NMR structural characterisation of a hairpin DNA with two-base loop: solution conformation of d-GGTACIAGTACC.

An inosine-adenosine mismatched base-pair oligonucleotide, d-GGTACIAGTACC has been studied by 1H and 31P NMR spectroscopy. Almost complete 1H and 31P resonance assignments of the oligomer at 0.90 mM concentration and 310 K have been achieved. NMR results demonstrate that the oligomer adopts a hairpin conformation, which has a structure with two purines I6 and A7 forming a two-base loop on a B-DNA stem. Stacking is continued on the 5'-side of the loop, with the I6 stacked upon C5. The base A7, on the 3'-side of the loop stacks partially with I6. All the bases are in anti conformation with respect to their respective sugar moiety.

Evidence for A+(anti)-G(syn) mismatched base-pairing in d-GGTAAGCGTACC.

Two-dimensional NMR spectroscopy has been used to study the structure and hydrogen bonding scheme of A:G mismatched base pairing in d-GGTAAGCGTACC at pH 5.8. Under the conditions of our study, the molecule forms a B-DNA helix, with the mismatched bases in the A+ anti)-G(syn) conformation. The adenosine exists in the protonated form. The NOESY spectrum in 90% H2O + 10% 2H2O has been used to assign all observable imino and amino protons including those involved in the A+(anti)-G(syn) base pair. Both the proton donors in the A:G mismatched inter-base hydrogen bonding are situated on adenosine.

The poly(dT).2poly(dA) triple helix.

A new homopolynucleotide triple helix, (dT)n.2(dA)n, detected by circular dichroism mixing curves, is the product of an endothermic reaction of (dA)n.(dT)n with (dA)n at moderate temperatures and high salt concentrations ([NaCl] > or = 2.6 M): (dA)n.(dT)n + (dA)n<-->(dT)n.2(dA)n. At higher temperatures (dT)n.2(DA)n forms from the (dA)n.(dT)n duplex alone: 3[(dA)n.(dT)n]<-->(dT)n.2(dA)n + (dA)n.2(dT)n. Upon further heating, (dT)n.2(dA)n is converted to the triplex (dA)n.2(dT)n: 2[(dT)n.2(dA)n]<-->(dA)n.2(dT)n + 3(dA)n. (dT)n.2(dA)n forms owing to a favorable entropy change; delta Hm is unfavorable, ranging from 1 to 2.5 kcal mol-1, depending upon [NaCl]. The formation reaction is associated with a negative change in heat capacity. (dT)n.2(dA)n is an extremely weak complex with a free energy of stabilization, delta Go < or = 100 cal mol-1. Tm values of ultraviolet, circular dichroism, and differential scanning calorimetry transition curves for the formation of (dT)n.2(dA)n decrease with increasing [NaCl], reflecting, in part, the net uptake of cations. The values of (dTm/d ln a +/- )(delta Hm/RTm2) can be accounted for in terms of the charge spacing and cylindrical dimensions of the polynucleotides. The ionic strength dependence of this quantity is consistent with interaction of anions with (dAn). High concentrations of the anions Cl-, Br-, and ClO4- decrease the stability of (dT)n.2(dA)n according to the lyotropic series. The highly polarizable anion, ClO4-, entirely prevents the formation of (dT)n.2(dA)n. Phase diagrams of the (dA)n,(dT)n system in solutions of NaCl, NaBr, and NaClO4 are presented. A bonding scheme for (dT)n.2(dA)n is proposed, and implications of this work for Py.Pu.Pu triple helices are discussed.

Structure of d(T)n.d(A)n.d(T)n: the DNA triple helix has B-form geometry with C2'-endo sugar pucker.

