NMR and model-building studies were carried out on the duplex d(CTGGTGCGG).d(CCGCCCAG), referred to as (9+8)-mer, which contains an unpaired thymidine residue. Resonances of the base and of several sugar protons of the (9+8)-mer were assigned by means of a NOESY experiment. Interresidue NOEs between dG(4) and dT(5) as well as between dT(5) and dG(6) provided evidence that the extra dT is stacked into the duplex. Thermodynamic analysis of the chemical shift vs temperature profiles yielded an average TmD value of 334 K and delta HD of -289 kJmol-1 for the duplex in equilibrium random-coil transition. The shapes of the shift profiles as well as the thermodynamic parameters obtained for the extra dT residue and its neighbours again indicate that the unpaired dT base is incorporated inside an otherwise intact duplex. This conclusion is further supported by (a) the observation of an imino-proton resonance of the unpaired dT; (b) the relatively small dispersion in 31P chemical shifts (approximately 0.5 ppm) for the (9+8)-mer, which indicates the absence of t/g or g/t combinations for the phosphate diester torsion angles alpha/zeta. An energy-minimized model of the (9+8)-mer, which fits the present collection of experimental data, is presented.

Model-building studies were carried out on the trimer AUA. Bulge-out structures which allow incorporation into a continuous RNA helix were generated and energy-minimized. All geometrical features obtained by previous NMR studies on purine-pyrimidine-purine sequences are accounted for in these models. One of the models was used to fit into a double helical fragment. Only minor changes were necessary to construct a central bulge-out in an otherwise intact duplex. NMR and model-building studies were performed on the duplex (CUGGUGCGG).(CCGCCCAG) which contains an unpaired uridine residue. NOE data, chemical-shift profiles and imino-proton resonances provided evidence that the extra U is bulged out of the duplex. The relatively small dispersion in 31P chemical shifts (approximately equal to 0.7 ppm) indicate the absence of t/g or g/t combinations for the phosphodiester angles zeta/alpha. An energy-minimized model of the duplex, which fits the present collection of data, is presented.

NMR studies were carried out on some alternating pyrimidine-purine sequences : the single-stranded tetramers CACA and UGUG and the self-complementary octamer CACAUGUG. Assignments, based upon COSY, homonuclear Hartmann-Hahn, and NOESY experiments, are given for the resonances of all base protons and of several sugar protons. Chemical shift vs temperature profiles were used to obtain thermodynamic parameters for the single-stranded stack P random coil and the duplex $ random coil equilibria. The populations of N-type conformer of the ribose rings were estimated from the observed .Ilf2,. Comparisons with another alternating pyrimidine-purine sequence Um;AUm$A and with the deoxyribose counterparts d(CACA), d(TGTG) and d(CACATGTG) are given.Previous 'H-NMR investigations of UmZAUmgA revealed that the population of bulge-out structure diminishes compared to m;AUm$A due to the U(l)-m;A(2) stacking interaction. In CACA a strong stacking proclivity (T, = 310 K) together with a clear preference for N-type ribose is observed. However, the stacking interactions in UGUG are relatively less stable (T, = 288 K) and a bias towards S-type sugar is present. Besides a small amount of stack, a significant contribution of bulge-out structure is proposed for UGUG. We conclude that the nature of the pyrimidine base mainly determines the formation of bulge-out structures. The poor stacking properties of uracil now appear to be mainly responsible for this phenomenon. Comparison with the deoxyribose counterparts shows a reasonable agreement between the T, values of CACA and d(CACA), whereas the T, of UGUG (288 K) is much lower than the T , of d(TGTG) (315 K). It is suggested that the absence of bulge-out structures in DNA purine-pyrimidine-purine sequences is related to the relatively strong stacking proclivity of dT residues compared to that of U residues.The T, values (average 341 K) for the duplex + random coil transition obtained for each residue of CACAUGUG appear very similar. All ribose rings, except the G(8), adopt a pure N conformer in the duplex. This is taken to mean that the differences in conformational behaviour of the constituent tetramers disappear upon duplex formation.NMR and CD studies on short RNA fragments have provided much information about the preferred conformation of these fragments in solution. For fragments containing a purine-pyrimidine-purine base sequence (R-Y-R) the existence of a particular structure, denoted bulge-out, was proposed by Lee and Tinoco [l]. In this structure the pyrimidine residue is thought to reside outside the stacking influence of its neighbouring purines and at the same time a next-nearestneighbour interaction between the two purines is present. Earlier CD experiments, performed by Gray et al. [2], support the idea of a decreased nearest-neighbour stack in R-Y-R sequences. Also, the NMR experiments performed in our laboratory on fragments containing AUA sequences (A = m6,A) [3-61 generally agree with such a bulge-out model, albeit the bulge-out is visualized as a mixture of states.How...

