Parallel stranded DNA.

A series of four hairpin deoxyoligonucleotides was synthesized with a four-nucleotide central loop (either C or G) flanked by the complementary sequences d(T)10 and d(A)10. Two of the molecules contain either a 3'-p-3' or 5'-p-5' linkage in the loop, so that the strands in the stem have the same, that is, parallel (ps) polarity. The pair of reference oligonucleotides have normal phosphodiester linkages throughout and antiparallel (aps) stem regions. All the molecules adopt a duplex helical structure in that (i) the electrophoretic mobilities in polyacrylamide gels of the ps and aps oligomers are similar. (ii) The ps hairpins are substrates for T4 polynucleotide kinase, T4 DNA ligase, and Escherichia coli exonuclease III. (iii) Salt-dependent thermal transitions are observed for all hairpins, but the ps molecules denature 10 degrees C lower than the corresponding aps oligomers. (iv) The ultraviolet absorption and circular dichroism spectra are indicative of a base-paired duplex in the stems of the ps hairpins but differ systematically from those of the aps counterparts. (v) The bis-benzimidazole drug Hoechst-33258, which binds in the minor groove of B-DNA, exhibits very little fluorescence in the presence of the ps hairpins but a normal, enhanced emission with the aps oligonucleotides. In contrast, the intercalator ethidium bromide forms a strongly fluorescent complex with all hairpins, the intensity of which is even higher for the ps species. (vi) The pattern of chemical methylation is the same for both the ps and aps hairpins. The combined results are consistent with the prediction from force field analysis of a parallel stranded right-handed helical form of d(A)n.d(T)n with a secondary structure involving reverse Watson-Crick base pairs and a stability not significantly different from that of the B-DNA double helix. Models of the various hairpins optimized with force field calculations are described.

Conformational analysis of the hydrophobic peptide alphas1-casein(136-196).

Hydrophobic interactions are important in the self-association of milk proteins, including alphas1-casein. The extent to which casein interaction sites are influenced by local secondary structure is not widely known. Both primary amino acid sequence and local secondary structure are shown to affect the self-association of the hydrophobic peptide alphas1-casein(136-196). The peptide is aggregated at low concentrations (7 microM and above), as determined by 1H nuclear magnetic resonance (NMR) measurements at pH 6.0 in phosphate buffer. Increase in temperature is shown to induce side chain mobility (melting) as indicated by both 1H NMR and near-UV circular dichroism (CD) measurements. As determined by far-UV CD, there is also a loss in the global amount of extended structure with increasing temperature, while beta-turn structures and some aromatic dichroism are conserved at temperatures as high as 70 degrees C. Similar retention of structure occurs at pH 2 and in 6 M guanidine HCl. The observed stability of beta-turns and some side chains in alphas1-casein(136-196) supports previous assumptions that hydrophobic, proline-based turns are important interaction sites in the self-association of alphas1-casein, and possibly in the formation of the calcium transport complexes, the casein micelles. It may be speculated that these areas of the peptide represent a 'molten globule-like', heat stable, core structure for alphas1-casein.

Solution conformation of purine-pyrimidine DNA octamers using nuclear magnetic resonance, restrained molecular dynamics and NOE-based refinement.

The solution structures of two alternating purine-pyrimidine octamers, [d(G-T-A-C-G-T-A-C)]2 and the reverse sequence [d(C-A-T-G-C-A-T-G)]2, are investigated by using nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations. Chemical shift assignments are obtained for non-exchangeable protons by a combination of two-dimensional correlation and nuclear Overhauser enhancement (NOE) spectroscopy experiments. Distances between protons are estimated by extrapolating distances derived from time-dependent NOE measurements to zero mixing time. Approximate dihedral angles are determined within the deoxyribose ring from coupling constants observed in one and two-dimensional spectra. Sets of distance and dihedral determinations for each of the duplexes form the bases for structure determination. Molecular dynamics is then used to generate structures that satisfy the experimental restraints incorporated as effective potentials into the total energy. Separate runs start from classical A and B-form DNA and converge to essentially identical structures. To circumvent the problems of spin diffusion and differential motion associated with distance measurements within molecules, models are improved by NOE-based refinement in which observed NOE intensities are compared to those calculated using a full matrix analysis procedure. The refined structures generally have the global features of B-type DNA. Some, but not all, variations in dihedral angles and in the spatial relationships of adjacent base-pairs are observed to be in synchrony with the alternating purine-pyrimidine sequence.

