Conformation of double-stranded polydeoxynucleotides in solution by proton two-dimensional nuclear Overhauser enhancement spectroscopy

Proton 2D-NOE spectroscopy has been used to investigate the three-dimensional conformations of several sonicated polydeoxynucleotides in solution. The observed pattern of cross peaks indicate that poly(dA-dT) · poly(dA-dT) in all salt concentrations studied (up to 6.6M CsF), and poly(dG-m⁵dC) · poly(dG-m⁵dC) in low salt (0.1M NaCl) are righthanded B-structures. Poly(dG-m⁵dC) · poly(dG-m⁵dC) in Mg²⁺ (3 mM) solution exhibits a pattern characteristic of the left-handed Z-form. These results for poly(dA-dT) · poly(dA-dT) are in contrast to suggestions that this copolymer exists as a left-handed form, either in low or high salt. We present pure absorption-mode 2D-NOE spectra that enable us to compare several distances and define the conformations of these polydeoxynucleotides in solution.     

Cyclic pentapeptides as models for reverse turns: determination of the equilibrium distribution between type I and type II conformations of Pro-Asn and Pro-Ala beta-turns

Cyclic pentapeptides are excellent models for reverse turns and have been used extensively in our laboratory to explore the influence of different amino acid sequences on turn preference. This paper is divided into two parts: In the first, we review our previous studies of cyclic pentapeptides. We summarize work that demonstrates the range of conformations possible within the cyclic pentapeptide backbone, the importance of sequence chirality in determining the backbone fold, and the utility of these cyclic pentapeptides as models for various turns. In the second, we present new results on two cyclic pentapeptides that contain beta-turns with Pro-Ala or Pro-Asn sequences in the i + 1 and i + 2 positions. By stereochemical criteria, a type I beta-turn is expected to be preferred by such L-L sequences. On the other hand, in proteins Asn occurs frequently in the i + 2 position of type II turns. We asked whether the same propensity would be manifest in an isolated model peptide, and if so, what the interactions were that influenced the relative stability of the type I and type II turns. To address these questions we have compared the conformational behavior of two peptides: cyclo(Gly-Pro-Ala-D-Phe-Pro) and cyclo(D-Ala-Pro-Asn-Gly-Pro). From previous studies, we anticipated that both peptides would contain an inverse gamma-turn and a beta-turn which consisted of either Gly-Pro-Ala-D-Phe or D-Ala-Pro-Asn-Gly in positions i to i + 3, respectively. Nuclear magnetic resonance analysis confirms this overall backbone conformation. Furthermore, quantitative nuclear Overhauser effect measurements in combination with molecular dynamics simulations and torsionally-forced energy minimizations have enabled us to determine that both type I and type II beta-turns are present in equilibrium in these peptides. The introduction of Asn in position i + 2 shifts this equilibrium significantly towards type II. We have done preliminary assessment of the possible side-chain/backbone conformations that contribute to the shift in populations.     

Structure investigation of amphiphilic cyclopeptides in isotropic and anisotropic environments-A model study simulating peptide-membrane interactions.

It has been proposed that the membrane allows a much more efficient binding of certain small or medium-sized amphiphilic messenger molecules to their receptor, not only by accumulation of the drug, but also by induction of orientations and conformations that are much more favorable for receptor docking than structures adopted in isotropic phases. A series of eight amphiphilic cyclic peptides containing lipophilic (L-alpha-aminodecanoic acid = Ada, L-alpha-aminohexadecanoic acid = Ahd, Nhdg = N-hexadecylglycine) and hydrophilic (Lys, Asp) amino acids were synthesized and examined by means of NMR spectroscopy and molecular dynamics (MD) simulations in isotropic (CDCl3) and membrane-mimicking anisotropic (SDS/H2O) solvents to study the influence of the environment on their individual conformations. NMR data of cyclo(-Gly1-D-Asp2-Ahd3-Ahd4-Asp5-Gly6+ ++-) (C4), cyclo(-Lys1-D-Pro2-Lys3-Ada4-Pro5-Ada6-) (C5) and cyclo(-Lys1-Pro2-Lys3-Ada4-D-Pro5-Ada6-) (C6) clearly indicate that those compounds are too rigid to perform a conformational change upon transition from an isotropic to an anisotropic environment. On the other hand, the experimental data of cyclo (-Gly1-Asp2-Ahd3-Ahd4-Asp5-Gly6-) (C1), cyclo(-Asp1-Ala2-Nhdg3-Ala4-D-Asp5-) (C7), and cyclo(-D-Asp1-Ala2-Nhdg3-Ala4-Asp5-) (C8) suggest highly flexible unstructured molecules in both environments. However, for cyclo(-Asp1-Asp2-Gly3-Ahd4-Ahd5-Gly6-) (C2) we observed a structure inducing effect of a membrane-like environment. The compound populates three different conformations in SDS/H2O, whereas in CDCI3 no preferred conformation can be detected. cyclo(-D-Asp1-Asp2-Gly3-Ahd4-Ahd5-Gly6-) (C3) clearly exhibits two different conformations with a shifted beta,beta-turn motif in CDCI3 and SDS/H2O solutions. The conformational change could be reproduced in a restraint-free MD simulation using the biphasic membrane mimetic CCl4/H2O. Our results give clear evidence that membrane interactions may not only lead to structure inductions, but can also induce major conformational changes in compounds already exhibiting a defined structure in isotropic solution.     