The polynucleotide helix d(T)n.d(A)n.d(T)n is the only deoxypolynucleotide triple helix for which a structure has been published, and it is generally assumed as the structural basis for studies of DNA triplexes. The helix has been assigned to an A-form conformation with C3'-endo sugar pucker by Arnott and Selsing [1974; cf. Arnott et al. (1976)]. We show here by infrared spectroscopy in D2O solution that the helix is instead B-form and that the sugar pucker is in the C2'-endo region. Distamycin A, which binds only to B-form and not to A-form helices, binds to the triple helix without displacement of the third strand, as demonstrated by CD spectroscopy and gel electrophoresis. Molecular modeling shows that a stereochemically satisfactory structure can be build using C2'-endo sugars and a displacement of the Watson-Crick base-pair center from the helix axis of 2.5 A. Helical constraints of rise per residue (h = 3.26 A) and residues per turn (n = 12) were taken from fiber diffraction experiments of Arnott and Selsing (1974). The conformational torsion angles are in the standard B-form range, and there are no short contacts. In contrast, we were unable to construct a stereochemically allowed model with A-form geometry and C3'-endo sugars. Arnott et al. (1976) observed that their model had short contacts (e.g., 2.3 A between the phosphate-dependent oxygen on the A strand and O2 in the Hoogsteen-paired thymine strand) which are generally known to be outside the allowed range.(ABSTRACT TRUNCATED AT 250 WORDS)

Thermodynamics of antiparallel hairpin-double helix equilibria in DNA oligonucleotides from equilibrium ultracentrifugation.

Five highly palindromic DNA dodecamers, four of which may form G-A or I-A purine-purine mispairs at either the 5.8 or 6.7 positions, have been studied at sedimentation equilibrium in the analytical ultracentrifuge. Each DNA oligonucleotide forms an equilibrium mixture of ordered antiparallel hairpin and double-stranded helical structures in solutions of 0.1 or 0.5 M NaCl between 5 and 40 degrees C. The dimeric duplex is favored by conditions of high salt and low temperature. The monomer-dimer equilibrium constants vary from 5 x 10(6) to 5 x 10(3) and are unique for each DNA dodecamer. Analysis of the temperature dependence of the equilibrium constants shows that the double helix to hairpin conversion is driven by a positive entropy change and is associated with an endothermic enthalpy change. The mispair substitutions at the 5.8 positions and the IA(6.7) mispair have the greatest tendency toward hairpin formation and exhibit significantly larger entropy changes than the nonmispaired dGGTACGCGTACC parent sequence and the thermodynamically similar GA(6.7) DNA. The consequences of such hairpin-double helix equilibria must be considered in the interpretation of other kinds of experiments carried out on oligonucleotides at different concentrations.

Hairpin formation in the self-complementary dodecamer d-GGTACGCGTACC and derivatives containing GA and IA mispairs.

The dodecamer d-GGTACGCGTACC and four derivatives with GA and IA mispairs in the 6,7 and 5,8 positions have been examined in dilute solution and 0.01-0.1 M sodium chloride. Concentration dependence of Tm, gel electrophoresis, and equilibrium centrifugation indicate that these self-complementary oligomers can form hairpins under the present conditions. Thermal transitions measured in the ultraviolet primarily represent melting of hairpin to coil [cf. Scheffler et al. (1968, 1970)]. The Tm values show little or no depression for 6,7 substitution but rather large depression for 5,8 replacement. We interpret the results to indicate that the 6,7 sequences have two-base loops and five base pair stems and that the 5,8 sequences have four-base loops and four base pair stems. A concurrent theoretical modeling study [Raghunathan et al. (1991) Biochemistry (following paper in this issue)] provides support for this interpretation.

Poly(2-amino-8-methyldeoxyadenylic acid): contrasting effects in deoxy- and ribopolynucleotides of 2-amino and 8-methyl substituents.

Poly(2-amino-8-methyldeoxyadenylic acid) interacts readily with pyrimidine polynucleotides to form double helices only slightly less stable than those in which the purine polymer lacks the 8-Me group. In the ribo series, by contrast, complexes formed with poly(2-amino-8-methyladenylic acid) are very strongly destabilized by the 8-Me group, despite a larger stabilizing effect of the 2-NH2 group in the ribo series. These results are interpreted in terms of a smaller steric interference of the 8-Me group with 2'-CH2 than with 2'-CHOH, leading to a smaller population of syn structures in the deoxy chain and a consequent lower interference with homopolymer duplex formation. UV, circular dichroism (CD), and IR spectra of the new polymer and its complexes are reported and related to structural and energetic characteristics of the molecules. Since direct synthesis of 2-amino-8-methyldeoxyadenosine was not feasible, the corresponding riboside was prepared, the 3'- and 5'-positions were protected with a disilyloxy group, and a 2'-[(imidazol-1-yl)thiocarbonyl] group was introduced. Reduction with tributyltin hydride followed by deprotection gave the nucleoside, which was then converted to the triphosphate by standard methods. The homopolymer was prepared with terminal deoxynucleotidyl transferase.