Antiparallel DNA duplex formation between alternating alpha d(GA)n and beta d(GA)n sequences.

Alternating polypurine d(GA)n, sequences exhibit a considerable polymorphism. Here we report that alpha d(GA) x d(GA) sequences form an antiparallel stranded duplex DNA at neutral pH. The spectroscopic, electrophoretic and thermodynamic properties of the alpha/beta chimeric oligodeoxynucleotide, 5'-d(GA)4(T)4 alpha d(AG)4T-3', support the formation of a hairpin structure with antiparallel strands in the stem. The optical properties of this novel antiparallel structure are different from the parallel stranded homoduplex formed by d(GA)G7. This alpha/beta hairpin has a remarkably high Tm of 44.5 degrees C in 0.4 M NaCl with a van't Hoff enthalpy comparable to that of a parallel d(GA)n duplex. Base pairing was confirmed by T4 polynucleotide ligase catalyzed joining of the alpha/beta hairpin to an antiparallel bimolecular duplex and by non-denaturing gel electrophoresis using duplexes containing sequence constraints. Both support the presence of alphaG-G and alphaA-A base pairing in the antiparallel 5'-d(GA)4(T)4 alpha d(AG)4T-3' intramolecular duplex. This study adds to the polymorphic nature of alternating d(GA)n sequences as well as providing novel homopurine base pairing approaches for probing polypurine polypyrimidine sequences.

Homooligomeric dA.dU and dA.dT sequences in parallel and antiparallel strand orientation: consequence of the 5-methyl groups on stability, structure and interaction with the minor groove binding drug Hoechst 33258.

Oligodeoxyribonucleotides containing dA.dU base combinations were shown to form parallel stranded DNA. CD spectra and hyperchromicity profiles provide evidence that the structure is very similar to that of a related parallel stranded dA.dT oligomer. Thermal denaturation studies show that these parallel dA.dU sequences are significantly less stable than their dA.dT analogues in either antiparallel or parallel stranded orientations. The stabilizing effect of the 5-methyl group is similar for parallel and antiparallel sequences. The minor groove binding drug Hoechst 33258 binds with similar affinity to APS dA.dT and APS dA.dU sequences. However, binding to the PS dA.dT hairpin is significantly impaired as a consequence of the different groove dimensions and the presence of thymine methyl groups at the binding site. This results in an 8.6 kJmol-1 reduced free energy of binding for the PS dA.dT sequence. Replacement of the bulky methyl group with a hydrogen (ie. T-->U) results in significantly stronger Hoechst 33258 binding to the parallel dA.dU sequences with a penalty of only 4.1 kJmol-1. Our data demonstrate that although Hoechst 33258 detects the altered groove, it is still able to bind a PS duplex containing dA.dU base pairs with high affinity, despite the large structural differences from its regular binding site in APS DNA.

Structural, dynamic, and enzymatic properties of mixed alpha/beta-oligonucleotides containing polarity reversals.

We employ NMR structure determination, thermodynamics, and enzymatics to uncover the structural, thermodynamic and enzymatic properties of alpha/beta-ODNs containing 3'-3' and 5'-5' linkages. RNase H studies show that alpha/beta-gapmers that are designed to target erbB-2 efficiently elicit RNase H activity. NMR structures of DNA.DNA and DNA.RNA duplexes reveal that single alpha-anomeric residues fit well into either duplex, but alter the dynamic properties of the backbone and deoxyriboses as well as the topology of the minor groove in the DNA.RNA hybrid.

NMR studies of DNA duplexes containing alpha-anomeric nucleotides and polarity reversals.

We present a summary of our research to date on a family of self-complementary DNA decamers containing single alpha-anomeric nucleotides flanked by 3'-3' and 5'-5' phosphodiester linkages from the perspective of the salient NMR techniques employed to shed light on the structural and dynamic properties of these sequences. Research into this class of synthetic alpha-/beta-oligonucleotides containing mixed strand disposition may have medical relevance given their recently documented efficacy as antisense therapeutics.

Solution structure of a DNA.RNA hybrid containing an alpha-anomeric thymidine and polarity reversals: d(ATGG-3'-3'-alphaT-5'-5'-GCTC). r(gagcaccau).