Cyclic hexapeptides bearing carboxyl groups. Interaction with metal ions and lipid membrane

Three cyclic hexapeptides bearing carboxyl groups, cyclo(L-Asp-L-Phe-L-Pro)2, cyclo(L-Aad-L-Phe-L-Pro)2 (Aad represents alpha-amino adipic acid residue), and cyclo(D-Asp-D-Phe-L-Pro)2 were synthesized and investigated on conformation, complexation with metal ions, and interaction with lipid membrane. These cyclic hexapeptides were found to take several different conformations, though their reference compounds, cyclo(L-Leu-L-Phe-L-Pro)2 and cyclo[D-Asp(OMe)-D-Phe-L-Pro]2, took a single C2 symmetric conformation. Cyclo(D-Asp-D-Phe-L-Pro)2 formed complexes with Ba2+ and Ca2+, whereas cyclo(L-Asp-L-Phe-L-Pro)2 and cyclo(L-Aad-L-Phe-L-Pro)2 did not. The latter two take amphiphilic structures and were distributed to lipid membrane more easily than cyclo(D-Asp-D-Phe-L-Pro)2. Cyclo(D-Asp-L-Phe-L-Pro)2 binds Ca2+ on the lipid membrane.     

Conformation of a neurokinin antagonist in solution. 2D NMR and restrained molecular dynamics study.

The hexapeptide [cyclo(Leu1 psi(CH2NH2)Leu2-Gln3-Trp4-Phe5-Gly6)]+1 is a potent antagonist of neurokinin A activity in tissues of hamster urinary bladder. The solution conformation of this cyclic hexapeptide has been characterized by the combined use of two dimensional nuclear magnetic resonance spectroscopy and restrained molecular dynamics. The proton spectrum of the peptide was fully assigned by the sequential assignment procedure. Interproton distances were derived from crosspeak volumes in two dimensional Nuclear Overhauser Effect spectra, and dihedral angles were calculated from appropriate coupling constants. Temperature coefficients of the amide protons were determined. Restrained molecular dynamics simulations were carried out using the backbone interproton distances as constraints. During 210 ps of restrained molecular dynamics the peptide interconverted among three closely related families of conformations. These interconversions occurred at picosecond timescales under the simulation conditions.     

Structural characterization of manganese(II)-nucleotide complexes bound to yeast 3-phosphoglycerate kinase: 13C relaxation measurements using [U-13C]ATP and [U-13C]ADP

