The temperature dependence of the circular dichroism (CD) spectra of a series of deoxyadenylates (dA),,, n = 2, 3, 6, 9, 12, cxi, in aqueous solution was studied. The data were interpreted on the basis of a new conformational model for the stacked state suggested by our previous proton N M R studies on (dA)2 and (dA)3 Biochmz. 112, this model the stacked regions of the single-stranded oligomers consist of residues taking up a geometry resembling that of the B-DNA genus of structures (all sugars S or C2'-enrb) except those residues at the 3' end that do not 'feel' a following stacking interaction. The deoxyribose rings in the latter residues retain (or regain when melting out removes a stacking interaction somewhere along the chain) the conformational freedom (S N, N = C3'-endo) that these rings possess in the monomers 2'-deoxyadenosine 5'-methylphosphate or in 2'-deoxyadenosine 3',5'-bis(methylphosphate), as the case may be. It is shown that this model allows (a) construction of the C D spectra of (dA),, n = 3, 6, 9, 12, from those of the dimer and the polymer; (b) the separation of the weak C D displayed by the regular S-S stacking mode and the far stronger C D exhibited by the 3'-end S-N stacking (the latter C D resembles that of the A-DNA genus of structures); (c) delineation of the thermodynamics of stacking. The melting temperature remains constant and independent of chain length (about 50 "C) whercas AH" and AS" show a slight increase in absolute values on increasing n from 2 to x owing to small cooperativity effects. Near 0°C the dimer occurs for about 90% in the stacked form, the oligomers attain even higher conformational purities. It is suggested that premelting phenomena observed in the C D spectra of double-helical DNAs may also involve local transitions from the normal B-like ----S-S-S----stacking mode to an A-like ----S-S-N----stacking geometry.The correlation between the mode of pucker of the ribose (or deoxyribose) ring, the phosphate diester backbone conformation and vertical base-base stacking in oligonucleotides to form a specific type of single helix has been the subject of several studies conducted in our laboratory [l-91. We now present a conformational study of a series of oligodeoxyadenosine nucleotides (dA),, n = 2, 3, 6, 9, 12, a, (cf. Fig. 1) carried out by means of temperature-dependent circular dichroism. The reasons for this study are manifold; however, three main objectives may be discerned: (a) the need for a quantitative insight into the thermodynamics of stacking of single helices; (b) to find an explanation for the curious C D spectral properties displayed by poly(dA) at neutral pH, which in no way resembles that of the dimer (dA)2; (c) to explore a possible correlation between the C D properties and the unexpected conformational behaviour of (dA)z and (dAh recently discovered by means of NMR spectroscopy [7]. It was discovered that these three objectives This paper is part XVII in the series on Nucleic Acid Constituents from this laboratory; part XVI appeared earlier in this j...

Proton NMR studies at 360 MHz were carried out on the trinucleoside diphosphate dApdApdA, the dinucleoside monophosphate dApdA and the methylphosphate esters of the monomers pdA, pdAp and dAp. The compounds were also investigated by means of circular dichroism at varying temperatures. Complete NM R spectral assignments are given. The thermodynamics of stacking derived from the circular dichroic spectra was used to extrapolate the observed coupling constants, measured at a range of temperatures, to values appropriate to the pure stacked states of the dimer and the trimer.The data were interpreted in terms of the N and S deoxyribose pseudorotational ranges [Altona, C. and Sundaralingam, M. (1972) J . Am. Clzem. Soc. 94,8205 -82121 and it is shown that the right-handed single helix of the trimer occurs in two distinct forms : the major one corresponds to a B-DNA-like structure, S-S-S ; the minor one (30 '%;) has the sugar rings in a hitherto unsuspected S-S-N conformational sequence. The dimer behaves quite similarly, a mixture of 70% S-S and 30% S-N stacks being indicated. The detailed geometry of the S-type sugar rings is not invariable but appears to undergo a slight shift when another base stacks on top of a given nucleotide unit. The backbone angles of the fully stacked B-DNA-like single helix in solution are (starting the notation at P) /3 = 187", y = 50°, 6 = 138", E = 186".