We report the thermodynamic and structural properties of an alpha-containing DNA.RNA nonamer hybrid duplex, d(ATGG-3'-3'-alphaT-5'-5'-GCTC).r(gagcaccau). The RNA strand corresponds to the core of the initiation sequence for the transcript of the erbB-2 oncogene. The tandem anomeric and polarity changes in the DNA strand result in a slight decrease in thermostability (DeltaT(m) = -2.8 degrees C) compared to the unmodified control hybrid. The three-dimensional solution structure determination of the alpha-containing DNA.RNA hybrid, conducted via restrained molecular dynamics using interproton distance (nuclear Overhauser enhancement) and furanose ring torsion angle (J-based) restraints, converged to a final ensemble of structures from unique starting models. In agreement with hyperchromicity and circular dichroism data, the final average structure derived from this ensemble is consistent with an overall A-like motif featuring Watson-Crick base pairing and base stacking across the entire sequence, albeit with localized B-like traits within the DNA strand. Comparative pseudorotation analyses of the J-coupling data for this hybrid and its unmodified control reveal a surprising increase in S-puckering for two nucleotides immediately upstream of the 3'-3' linkage, and the associated narrowing of the minor groove in this portion of the hybrid. Other structural perturbations are localized to and diagnostic of the central alpha-nucleotide and juxtaposed polarity reversals. The structural information presented here has direct relevance to the design of future antisense oligonucleotides composed of these modifications.

Following plant metabolism in vivo and in extracts with heteronuclear two-dimensional nuclear magnetic resonance spectroscopy.

Limits of sensitivity and spectral resolution currently restrict the application of nuclear magnetic resonance (NMR) spectroscopy in plant metabolism. This study shows that these limits can be substantially expanded through the application of heteronuclear single- and multiple-quantum two-dimensional (2D) spectroscopic methods using pulsed field gradients both in vivo and in extracts. The course of metabolism in approximately 0.2 g of maize (Zea mays L.) root tips labeled with [1-13C]glucose was followed with 1 min time resolution using heteronuclear multiple quantum coherence (HMQC) 13C-1H spectroscopy in vivo. The timing of alanine, lactate, and ethanol synthesis was followed during the transition from normal to hypoxic conditions. In extracts of labeled maize root tips, 13C-1H heteronuclear single quantum coherence and heteronuclear multiple quantum coherence (HMBC) spectra acquired in 2-3 h allowed the detection and assignment of resonance that are not seen in one-dimensional (1D) 13C NMR spectra of the same samples taken in 12 h. In root tips labeled with 15NH4+, 15N-(1)H HMQC spectra in vivo showed labeling in the amide of glutamine. In extracts, 15N labeling in amines and amides was detected using 15N-1H HMBC spectra that is not seen in 1D 15N spectra of the same sample.

NMR solution structures of [d(GCGAAT-3'-3'-alphaT-5'-5'-CGC)2] and its unmodified control.

We present the high-resolution solution structures of a self-complementary DNA decamer duplex featuring a single alpha-anomeric nucleotide per strand encompassed by a set of 3'-3' and 5'-5' phosphodiester linkages, d(GCGAAT-3'-3'-alphaT-5'-5'-CGC)2, alphaT, and its unmodified control, d(GCGAATTCGC)2, obtained by restrained molecular dynamics. Interproton distance and deoxyribose ring torsion angle restraints were deduced from homonuclear NOESY and DQF-COSY data, respectively. For both the control and alphaT duplexes, excellent global convergence was observed from two different (A- and B-) starting models. The final average structures of the two duplexes are highly homologous, and overall possess the traits characteristic of right-handed B-DNA duplexes. However, localized differences between the two structures stem from the enhanced conformational exchange in the deoxyribose ring of the cytidine following the 5'-5' linkage, the C3'- exo pseudorotation phase angle of the alpha-nucleotide, and unusual backbone torsions in the 3'-3' and 5'-5' phosphodiester linkages. The structural data reported here are relevant to the design of antisense therapeutics comprised of these modifications.

Conformational dynamics in mixed alpha/beta-oligonucleotides containing polarity reversals: a molecular dynamics study using time-averaged restraints.