A complete characterization of the conformations of Mn.ADP and Mn.ATP bound to the active site of yeast 3-P-glycerate kinase is presented. These conformations have been deduced on the basis of paramagnetic effects on 13C spin-lattice relaxation rates in [U-13C]nucleotides due to Mn(II), used as a substituent activating cation. The 13C relaxation measurements were performed on exclusively enzyme-bound complexes E.Mn.[U-13C]ATP and E.Mn.[U-13C]ADP at three distinct 13C NMR frequencies: 75.4, 125.7, and 181 MHz. The frequency dependence of the relaxation data has been analyzed in an effort to evaluate distances from the cation for all 10 13C nuclei in the adenosine moieties of E.Mn.ATP and E.Mn.ADP. These distance data, taken along with previously published cation-31P distances, have been used as constraints in the molecular modeling program Quanta, in which molecular dynamics simulations and energy minimization have been performed to determine the conformations that are compatible with the distance data. It was possible to model the distances on the basis of a single enzyme-bound conformation for each of the nucleotides. The details of the enzyme-bound Mn.ATP and Mn.ADP conformations are distinguishably different from each other, indicating that structural alterations occur in the enzyme-bound reaction complex as the enzyme turns over. For example, when the adenosine moieties in the bound structures of Mn.ATP and Mn.ADP are superposed, the cation is found to be displaced by approximately 2.4 A between the two conformations, suggesting that these structural changes may involve movements with significant amplitudes. Furthermore, the NMR-determined structures of enzyme-bound Mn.ATP and Mn.ADP are significantly different from those in published X-ray crystal structures of the enzyme-nucleotide complexes.     

A 1H-NMR study of the solution conformation of cyclo(GRGDSPA): conformational effects on the physiological activity.

Solution conformations of cyclo(GRGDSPA) have been analyzed by the use of two-dimensional proton nuclear magnetic resonance spectroscopy and the dynamical simulated annealing calculation. It has been shown that the RGDS segment in cyclo(GRGDSPA) takes a beta-turn conformation. We have concluded that this beta-turn conformation is essential for the physiological activity of cyclo(GRGDSPA).     

Solution-phase synthesis of Aib-containing cyclic hexapeptides

The 18-membered Aib-containing cyclohexapeptides, cyclo(-Gly-Aib-Aib-Gly-Aib-Phe-) (22), cyclo(-Gly-Aib-Phe(2Me)-Gly-Aib-Aib-) (24a), cyclo(-Gly-Phe(2Me)-Aib-Gly-Aib-Aib-) (24b), and cyclo(-Gly-Phe(2Me)-Aib-Gly-Aib-Phe-) (25), have efficiently been synthesized by solution-phase techniques. The linear precursors 1a-1d were prepared by combining the 'azirine/oxazolone method' for incorporation of alpha,alpha-disubstituted alpha-amino acids (Aib, Phe(2Me)) into the peptide chains by classical peptide coupling methods for segment condensations. Deprotection of the amino and carboxy termini of 1a-1d, followed by cyclization with DEPC as the coupling reagent, gave the above-mentioned cyclic hexapeptides 22, 24a, 24b, and 25 in good yields (26-57%). The solid-state conformations of the linear hexapeptides 1d, 16 and 27, and of the cyclohexapeptides 22 and 25 have been established by X-ray crystallography.     

Conformations of cyclo(L-orD-Phe-L-Pro-Aca) and cyclo(L-Pro-L- or D-Phe-Aca). Cyclized dipeptide models for specific types of beta-bends

Conformational analyses on four cyclic model peptides of the beta-bend, cyclo(L- or D-Phe-L-Pro-epsilon-aminocaproyl(Aca] and cyclo(L-Pro-L- or D-Phe-Aca), were carried out both experimentally and theoretically. Cyclo(D-Phe-L-Pro-Aca) was shown to exist as a single conformer taking the type II' beta-bend. The comparison of its CD spectra with those of cyclo(L-Ala-L-Ala-Aca) revealed that type I and II' beta-bends, both with alpha-helix-like CD spectra, can be distinguished. Cyclo(L-Phe-L-Pro-Aca) was shown to exist as a single conformer with a cis L-Phe-L-Pro peptide bond, taking the type VI beta-bend. Its CD spectrum has thus been observed for the first time for the bend containing a cis peptide bond. Cyclo(L-Pro-L-Phe-Aca) was shown to exist as a mixture of two conformers, the major one taking the type I beta-bend with a trans Aca-L-Pro peptide bond and the minor one with a cis Aca-L-Pro peptide bond. Cyclo(L-Pro-D-Phe-Aca) was suggested to exist as a mixture of two conformers, the major one taking the type II beta-bend with a trans Aca-L-Pro peptide bond and the minor one with a cis Aca-L-Pro peptide bond.     

Mimicry of beta II'-turns of proteins in cyclic pentapeptides with one and without D-amino acids.