Proton NMR studies at 360 MHz are reported on the adenine dinucleoside monophosphates N6-dimethyladenyly(3'-5')-N6-dimethyladenosine (m(6)(2)Apm(6)(2)A), ApA, rApdA, dAprA and on the methyl phosphate esters of the monomers m(6)(2)Ap, pm(6)(2)A, Ap and pA. Complete 1H-NMR spectral assignments are given. The dimers were also investigated by means of circular dichroism to obtain accurate thermodynamic parameters of the stacking equilibrium. With the aid of the thermodynamic data NMR coupling constants are extrapolated to values appropriate to the stacked conformers. A modernized version of pseudorotation analysis is used to delineate the conformational behaviour of the ribose and 2'-deoxyribose rings. It is shown that the unmethylated dimers can be arranged in two groups (dApdA/dAprA vs ApA/rApdA) according to their melting temperatures. ApA and the fully N6-methylated dimer m(6)(2)Apm(6)(2)A prefer to adopt the classical right-handed N-N stacked conformation. Both dimers with a 2'-deoxyribose ring at the 5'-OH end (dApdA and dAprA) behave similarly and occur in solution as a 75:25 mixture of S-S and S-N stacked states. The fully stacked hybrid dimer rApdA displays an unexpectedly large amount of S conformers (greater than 40%) in both sugar rings. This finding is rationalized by the postulation of a right-handed helical S-S stacked state on the basis of NMR and circular dichroic data.

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The temperature dependence of the circular dichroism spectra of two adenylyladenosine and three adenylyluridine dinucleotides in aqueous solution have been studied. The effect of methylation of the N-6 position of the adenylyl moieties on the thermodynamic parameters of the stacking equilibrium was investigated applying a two-state model for the thermodynamic analysis of the experimental data. It is shown that the methylated compounds allow a more accurate analysis of the data and that methylation stabilizes the stacking interaction by a relative decrease of the entropy of unstacking.The rather unique vertical base-base stacking interaction exhibited by polynucleotides to form a single helix is understood to be one of the major driving forces in the formation of stable three-dimensional structures of RNA and DNA (e.g. double helices, various internal loops, hairpin loops and so forth). However, before the conformational properties of polymers can be understood in some measure, the stacking interaction must be investigated in the greatest possible detail in simple model compounds. Even if extrapolation of experimental findings in dinucleotides to properties of polynucleotides may prove to be of partial validity, one may be certain that knowledge of the differences in conformational behaviour between dinucleotides, trinucleotides and tetranucleotides will in the long run enable us to build a consistent picture of factors governing the equilibrium between helix and random coil forms in polynucleotides. For these reasons the conformational analysis of dinucleoside phosphates has received much attention during the past two decades. Most of the early papers on this subject concerned studies of the geometry and the thermodynamics of the stacking interaction by means This paper is number XV in a series from this laboratory entitled Nucleic Acid Constituents; the previous paper appeared elsewhere [C.A. G Ahhrevicrtions. CD, circular dichroism; ApU, adenylyl(3'-5')-uridine; ApA, adenylyl(3'-5')adenosine: m6ApU, N6-methyl-adenylyl(3'-5')uridine; m:ApU, N6-dimelhyl-adenosy1y1(3'5')uridine; mpApm:A, N6-dimethyl-adenylyl(3'-5')N6-dimethyl-adenosine.of optical methods viz. ultraviolet spectroscopy, optical rotatory dispersion and circular dichroism (CD) [l -51. Later, the advent of high-field Fourier-transform nuclear magnetic resonance (NMR) instrumentation allowed the complete assignment and analysis of the spectra of a wide variety of dinucleotides, providing information about the conformational changes in the dinucleotide backbone as a function of temperature [6 -101. From that time the interest for optical measurements receded to the background, presumably because of the lack of reproducibility and precision of the derived energy parameters, strikingly illustrated by the wide spread in published enthalpy values of unstacking (21 -42 kJ/mol) in ApA [ll].In previous studies [ 12 -141 we have already indicated the importance of the application of a combination of experimental techniques, i.e. C D and NMR, in the approach of ...