A 1H-NMR investigation was carried out on the tetranucleotides U-m6(2)A-U-m6(2)A and m6(2)A-m6(2)A-U-m6(2)A (m6(2) = N6-dimethyladenosine) as well as on the hybrid trinucleotide dA-r(U-A). An extensive comparison with m6(2)A-U-m6(2)A and other relevant compounds is made. Previous proton NMR studies on trinucleotides have shown that purine-pyrimidine-purine sequences prefer to adopt a mixture of states which have as a common feature that the interior pyrimidine residue bulges out, whereas the flanking purine residues stack upon each other. A stacking interaction on the 3' side of the bulge is known to have no measurable effect on the bulge population. Chemical-shift data, ribose ring conformational analysis and information from NOE experiments now show unambiguously that the moderate U(1)-m6(2)A(2) stack in U-m6(2)A-U-m6(2)A diminishes the population of bulged-out structures in favour of a regular stack. This tendency towards conformational transmission in the downstream 5'----3' direction is fully confirmed by the fact that the strong m6(2)A(1)-m6(2)A(2) stack in the tetranucleotide m6(2)A-m6(2)A-U-m6(2)A virtually precludes the formation of bulged-out structures. The conformational characteristics of dA-r(U-A) appear comparable with those of m6(2)A-U-m6(2)A, which indicates that the presence of a 2'-hydroxyl group in the first purine residue is not a necessary prerequisite for the formation of a bulge.

NMR and CD studies were carried out on the dinucleotides 5'-methylphospho-N 6-dimethyladenylyl-uridine (mpmf-U) and 5'-methylphospho-uridylyl-N6-dimethyladenosine (mpU-mZ A) and on the trinucleotide U-mzA-U.A detailed comparison is given of the conformational features of mpmfA-U and mpU-m$A with the corresponding 5'-nonphosphorylated dinucleotides mqA-U and U-mqA, respectively. The behaviour of the trinucleotide U-mzA-U is compared with the properties of the constituent dinucleotides U-mqA and mpmq A-U.Chemical-shift and CD data were used to determine the amount of stacking interactions. For each compound NMR spectra were recorded at two or three sample concentrations in order to separate intermolecular and intramolecular base-base interactions. The coupling constants of the ribose ring are interpreted in terms of the N/S equilibrium, and population distributions along the backbone angles fl, y and E are presented.The combined data indicate a strong similarity between mpmqA-U and m$A-U both in degree and in mode of stacking. In contrast, the existence of different types of stacking interactions in mpU-m$A and U-mzA is suggested in order to explain the NMR and CD data. It is concluded that dinucleoside bisphosphates serve better as a model for the behaviour of trinucleotides than dinucleoside monophosphates.The trinucleotide U-mqA-U adopts a regular single-stranded stacked RNA structure with preference for N-type ribose and y+ and p' backbone torsion angles. The difference in behaviour between the U-mfA-part of U-mfA-U and the dimer U-mzA is seen as a typical example of conformational transmission.The biological activity of polynucleotides is determined mainly by the three-dimensional structure of these polymers. It is well known that the vertical base-base stacking constitutes a dominant driving force in the formation of stable RNA and DNA structures. For example, in the case of the codonanticodon interaction, the stacking energies of the participant bases are crucial for the conformation of the anticodon loop and the codon-anticodon complex [l, 21. This might be an important feature for the translation activity.Seen in this light, studies of the conformational behaviour of single-stranded oligonucleotides pursued in this laboratory and elsewhere are quite useful because these yield valuable information about stacking properties. Of special interest are differences in behaviour between dinucleoside monophosphates, trinucleotides, tetranucleotides and longer oligomers.Correspondence to C. Altona, Gorlaeus Laboratories, P.O. Box 9502, NL-2300 RA Leiden, The Netherlands Note. This is part 54 of the series Nucleic Acid Constituents from this laboratory, part 53 will appear later [Orbons, L. P. M., van Such studies may provide insight into the factors important for base-base stacking, like base sequence, electrostatic repulsion between the phosphate groups, conformational preferences of the sugar ring and backbone chain, and conformational transmission effects.For example, 'H-NMR [3 -51 and CD [6] investigations c...