The solution structure of eight cyclic pentapeptides has been determined by two-dimensional 1H-NMR spectroscopy combined with spectra simulations and restrained molecular dynamic simulations. Six of the cyclic pentapeptides were derived from the C-terminal cholecystokinin fragment CCK-4 enlarged with Asp1 resulting in the sequence (Asp-Trp-Met-Asp-Phe), one L-amino acid after the other was substituted by its D-analog. In addition, two peptides, including an all-L-amino-acid-containing cyclic pentapeptide, cyclo(Asp-Phe-Lys-Ala-Thr) and cyclo(Asp-Phe-Lys-Ala-D-Thr) were investigated. All D-amino-acid-containing peptides show beta II'-turn conformations with the D-amino acid in the i + 1 position, excepting the D-aspartic-acid-containing peptides. These two peptides are characterized by the lack of beta-turns at pH values less than 4, suggesting that D-aspartic acid in the full-protonized state avoids the formation of beta-turns in these compounds. At pH values greater than 5, a conformational change into the beta II'-turn conformation was also observed for these peptides. Conformations without beta-turns are expected for cyclic all-L pentapeptides, but both cyclo(Asp-Phe-Lys-Ala-Thr) and the D-Thr analog cyclo(Asp-Phe-Lys-Ala-D-Thr) exhibit beta II'-turn conformations around Thr-Asp and D-Thr-Asp. Thus cyclic all-L pentapeptides and those with one D-amino acid are able to form similar structures preferably with a beta II'-turn. The beta-turn formation in cyclic pentapeptides containing a D-aspartic acid is dependent on the ionization state. The relevance of the work to the design of beta'-turn mimetics is discussed.     

Theoretical pi-pi absorption and circular dichroic spectra of beta-turn model peptides

The dipole interaction model, treated by the partially dispersive normal mode method, is used to calculate pi-pi absorption and circular dichroic spectra of beta-turn model peptides in certain conformations. These include Ac-Gly-Gly-NHMe, Ac-L-Ala-L-Ala-NHMe, and Ac-L-Ala-Gly-NHMe in the standard beta-turn conformations I, II, and III of Venkatachalam and cyclo(L-Ala-Gly-epsilon-aminocaproyl), cyclo(L-Ala-L-Ala-epsilon-aminocaproyl), and cyclo(L-Ala-D-Ala-epsilon-aminocaproyl) in the minimum-energy conformations of Nemethy et al. Boltzmann average circular dichroic spectra of the cyclic compounds agree with experimental spectra in most respects. The results are compared with previous theoretical CD spectra for these molecules and with conformational assignments based on other evidence. Absorption spectra in the pi-pi band are predicted to be moderately sensitive to conformation.     

Yeast tRNAAsp: codon and wobble codon-anticodon interactions. A transferred nuclear Overhauser enhancement study

The conformations of the ribotrinucleoside bisphosphates GpApC and GpApU, the codon and wobble codon for aspartic acid respectively, bound to yeast tRNAAsp in solution, have been examined by means of time-dependent transferred nuclear Overhauser enhancement measurements to determine distances between bound ligand protons. The conformations of the two bound ribotrinucleoside bisphosphates are shown to be very similar with an overall root-mean-square difference in interproton distances of 0.03 nm. The ribose conformations of all the residues are 3'-endo; the glycosidic bond torsion angles of the A and C residues of GpApC and of the A and U residues of GpApU are in the low anti range. These features are typical of an A-RNA type structure. In contrast, the G residue of both GpApC and GpApU exists as a mixture of syn and anti conformations. The overall conformation of the two bound ribotrinucleoside bisphosphates is also similar to A-RNA and the stability of the complexes is enhanced by extensive base-base stacking interactions. In addition, it is shown that the binding of the codon GpApC to tRNAAsp induces self-association into a multicomplex system consisting of four GpApC-tRNAAsp complexes, whereas the wobble codon GpApU fails to induce any observable self-association.     

Conformational studies of proline-, thiaproline- and dimethylsilaproline-containing diketopiperazines.

As proline plays an important role in biologically active peptides, many analogues of this residue have been developed to modulate the proportion of cis and trans conformers. A correlation between the pyrrolidine ring shape and structural properties of proline has been established. Diketopiperazine (DKP) is the model of choice to study the influence of the proline ring modification. In this contribution, cyclo(Gly-Pro) and two analogues cyclo(Sip-Pro) and cyclo(Thz-Pro) have been studied with proton NMR. We showed that both analogues with heteroatoms in gamma position, silicon and sulfur respectively, display a more rigid five-member ring. The usual flexibility of proline ring is restrained in both cases and only the two C(beta)-exo and C(beta)-endo conformations are observed.     