Nucleic acid duplexes featuring a single alpha-anomeric thymidine inserted into each DNA strand via 3'-3' and 5'-5' phosphodiester linkages exhibit local conformational dynamics that are not adequately depicted by conventional restrained molecular dynamics (rMD) methods. We have used molecular dynamics with time-averaged NMR restraints (MDtar) to explore its applicability to describing the conformational dynamics of two alpha-containing duplexes--d(GCGAAT-3'-3'-alphaT-5'-5'-CGC)2 and d(ATGG-3'-3'-alphaT-5'-5'-GCTC) x r(gagcaccau). In contrast to rMD, enforcing NOE-based distance restraints over a period of time in MDtar rather than instantaneously results in better agreement with the experimental NOE and J-data. This conclusion is based on the dramatic decreases in average distance and coupling constant violations (delta d(av), J(rms), and delta J(av)) and improvements in sixth-root R-factors (R(X)). In both duplexes, the deoxyribose ring puckering behavior predicted independently by pseudorotation analysis is portrayed remarkably well using this approach compared to rMD. This indicates that the local dynamic behavior is encoded within the NOE data, although this is not obvious from the local R(X) values. In both systems, the backbone torsion angles comprising the 3'-3' linkage as well as the (high S-) sugars of the alpha-nucleotide and preceding residue (alpha - 1) are relatively static, while the conformations of the 5'-5' linkage and the sugar in the neighboring beta-nucleotide (alpha + 1) show enhanced flexibility. To reduce the large ensembles generated by MDtar to more manageable clusters we utilized the PDQPRO program. The resulting PDQPRO clusters (in both cases, 13 structures and associated probabilities extracted from a pool of 300 structures) adequately represent the structural and dynamic characteristics predicted by the experimental data.

RNA facilitates RecA-mediated DNA pairing and strand transfer between molecules bearing limited regions of homology.

The RecA protein of Escherichia coli catalyzes homologous pairing and strand exchange between a wide range of molecules showing nucleotide sequence complementarity, including a linear duplex and a single-stranded DNA molecule. We demonstrate that RecA can promote formation of joint molecules when the duplex contains an RNA/DNA hairpin and a single-stranded circle serves as the pairing partner. A chimeric RNA/DNA hairpin can be used to form stable joint molecules with as little as 15 bases of shared homology as long as the RNA stretch contains complementarity to the circle. The joint molecule bears some resemblance to a triple helical structure composed of RNA residues surrounded by two DNA strands which are in a parallel orientation. Evidence is presented that supports the notion that short stretches of RNA can be used in homologous pairing reactions at lengths below that required for DNA-DNA heteroduplex formation.

Relative stability of parallel- and antiparallel-stranded duplex DNA.

We have recently shown that DNA containing homopolymeric A-T base pairs can form a parallel-stranded intramolecular duplex [van de Sande et al. (1988) Science (Washington, D.C.) 241, 551-557]. In the present paper, we demonstrate that parallel-stranded DNA can also be formed in unconstrained bimolecular DNA of appropriate sequence homology. Three deoxyoligonucleotides, a 21-mer [dCCCATATATATTTTTTTTCCC], a ps-15-mer [dTATATATAAAAAAAA], and an aps-15-mer [dAAAAAAAATATATAT], have been synthesized. Annealing of 21-mer and aps-15-mer results in the formation of a conventional antiparallel duplex (aps); however, the combination of 21-mer and ps-15-mer forms a duplex in which the two strands are in a parallel orientation (ps). The parallel-stranded structure was established from the following criteria: (i) The parallel-stranded structure shows a 1:1 stoichiometry of the constituent strands. (ii) Gel electrophoretic mobility of the ps and aps duplexes are similar under native conditions. (iii) Spectroscopic properties of the ps duplex are characteristics for a base-paired structure but are different from the aps structure. (iv) Both duplexes undergo a thermally induced helix to coil transition; however, the melting temperature for the ps duplex is 22 degrees C lower. (v) The minor groove binding drug Hoechst 33258 shows a reduced affinity for the ps compared to the aps duplex. (vi) The parallel-stranded duplex is not a substrate for DNA Escherichia coli polymerase I (Klenow fragment) or AMV reverse transcriptase. Parallel-stranded DNA can exist under normal solution conditions, but competition experiments show it to be thermodynamically less favorable than the conventional antiparallel form.

