A graphical method is presented for the conformational analysis of the sugar ring in DNA fragments by means of proton-proton couplings. The coupling data required for this analysis consist of sums of couplings, which are referred to as sigma 1' (= J1'2' + J1'2''), sigma 2' (= J1'2' + J2'3' + J2'2''), sigma 2'' (= J1'2'' + J2''3' + J2'2'') and sigma 3' (= J2'3' + J2''3' + J3'4'). These sums of couplings correspond to the distance between the outer peaks of the H1', H2', H2'' and H3' [31P] resonances, respectively, (except for sigma 2' and sigma 2'' in the case of a small chemical shift difference between the H2' and H2'' resonances) and can often be obtained from 1H-NMR spectra via first-order measurement, obviating the necessity of a computer-assisted simulation of the fine structure of these resonances. Two different types of graphs for the interpretation of the coupling data are discussed: the first type of graph serves to probe as to whether or not the sugar ring occurs as a single conformer, and if so to analyze the coupling data in terms of the geometry of this sugar ring. In cases where the sugar ring does not occur as a single conformer, but as a blend of N- and S-type sugar puckers, the second type of graph is used to analyze the coupling data in terms of the geometry and population of the most abundant form. It is shown that the latter type of analysis can be carried out on the basis of experimental values for merely sigma 1',sigma 2' and sigma 2'', without any assumptions or restrictions concerning a relation between the geometry of the N- and S-type conformer. In addition, the question is discussed as to how insight can be gained into the conformational purity of the sugar ring from the observed fine structure of the H1' resonance. Finally, a comparison is made between experimental coupling data reported for single-stranded and duplex DNA fragments and covalent RNA-DNA hybrids on the one hand and the predicted couplings and sums of couplings presented in this paper on the other hand.

. Assignments, based upon one-dimensional NOE and homonucleardecoupling and two-dimensional correlated and NOE spectroscopies are given of the resonances of most of the base and sugar protons. Chemical shift vs temperature profiles, constructed for all samples, yielded insight into the temperature-and concentration-dependent conformational behaviour of the compounds and were used to obtain thermodynamic parameters pertaining to the stacked-single-strand + random-coil and duplex randomcoil equilibria. Vicinal proton-proton couplings were analyzed in terms of the conformation of the deoxyribose rings in the single-stranded tetramers and duplexed octamers.The NOE patterns, chemical shift profiles, imino-proton resonances and coupling data revealed that the compounds adopt B-DNA-like structures. The ratio duplexed/stacked-single-strand/random coil depends upon external conditions as well as upon base sequence. The thermodynamic data indicate that: (a) in terms of singlehelical stacking, the R-R steps ( T , 321 -328 K) appear more stable than the Y-R or R-Y steps (T, 308 -316 K) and the Y-Y steps score least (T, 290-300 K), and (b) the duplexes consisting of alternating, d(Y-R),, strands are more stable, in terms of AH", compared to the d(R-R), . d(Y-Y)" duplexes. The analyses of the couplings demonstrated that the sugars of the single-stranded tetramers and duplexed octamers occur as a blend of N -and S-type conformers, with a preference for the S-type (C2'-endo) sugar conformation: upon duplex formation, no significant shift in the N-type/S-type ratio was observed. The fraction S-type sugar conformation of a given residue, % S , in the stacked-single strands was found to depend upon the nature of its own base and that of the adjacent residues: sugars in an R-R stretch display high values of % S (90-loo), whereas those in Y-Y stretches show relatively low values (z 65). It appears that a good stacking interaction between bases goes hand-in-hand with high values of % S.

solution. NMR spectra were observed at 10 mM sample concentration over the temperature range 273 -368 K. Assignments are given of the base, Hl', H2', H2", H3' and of some H4' resonances, based upon a combination of two-dimensional correlation spectra (COSY) and two-dimensional nuclear Overhauser effect spectra (NOESY); imino-proton resonances were assigned with the aid of a two-dimensional NOE experiment. Chemical shift vs temperature profiles were constructed in order to gain insight into the influence of N6-methylation of residue A(5) on the temperature-dependent conformational behaviour of the decamer and to determine thermodynamic parameters for the duplex-to-coil transition. The NOESY spectra, the imino-proton spectra and the shift profiles of the two compounds, under conditions where each forms a B-DNA-type duplex, are very similar. This is taken to indicate that the influence of N6-methylation of residue A(5) on the local structure of the duplex must be small. However, the temperature dependence of the (non-)exchangeable proton resonances of the two compounds reveals that methylation slows down the duplex -single-strand exchange. Furthermore, a thermodynamic analysis of the two compounds indicates that N6-methylation slightly decreases the stability of the duplex relative to the monomeric forms (T, is reduced from 332 K down to 325 K at 10 mM sample concentration). Proton-proton couplings were obtained by means of one-dimensional and two-dimensional NMR experiments and were used in a conformational analysis of the sugar ring of each residue of the two compounds in the duplex form. The analysis indicated that all sugar rings display conformational flexibility in the intact duplex : population S-type sugar conformation ranges from 70% to 100%. A more refined analysis of the sugar rings of the parent compound revealed a sequencedependent variation of the sugar geometry. This variation does not follow well the trend predicted by the Calladine/ Dickerson Z3-sum rule [Dickerson, R. E. (1983) J . Mol. Biol. 166,419-441;Calladine, C. R. (1982) J. Mol. Biol. 161,; moreover the actual variations appear to be smaller in solution than those expected on the basis of known X-ray structures.There is considerable interest in the sequence-dependent properties of DNA and in the possible role that these variations play in the biological functions of DNA. Thus far, much specific information is supplied by X-ray diffraction studies of single crystals of DNA oligomers [l]. Such studies have furnished interesting information concerning the possible interplays between base sequence on the one hand and geometrical variations of backbone and sugar conformation on the other [2, 31. However, in considering such results, one should keep in mind the existence of several complicating factors.Correspondence to C. Altona, Gorlaeus Laboratoria der Rijksuniversiteit te Leiden, Postbus 9502, NL-2300 RA Leiden, The Nethcrlands This paper is number 47 in a series on Nucleic Acid Constituents from this laboratory; part 46 appeared earlier in ...