Conformational energy analysis of melanostatin

Conformational energy calculations were carried out on the hypothalamic hormone melanostatin, a tripeptide with the primary structure H-L-Pro-L-Leu-Gly-NH2. The calculated lowest energy conformation was a type II beta bend, very similar to that reported in an X-ray crystal study. This conformation, however, was only one of 109 low-energy structures (less than or equal to 3 kcal/mol above the global minimum), indicating that the molecule in solution exists as an ensemble of conformations and is very flexible, in agreement with relaxation data from n.m.r. measurements. A statistical analysis yielded an average end-to-end distance of 6.8 A and a bend probability of 0.62, suggesting that, in nonpolar solvents, bend structures predominate within the statistical ensemble. The statistical analysis, however, also yielded a probability of only 0.11 for the occurrence of a 4 leads to 1 hydrogen bond. Hence, the calculations show that, although bend conformations predominate, bends would be difficult to observe in solution if the experiments were designed only to detect 4 leads to 1 hydrogen bonds.     

Molecular dynamics and quantum chemical studies of solvent effects on cyclo glycylglycine and glycylalanine dipeptides

Hydrogen bond (H-bond) interactions between the two cyclo dipeptides, cyclo(glycyl-glycine) (CGG) and cyclo(glycyl-alanine) (CGA), and water have been studied using molecular dynamics (MD) and quantum chemical methods. The MD studies have been carried out on CGG and CGA in water using fixed charge force field (AMBER ff03) for over 10 ns with a MD time step of 2 fs. The results of this study show that the solvation pattern influences the conformations of the cyclo dipeptides. Following molecular simulations, post Hartree–Fock and density functional theory methods have been used to explore the molecular properties of the cyclo dipeptides in gaseous and aqueous phase environments. The self-consistent reaction field theory has been used to optimise the cyclopeptides in diethyl ether (? = 4.3) and water (? = 78.5), and the solvent effects have been analysed. A cluster of eight water molecules leads to the formation of first solvation shell of CGG and CGA and the strong H-bonding mainly contributes to the interaction energies. The H-bond interactions have been analysed by the calculation of electron density ρ(r) and its Laplacian ▽²ρ(r) at bond critical points using atoms in molecules theory. The natural bond orbital analysis was carried out to reveal the nature of H-bond interactions. In the solvated complexes, the keto carbons registered the maximum NMR chemical shifts.     

Conformational analysis of r(CGCGCG) in aqueous solution: an A-type double helical conformation studied by two-dimensional nuclear Overhauser effect spectroscopy.

The conformation of the hexanucleoside pentaphosphate r( CGCGCG ) in aqueous solution was studied by circular dichroism, 1H- and 31P-NMR spectroscopy. The base-, H1'- and H2'-proton resonances were assigned by means of 2D-NOE spectroscopy. The base- and H1'-proton chemical shifts were studied as a function of temperature. Proton-proton distances are computed in A- and A'-RNA as well as in A-, B- and Z-DNA. A qualitative interpretation of the observed 2D-NOE intensities shows that r( CGCGCG ) adopts a regular A-type double helical conformation under our experimental conditions. The CD- and 31P-NMR experiments described in this paper are in agreement with this structure both under low- and high-salt conditions.     

Conformation of the common purine (beta) ribosides in solution: further evidence for a correlation between N-S state of the ribose moiety and syn-anti equilibrium.

The solution conformations of adenosine, guanosine and inosine in liquid ND3 have been determined by NMR. Comparison of the Karplus analysis of the proton HR spectra of the ribose moiety obtained in this solvent with the data from aqueous solutions of A and I proves that the conformations of the nucleosides are very similar in both liquids. From the analysis of the vicinal coupling constants of the ring protons it has been deduced that the S state C(2')-endo is slightly preferred. The mole fraction in S approximates 0.6 for all three nucleosides. C-13 relaxation measurements have been applied in the determination of the correlation times for rotational diffusion. Only at temperatures below - 40 degrees C is the pseudo-rotation of the furanoside ring slowed down sufficiently for it not to contribute to the measured relaxation rates. From NOE studies and T1 measurements on the individual protons it is derived that the N, C(3')-endo, form of the ribose is correlated with an anti conformation of the base (Y approximately 210 degrees to 220 degrees) and the S, C(2')-endo, form of the ribose with a syn conformation of the base (Y approximately 30 degrees to 50 degrees). The glycosyl torsion angles derived for the two conformations of A, G, and I are equal within the limits of accuracy.     

