Chemical shifts of base and sugar protons of the modified ribodinucleoside monophosphate Nh-dimethyladenylyl(3'-5')uridine (m4A-U) were measured at 100, 360 and 400 MHz in aqueous solution. Seven different samples were used with concentrations ranging from 0.28 mM to 32.7 mM. The temperature was varied from -5 "C to 105 "C. An internal temperature calibration was used. The effects of intermolecular self-association and of intramolecular stacking on the chemical shifts were quantitatively separated by means of a new approach: differential concentration/temperature profiles (DCTP). Several computational models were tested and the analysis allowed deeper insight into the behaviour of mgA-U at the molecular level. The simple two-state approach for both self-association and stacking already afforded a significant improvement over models in which the association is entirely neglected. A computer least-squares analysis of the chemical shift behaviour of each individual proton yielded thermodynamic parameters for self-association and stacking. However, the two-state model did not suffice to reproduce accurately all of the observations. A satisfactory fit required two additional assumptions : (a) the aromatic protons experience different association shifts in stacked and in unstacked molecules ; (b) a temperature-dependent conformational equilibrium exists between sets of unstacked microstates. The stacked state is taken to represent a single conformational species. The implementation of this extended model in the leastsquares optimization allowed the reproduction of over one thousand chemical shift observations within experimental error. Thermodynamic equilibrium parameters deduced for intramolecular stacking are : AH; = -28.8 kJ rnol-', A S ; = -93 J mol-' K-'. These numbers agree well with those obtained earlier by us from circular dichroism spectra. The equilibrium enthalpy and entropy values deduced for the association process are : AH; = -35 kJ mol-' and AS2 = -95 J mol-' K-'.The question of whether short oligonucleotides are really suitable as models for longer regions in nucleic acids has been raised [l], but cannot be answered until many careful systematic studies are carried out with experimental probes at atomic resolution. The most potent approach is offered by nuclear magnetic resonance (NMR) measurements, supplemented with thermodynamic information from independent techniques such as circular dichroism (CD) and ultraviolet hypochromism, and guided by information on geometrical details by X-ray methods.The shortest nuclei acid fragments available for studies of these properties, the dinucleoside monophosphates or dimers (L-M) represent at the same time 3'-terminal and 5'-terminal fragments of RNA (or DNA); therefore; these fragments constitute a limiting case. Therefore, attempts at This paper is part 24 in the series Nucleic Acid Cotzstituents from this laboratory; part 23 appeared elsewhere [4].Ahhrcviurions. CD, circular dichroism; NMR, nuclear magnetic resonance; Me4NC1, tetramethylammonium chloride; m$A...
The complete and unequivocal assignment of the 24 ribose proton signals of m+A(I)-U(2)-m §A(3)-U(4) by means of 500 MHz NMR spectroscopy at 170C is given. This assignment is based on scrupulous decoupling experiments carried out at various temperatures. Analysis of the observed chemical shifts and coupling constants of the tetramer shows that the two fragments -u4A(3)-U(4) comprising the 3'-end occur mainly in the classical right-handed stack conformati"on, whereas at the 5'-end the -U(2)-residue appears bulged ogt in favour of a-less well-defined stacking interaction between the bases m2A(I)-and -m4A(3)-. Conformational populations about each of the torsional degrees of freedom along the backbone are discussed. A modernized version of pseudorotation analysis is used to delindate the conformational behaviour of the four ribose rings.
NMR and CD studies were carried out on the dinucleotides 5'-methylphospho-N 6-dimethyladenylyl-uridine (mpmf-U) and 5'-methylphospho-uridylyl-N6-dimethyladenosine (mpU-mZ A) and on the trinucleotide U-mzA-U.A detailed comparison is given of the conformational features of mpmfA-U and mpU-m$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...
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