Proton NMR studies in H 2 0 and 'HzO were carried out on the self-complementary DNA octamer d(G-G-C-C-G-G-C-C) and compared with similar studies on the dimethylated analogue d(G-G-m5C-m5C-G-G-C-C) [Sanderson, M. R., Mellema, J.-R., van der Marel, G. A., Wille, G., van Boom, J. H. & Altona, C. (1983) Nucleic Acids Res. 11, 3333-33461. Base, Hl', H2' and H2" resonances were assigned by means of two-dimensional nuclear Overhauser spectroscopy (NOESY) and correlated spectroscopy (COSY) experiments. Chemical shift vs temperature profiles were used to analyze the temperature-dependent conformational behaviour and to extract thermodynamic parameters for the duplex-to-coil transition. Analysis of proton-proton couplings were used to discriminate between J1.2, and J1,2,, and between the H2' and H2" resonances as well as to obtain conformational parameters of the sugar rings. From the NOESY and COSY experiments, the temperature profiles and the analysis of the coupling data it is concluded that: (a) the compound adopts a B-DNA-type helix in solution; (b) the sugar rings in the intact duplex display limited conformational freedom; (c) methylation of the cytosine bases at the C5 position has no measurable effect on the conformational behaviour of the octamer, nor on the conformation of the sugar rings; however, methylation does increase the stability of the duplex in aqueous solution under conditions of low salt concentration.Recent advances in DNA synthesis have opened the possibility of studying oligonucleotides of known specific sequence. So far, X-ray crystallography and high-field NMR spectroscopy have proved powerful tools in the study of the crystalline and solution structure of oligonucleotides, respectively. 2D NMR techniques have opened the way to the study of long DNA fragments. 2D correlation (COSY) experiments have been found useful to establish scalar connectivities between protons. In this way sets of proton resonances can be distinguished, each set belonging to a different sugar residue. In addition, 2D NOE (NOESY) experiments allow one to find intra-and inter-residual connectivities between base and sugar protons and thus to assign many of the resonances in the proton spectra [l -171. NOESY signal intensities serve to determine the helical type [3, 151 and local structural features of oligonucleotide fragments [5, 7, 18, 191 and, finally, to find answers to questions concerning possible differences between Correspondence to C . Altona, Gorlaeus Laboratoria der Rijksuniversiteit te Leiden,

The solution conformations of the oligonucleotides d(C‐C‐G‐A‐A‐T‐T‐C‐G‐G) and d(C‐C‐G‐A‐m6A‐T‐T‐C‐G‐G) as a function of temperature and sample concentration were investigated by means of 1H‐NMR spectroscopy. The NMR spectra revealed that, at certain combinations of temperature and low sample and salt concentration, both compounds exist as a B‐DNA‐type duplex slowly (on the 1H‐NMR time scale) interconverting with a monomeric species. From chemical shift data and imino‐proton spectra, it is concluded that the monomeric species consists of a mixture of a hairpin form in rapid equilibrium with the random‐coil form. The double‐helical stem of the hairpin is formed by the six terminal cytidine and guanine residues, whereas the four core residues, ‐A‐(m6)A‐T‐T‐, partake in the loop. Thermodynamic analysis of the chemical shift of the resonances of the monomeric species vs temperature profiles of the two decamers and mutual comparison of these profiles indicate the following: (a) the influence of N6‐methylation of residue A(5) upon the local structure of the hairpin must be small; (b) methylation decreases the stability of the duplex relative to the monomeric species: the temperature at which the fraction duplex equals 0.5 was found to be 312 K for the parent compound and 305 K for the methylated decamer at 2 mM sample concentration; (c) methylation does not significantly alter the stability of the hairpin form relative to the random coil form: the Tm of the hairpin ⇌ random‐coil equilibrium is 308 K for the parent compound and 306 K for the methylated decamer. A higher fraction hairpin‐like structure for the N6‐methylated compound is observed under identical conditions of temperature and sample concentration: at 300 K, 2 mM sample concentration, the fraction hairpin form is 0.12 for d(C‐C‐G‐A‐A‐T‐T‐C‐G‐G) and 0.20 for d(C‐C‐G‐A‐m6A‐T‐T‐C‐G‐G). This finding appears to be a consequence of the reduced stability of the methylated dimeric species relative to the monomeric species, and to depend upon the sodium‐ion concentration: it becomes more pronounced under low‐salt conditions.