A general method for the purification of synthetic oligodeoxyribonucleotides containing strong secondary structure by reversed-phase high-performance liquid chromatography on PRP-1 resin.

Synthetic 5'-dimethoxytritylated oligodeoxyribonucleotides, which contained strong secondary structure, were satisfactorily denatured and purified by reversed-phase HPLC on PRP-1 columns when strongly alkaline conditions (0.05 M NaOH) were employed. This procedure was suitable for the purification of hairpin structures, e.g., d(CG)nT4(CG)n (n = 4, 5, 6), and oligo(dG) sequences, e.g., d(G)24, as well as oligodeoxyribonucleotide probes which contained degenerate base sites. Oligodeoxyribonucleotides as long as 50 bases in length were purified. Recovery of injected oligonucleotides was typically 90% or better. The high capacity of the PRP-1 resin also allowed purification to be performed on a preparative scale (2-8 mg per injection). Enzymatic degradation and HPLC analysis indicated that no modification of the heterocyclic bases occurred under the alkaline conditions described.

Spectroscopic and thermodynamic studies of DNA duplexes containing alpha-anomeric C, A, and G nucleotides and polarity reversals: coexistence of localized parallel and antiparallel DNA.

We present a thermodynamic, enzymatic, and spectroscopic study of three self-complementary DNA decamer duplexes, d[GCGAATT-3'-3'-(alphaC)-5'-5'-GC]2 (alphaC), d[GCG-3'-3'-(alphaA)-5'-5'-ATTCGC]2 (alphaA), and d[GC-3'-3'-(alphaG)-5'-5'-AATTCGC]2 (alphaG), which are identical in sequence but contain one alpha-anomeric nucleotide per strand in a parallel orientation via 3'-3' and 5'-5' phosphodiester bonds; the results are placed in the context of our recent studies on the other members of this series, namely alphaT, d[GCGAAT-3'-3'-(alphaT)-5'-5'-CGC]2, and the unmodified control [Aramini, J. M., et al. (1996) Biochemistry 35, 9355-9365]. On the basis of UV hyperchromicity and melting profiles as well as 1H and 31P nuclear magnetic resonance (NMR) spectroscopic data, we conclude that all five constructs form stable duplexes, with very comparable structural features that are consistent with an overall right-handed, antiparallel B-DNA motif and Watson-Crick base pairing throughout. However, each of the alpha-containing sequences exhibits unique thermodynamic and structural differences ascribed to the nature (and position) of the alpha-nucleotide. First, the thermostability of these duplexes decreases from the control to alphaC in the following series: control > alphaT approximately alphaA approximately alphaG > alphaC. Second, in each of the four alpha-duplexes, 1H and 31P chemical shift differences compared to those of the control duplex are largely confined to the region encompassing the alpha-nucleotide and unnatural phosphodiester linkages, as well as neighboring nucleotides. Surprisingly, for alphaC, these modifications result in a significant alteration to the backbone conformation at the phosphodiester group directly across from the 3'-3' linkage. Finally, spin-spin (J) coupling data, specifically Sigma1', indicate that the vast majority of the furanose rings in these duplexes display a high propensity for adopting the S pucker. However, in alphaC, alphaA, and alphaT (but not alphaG), the sugar ring conformation in the nucleotide immediately following the 5'-5' linkage is described by an approximately equal distribution between the N and S conformers.

Structure of d(GT)n.d(GA)n sequences: formation of parallel stranded duplex DNA.

Alternating polypurine sequences exhibit remarkable polymorphism. In this study, we report that dGA.dGT sequences form parallel stranded duplex DNA at neutral pH. Using two model hairpins, 3'-d(GT)3-5'5'-T4(AG)3-3' (I) and 3'-d(GT)4-5'5'-T4(AG)4-3' (II), containing 5'5' linkages which direct parallel strand formation, we systematically explored the spectroscopic and thermodynamic properties of parallel stranded d(GA)n.d(GT)n. The parallel stranded hairpins are remarkably stable structures with TM's of 41.5 and 47.5 degreesC (in 0.4 M NaCl) for the shorter and longer hairpins, respectively. The van't Hoff enthalpies of 80.7 and 114 kJ mol-1 are relatively low but are comparable to a parallel stranded d(GA)n duplex. On the basis of the spectroscopic and electrophoretic characteristics, we conclude that parallel strand formation is not restricted to hairpin systems, but also readily occurs in unconstrained dimeric duplexes with the appropriate sequence homologies. Both melting curves and electrophoretic analyses of parallel stranded heteroduplexes in which the sequence enforces specific base pairing demonstrate that G-G and A-T base pairs are formed in d(GA)n.d(GT)n segments.