NMR studies of the backbone protons and secondary structure of pentapeptide and heptapeptide substrates bound to bovine heart protein kinase

The conformations of enzyme-bound pentapeptide (Arg-Arg-Ala-Ser-Leu) and heptapeptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) substrates of protein kinase have been studied by NMR in quaternary complexes of the type (Formula: see text). Paramagnetic effects of Mn2+ bound at the inhibitory site of the catalytic subunit on the longitudinal relaxation rates of backbone Ca protons, as well as on side-chain protons of the bound pentapeptide and heptapeptide substrates, have been used to determine Mn2+ to proton distances which range from 8.2 to 12.4 A. A combination of the paramagnetic probe-T1 method with the Redfield 2-1-4-1-2 pulse sequence for suppression of the water signal has been used to measure distances from Mn2+ to all of the backbone amide (NH) protons of the bound pentapeptide and heptapeptide substrates, which range from 6.8 to 11.1 A. Paramagnetic effects on the transverse relaxation rates yield rate constants for peptide exchange, indicating that the complexes studied by NMR dissociate rapidly enough to participate in catalysis. Model-building studies based on the Mn2+-proton distances, as well as on previously determined distances from Cr3+-AMPPCP to side-chain protons [Granot, J., Mildvan, A.S., Bramson, H. N., & Kaiser, E. T. (1981) Biochemistry 20, 602], rule out alpha-helical, beta-sheet, beta-bulge, and all possible beta-turn conformations within the bound pentapeptide and heptapeptide substrates. The distances are fit only by extended coil conformations for the bound peptide substrates with a minor difference between the pentapeptides and heptapeptides in the phi torsional angle at Arg3C alpha and in psi at Arg2C alpha. An extended coil conformation, which minimizes the number of interactions within the substrate, would facilitate enzyme-substrate interaction and could thereby contribute to the specificity of protein kinase.     

Conformations of cyclic octapeptides. 4. Diastereomers of cyclo(Gly-Pro-Phe-Ala-Asn-Ala-Val-Ser)

Stereoisomers of cyclo(Gly-Pro-Phe-Ala-Asn-Ala-Val-Ser) were synthesized. NMR studies of their solution conformations, focusing on peptide N-H solvent exposure, were made. These indicated that a single proline residue in the cyclic octapeptide ring is insufficient constraint to stabilize the backbone conformations that were previously established for cyclo(Gly-Pro-Phe-Ala)2.     

Alkyl phosphotriester modified oligodeoxyribonucleotides. VI. NMR and UV spectroscopic studies of ethyl phosphotriester (Et) modified Rp-Rp and Sp-Sp duplexes, (d[GGAA(Et)TTCC])2.

1H NMR chemical shift assignments for the title compounds were made for all but a few H5' and H5" signals using two-dimensional nuclear Overhauser effect (2D-NOE) data, which was also used for the first time to assign absolute configuration at phosphorus. The chemical shifts were, in general, similar to those reported [Broido, M.S., et al. (1985) Eur. J. Biochem. 150, 117-128] for the B-like conformation of the unmodified, parent duplex, [d(GGAATTCC)]2. Differences in chemical shifts for corresponding protons were mostly localized to the AA(Et)TT region, and showed some stereochemical dependence. Unambiguous assignment of the phosphotriester 31P signals was achieved in a novel way using selective insensitive nucleus enhancement by polarization transfer (selective INEPT) NMR. The Rp-Rp duplex melted ca. 11 degrees C lower than either the Sp-Sp or parent duplexes, as evidenced by Tm and variable temperature 1H/31P NMR measurements. The 2D-NOE data for the Rp-Rp duplex suggested possible steric interactions between the ethyl group and the H3' of the flanking A residue. At low ionic strength, the Sp-Sp and parent duplexes had similar stability but at high ionic strength the Sp-Sp duplex was less stable.