]. Results from 1H NMR studies indicated that only three base pairs were involved in the junction and that one of these base pairs was highly distorted. Being interested in the nature of this distortion, we constructed DNA oligomers which have the potential to contain multiple B-Z junctions for polyacrylamide electrophoretic studies. We report that the mobilities displayed by these molecules through acrylamide gels in the absence and presence of cobalt suggest that these molecules run shorter than they actually are. This anomalous migration may be due to structural/dynamic properties of the DNA helix manifested by the periodic distortions of the potential B-Z junctions.

Energetics of B-Z junction formation in a sixteen base-pair duplex DNA.

We report analysis of the NaCl-induced B-Z transition in a 16 base-pair duplex DNA with sequence designed such that when NaCl is increased the left half of the molecule undergoes the B-Z transition while the right half remains in the B-form. An equilibrium thermodynamic model based on the body of available published experimental data and the recent theoretical work of Soumpasis, which indicate, in the salt range above approximately 0.9 M-NaCl, the transition free-energy of B-Z conversion in DNA is a linear function of the NaCl concentration, is employed. Analysis of the B-Z transition of the junction-containing molecule indicates the B-Z junction formed in this 16 base-pair DNA is composed of approximately three base-pairs and has a free energy of formation of approximately 4.7 kcal/mol junction. These values for the junction are in excellent agreement with published estimates of B-Z junction size and energy derived from much longer DNA pieces.

Temperature-dependent CD and NMR studies on a synthetic oligonucleotide containing a B-Z junction at high salt.

It is now accepted that two or more conformations may exist within the same DNA molecule, thereby generating conformational junctions. The presence of B-Z junctions between right- and left-handed DNA conformations has been detected in plasmids by a number of techniques. Preliminary characterization of the first example of a B-Z junction is a short DNA oligonucleotide has recently been reported [Sheardy, R. D. (1988) Nucleic Acids Res. 16, 1153-1167]. We report additional CD and NMR data that support the existence of the junction in this model oligomer. These studies indicate that only three base pairs are involved in the junction and only one of these is dramatically distorted. Furthermore, the NMR saturation-transfer experiments suggest the junction's internal motion is temperature dependent.

Preliminary spectroscopic characterization of a synthetic DNA oligomer containing a B-Z junction at high salt.

It is well known that the local conformation of a segment of DNA is dependent upon both the sequence of the segment and the conditions under which the DNA is prepared. In extreme cases, the DNA may contain regions of both right and left-handed conformations, mandating the existence of a conformational junction between the two. These B-Z junctions have been observed in plasmids but, to date, no model systems have been characterized to determine the molecular nature of these junctions. Preliminary CD, UV, and NMR studies on such a model are presented here. A 16 base pair oligonucleotide, containing a potential B-Z junction, has been synthesized and characterized by the above techniques. The results suggest that this molecule contains both right and left-handed conformations under condition of high salt, and thus a B-Z junction.