Conversions of poly(2-aminodeoxyadenylate-5-halodeoxyuridylate) from B to A forms in high salt. An NMR and circular dichroism study.

Proton one- and two-dimensional nuclear Overhauser enhancement (1D and 2D NOE) spectroscopy has been used to demonstrate that poly(d2NH2A-d5IU) and poly(d2NH2A-d5BrU) are converted from the B to the A conformation in high salt, as found previously for poly(d2NH2A-dT) [Borah, B., Cohen, J. S., Howard, F. B., & Miles, H. T. (1985) Biochemistry 24, 7456-7462]. The 2D NOE and 1D NOE spectra exhibit strong base proton (H8,H6)-H3' cross relaxation, suggesting short interproton distances. These results are indicative of a C3'-endo sugar pucker for both purine and pyrimidine residues in an A or closely related structure. The circular dichroism and UV spectra are consistent with the interpretation of an A conformation in high salt.

Poly(d2NH2A-dT): two-dimensional NMR shows a B to A conversion in high salt.

Poly(d2NH2A-dT) forms a structure in high salt that is clearly distinct from the B form present in low salt. Two-dimensional nuclear Overhauser effect (2D NOE) NMR spectra establish that the conformation of the high-salt form is not Z. Correlations of observed cross peaks in the 2D NOE spectra and estimated interproton distances of the common DNA conformations are consistent only with an A form or closely related structure. This interpretation is also consistent with the negative circular dichroic band observed in the high-salt form of poly(d2NH2A-dT) and in A-form ribohomopolymer helices containing 2NH2A.

Poly(2-amino-8-methyladenylic acid). Competing structural and energetic effects of substituents.

The ribopolynucleotide poly(2-amino-8-methyladenylic acid), (r2NH2(8)MeA)n, has been synthesized, and its physical and chemical properties have been examined. The study reveals competing effects on these properties of the 2-NH2 and 8-Me substituents. In marked contrast to the analogous (r8MeA)n, the new polymer readily interacts to form double helixes with complementary pyrimidine polynucleotides. Triple helixes are not formed. The 8-Me group is strongly destabilizing for helix formation (delta Tm approximately 65 degrees C), presumably by favoring a syn conformation, which blocks heteroduplex formation with ribohomopolymers. The 2-NH2 substituent stabilizes helixes in the ribo series by about 30 degrees C in Tm by forming a third interbase hydrogen bond. We suggest that the free energy from the 2-NH2 interaction drives the syn-anti equilibrium to the purine polymer to the anti form present in the double helix. CD spectra of the homopolymers (r2NH2A)n and (r2NH2(8)MeA)n are completely different, reflecting major differences of conformation. The double helixes formed by these polymers with (rT)n and (rBrU)n, on the other hand, have closely similar CD spectra, supporting our proposal of a major change in conformation of (2NH2(8)MeA)n on going from single strand to double helix.

2NH2A X T helices in the ribo- and deoxypolynucleotide series. Structural and energetic consequences of 2NH2A substitution.

Polynucleotide helices formed by the interaction of (d2NH2A)n, (r2NH2A)n, (dT)n, and (rT)n have been prepared and their physical and spectroscopic properties examined. Thermal transitions, dependence of Tm on salt concentration, stoichiometry, phase diagrams, and calculated enthalpies are reported. UV, CD, and IR spectra are reported. All of the deoxy-deoxy helices containing 2NH2A have positive CD first extrema near 290 nm and appear to have B-form structure. All the ribo-ribo or hybrid helices have negative first extrema in this region and appear to have A-form structure. Elevation of Tm by the 2-NH2 group of 2NH2A is much smaller in the deoxy than in the ribo series. We have applied an equation based on the electrostatic theory of Manning [Manning, G.S. (1972) Bopolymers 11, 937-949; Manning, G.S. (1978) Q. Rev. Biophys. 11, 179-246; Record M.T., Anderson, C.F., & Lohman, T.M. (1978) Q. Rev. Biophys. 11, 103-178] to calculate ethalpies of the helix-coil transitions of the complexes reported here. These calculated enthalpies are larger for 2NH2A X T than for A X T helices, but the difference is much smaller in the deoxy than in the ribo series. We attribute these effects on Tm and delta H in the deoxy series to loss of stabilization of the spine of hydration in B-form structures caused by interference of the 2-NH2 group in the minor groove of the helix [Dickerson, R.E., Drew, H.R., Conner, B.N., Wing, R.M., Fratini, A.V., & Kopka, M.L. (1982) Science (Washington, D.C.) 216, 475-485]. Complete phase diagrams for all 2NH2A,T systems and some A,T systems are reported. The diagrams differ widely and can be placed in four groups according to the number of transitions each system possesses.