Characterization of a parallel-stranded DNA hairpin.

Recently we have shown that synthetic DNA containing homooligomeric A-T base pairs can form a parallel-stranded intramolecular hairpin structure [van de Sande et al. (1988) Science (Washington, D.C.) 241, 551-557]. In the present study, we have employed NMR and optical spectroscopy to investigate the structure of the parallel-stranded (PS) DNA hairpin 3'-d(T)8C4(A)8-3' and the related antiparallel (APS) hairpin 5'-d(T)8C4(A)8-3'. The parallel orientation of the strands in the PS oligonucleotide is achieved by introducing a 5'-5' phosphodiester linkage in the hairpin loop. Ultraviolet spectroscopic and fluorescence data of drug binding are consistent with the formation of PS and APS structures, respectively, in these two hairpins. Vacuum circular dichroism measurements in combination with theoretical CD calculations indicate that the PS structure forms a right-handed helix. 31P NMR measurements indicate that the conformation of the phosphodiester backbone of the PS structure is not drastically different from that of the APS control. The presence of slowly exchanging imino protons at 14 ppm and the observation of nuclear Overhauser enhancement between imino protons and the AH-2 protons demonstrate that similar base pairing and base stacking between T and A residues occur in both hairpins. However, the small chemical shift dispersion observed in proton NMR spectra of the PS hairpin suggests that the stem of this hairpin is more regular than that of the APS hairpin. On the basis of NOESY measurements, we find that the orientation of the bases is in the anti region and that the sugar puckering is in the 2'-endo range.(ABSTRACT TRUNCATED AT 250 WORDS)

Length-dependent formation of parallel-stranded DNA in alternating AT segments.

Parallel-stranded DNA can be formed from alternating AT segments and is not restricted exclusively to homooligomeric AT sequences. DNA oligonucleotides 3'-d(AT)nxC4(AT)n-3' (where x indicates the location of the 5'-5' phosphodiester linkage) form parallel-stranded hairpin structures at micromolar strand concentration for n = 4 or 5 but not for n = 6, 7. The spectral properties of the parallel-stranded structures are similar to those of the hairpin structures containing homooligomeric AT stems. However, parallel-stranded structures formed in alternating AT segments are significantly less stable than either their corresponding antiparallel control or the homooligomeric parallel AT hairpins as evidenced by their lower helix-coil transition enthalpy, melting temperature, and stability constant. This results in a remarkable polymorphism which is most pronounced for 3'-d(AT)5xC4(AT)5-3'. This oligonucleotide can exist as a parallel-stranded hairpin, coil, or concatameric antiparallel structure(s), depending on temperature and strand concentration. These results suggest simple guidelines for the design of parallel-stranded DNA. In addition, we present a model for the assessment of the stability of parallel-stranded duplex structures formed from AT base pairs based on their sequence.

Right- and left-handed (Z) helical conformations of the hairpin d(C-G)5T4(C-G)5 monomer and dimer.

The partial self-complementary 24-mer oligodeoxynucleotide d(C-G)5T4(C-G)5 forms a hairpin which can be enzymatically dimerized to a dumbbell structure. The blunt-ended nature of the hairpin is indicated by its ability to inhibit the T4 DNA ligase catalyzed joining of phi X174 HaeIII fragments. The hairpin monomer and dimer (dumbbell) undergo a reversible B to Z transition as shown by ultraviolet, circular dichroism, and 31P NMR spectroscopy. The Z form of the hairpin monomer and dimer is supported by monovalent ions (Na+), divalent ions (Mg2+ but not Mn2+), and dehydrating (ethanol) conditions. The conformational transition of d(C-G)5T4(C-G)5 monomer requires higher ionic or dehydrating conditions than those necessary for the corresponding linear oligomer d(C-G)5. The contribution of the loop (-T4-) of the hairpin to the apparent free energy change for the B to Z conformational transition at the midpoint was calculated to be 3.8 kJ mol-1.