Poly(8-bromodeoxyadenylic acid): properties of the polymer and contrast with the ribopolynucleotide analogue.

Introduction of the bulky 8-bromo substituent into adenine residues of polynucleotides has strikingly different consequences in the deoxy- and ribopolynucleotide series. Poly(r8BrA) was found in earlier studies to form a very stable double-helical self-structure but not to undergo interaction with potentially complementary polynucleotides. We find that poly(d8BrA), in contrast, does not form an ordered self-structure in 0.1 M Na+ but appears to exist as an electrostatically expanded rigid rod with unusual circular dichroism (CD) properties at very low ionic strength. The deoxy polymer, moreover, readily forms double helices with either deoxy or ribo pyrimidine polynucleotides, studied by UV, CD, and IR spectroscopy. These complexes are destabilized, relative to those formed by poly(dA), possibly because energy is needed to convert the purine residues from a more stable syn to an anti conformation, required for heteroduplex formation. The CD spectrum of (d8BrA)n X (dT)n is similar to that of B DNA. The deoxy-ribo hybrids (d8BrA)n X (rU)n and (d8BrA)n X (rBrU)n have CD spectra resembling those of A DNA or RNA. Unlike other deoxy-deoxy pairs (d8BrA)n X (dBrU)n, however, has a CD spectrum resembling RNA and other helices having the A form.

Poly(d2NH2A-dT): effect of 2-amino substituent on the B to Z transition.

The effect of 2-amino substitution in adenosine (which permits formation of three hydrogen bonds to T) on the B to Z transition was examined in synthetic DNA of alternating purine-pyrimidine sequence. Results of CD, IR, and 31P NMR experiments show that (d2NH2A-dT)n undergoes a cooperative transition to a different helical structure under the same conditions as the B to Z transition of (dG-dC)n.

Copolymers of adenylic and 2-aminoadenylic acids. Effect of progressive changes in hydrogen bonding and stacking on interaction with poly(uridylic acid).

Random copolymers of adenylic acid and 2-aminoadenylic acid form both double and triple helices with poly(uridylic acid) [poly(U)]. Transition temperatures of two-stranded helices increase with 2NH2A content, exhibiting a slight positive departure from linearity and indicating that the contribution to helix stability arising from introduction of the 2-amino group does not significantly depend upon base sequence. We have shown previously that poly(2NH2A) . poly(U) does not undergo a disproportionation reaction (2 leads to 3 transition). Extrapolation from melting curves of 1:1 complexes between A,2NH2A copolymers and poly(U) indicates a Tm for the 2 leads to 3 transition of poly(2NH2A) . poly(U) which is too high to be observable under normal conditions. Addition of an organic solvent (50% ethylene glycol), however, lowers Tm by promoting unstacking of single-stranded poly(2NH2A) sufficiently to permit observation of the disproportionation of poly(2NH2A) . poly(U) for the first time. Transition breadths of 1:1 complexes of A,2NH2A copolymers with poly(U) are greater than those of either of the homopolymer complexes in the middle range of composition (67 and 48% A) but not at 25% A. These results are consistent with previous calculations on the effect of heterogeneity in base-pair stability on DNA transition breadths. In the poly(A), poly(U) system, Et4N+ counterion reduces the Tm of the double and triple helices by 26 and 41 degrees C, respectively. The larger depression in the latter case arises from the higher charge density of the triple helix and less effective counterion screening by Et4N+. In the poly(2NH2A), poly(U) system Tm,2 leads to 1 is reduced by 24 degrees C, but extrapolation of the copolymer results indicates a reduction of approximately 100 degrees C for Tm,3 leads to 2, accounting for previous failure to observe a triple helix in this system. CD spectra of A,2NH2A copolymers suggest that much of the spectral region can be regarded as a contribution of the CD spectra of the parent polymers poly(A) and poly(2NH2A) but that the region from 255 to 275 nm requires that contributions made by longer range interactions be taken into account.