Minor nucleosides in RNA: optical studies of dinucleoside phosphates containing inosine

From the study of circular dichroism (CD) spectra and hypochromism we conclude that the dinucleoside phosphates IpA, ApI, and IpI stack, while IpU stacks very little. Studies with various concentrations of IpI in low salt, and 1M NaCl indicate that the stacking geometry of this compound is sensitive to the ionic strength of solution. The CD of poly I is presented and compared to the data for IpI. Little change was found in the CD of poly (G,I) (1 : 1) with change of salt concentration, and we conclude that, unlike Poly I, there is no major structural change. From the CD of poly (G, I), IpI, and GpG, the CD of IpG plus GpI is calculated by using the nearest-neighbor approximation. From the calculated spectrum, we tentatively conclude that there is stacking in either IpG or GpI or both.     

Temperature-dependent properties of dinucleoside phosphates

The optical rotatory dispersion, hypochromism, and proton magnetic resonance were measured for a number of dinucleoside phosphates in both dilute salt solutions and in 25.2% LiCl (which freezes below ?70°C.) as a function of temperature. Two models for the “disordering” of dinucleoside phosphates: the two-state model and the oscillating dimer model, are used to analyze the data. Both models fit some of the data, but neither are completely satisfactory. Evidence is given for a dynamic structure of dinucleoside phosphates, in which the bases oscillate with respect to one another, remaining parallel to one another with no solvent in between, even at high temperatures. Implications of this model to the structures of single-strand sections of RNA's are discussed.     

Conformations of dinucleoside phosphates in aqueous solution

The conformations of dinucleoside phosphates have been reexamined by semiempirical potential energy calculations. Conformations I, II, and III, proposed by Lee & Tinoco [Lee, C. H., & Tinoco, I., Jr. (1977) Biochemistry 16, 5403], are possible species after refinement of their structures by potential energy minimization. These three conformers can represent three types of dinucleoside phosphate species in solution. Dhingra et al. [Dhingra, M. M., Sarma, R. H., Giessner-Prettre, C., & Pullman, B. (1978) Biochemistry 17, 5815] had concluded that conformations of type II and III were unlikely or impossible. They favored conformations g-g- (equivalent to I), g+g+,g+t, and tg+; the last three conformations have little stacking and are calculated to be energetically less favorable by more than 5 kcal/mol. Common structures of the types I, II, and III are found for dinucleoside phosphates with different purine-pyrimidine sequences. The sequence dependence of the potential energy of these three conformers has been calculated. The experimental nuclear magnetic resonance data of dinucleoside phosphates are consistent with these three conformations.     

RNA double helices generated from crystal structures of double helical dinucleoside phosphates.

The dinucleoside phosphates ApU and GpC form right-handed anti-parallel double helical fragments within their crystal lattices. Using a least squares procedure, we have generated the extended double helices which these fragments represent. ApU corresponds to a double helix with 11.9 residues per turn and a pitch of 28. 1Å. The GpC double helix has 10.4 residues per turn and a pitch of 26. 9Å.     

Conformation of dinucleoside phosphates: the conformation encoding hypothesis

The temperature dependence of the spin-lattice relaxation rate of nucleic bases protones and HI' of ApA, ApC, CpA and CpC (D2O, pH 7) were measured. The possible closed conformers of these dinucleoside phosphates (DNP) were computed by atom-atom potential method. On the basis of conformational calculation and experimental data the composition of closed state was determined. Besides the right-handed "canonic" conformers, the "non-canonic" right- and left-handed conformers were shown to be present in the solution of all DNP studied. It is important to note that, "canonic" conformers of DNP studied being equally probable, the possibility of the realization of "non-canonic" conformers is determined by the nucleotide sequence. It may be expected that different nucleotide sequences have unique "non-canonic" conformations. That type of dependence of the spatial organization of polynucleotides on its nucleotide sequence we call "the conformational encoding".     

Theoretical investigation on the hypochromism of dinucleoside phosphates]

The hypochromism of stacked dimers of the nucleotide bases taken as models of the dinucleoside phosphates and dinucleotides was studied with the use of the configuration interaction and pertubation theory methods. General expression for the hypochromism of the polynucleotides is given in the first order perturbation theory with three different ways for approximation of the matrix elements of the perturbation operator. This expression was used for calculation of the dimer hypochromism in terms of theoretically calculated monomer characteristics. Dependence of the hypochromism on the dimer conformation was investigated. The results obtained so far demonstrate that it is important to take into account the electronic transitions in the vacuum UV region. This approach will enable one to elucidate the contribution of neighbouring bases into the DNA hypochromism.     

Stacking and stability of RNA duplexes containing fluorobenzene and fluorobenzimidazole nucleosides

Six different fluorobenzene or fluorobenzimidazole ribonucleosides and one abasic site were incorporated in oligoribonucleotides. Individual contributions of base stacking and solvation of the modified nucleosides could be determined. In fluorobenzene.fluorobenzimidazole-modified base pairs a duplex stabilizing force was found that points to a weak F...H hydrogen bond. The lipophilicity of the unprotected nucleosides were investigated by determination of 1-octanol water partition coefficients.     

PPP calculations of the circular dichroism spectra of some dinucleoside phosphates

Circular dichriosm (CD) spectra have been calculated for serveral dinucleoside phosphates using a variant of the Pariser-Parr-Pople π-electron molecular orbital method. This method does not require the prior knowledge of the experimental absorption spectra of transition moments of the bases forming the dinucleoside phosphates. Calculated spectra were obtained in good agreement with experimental spectra for four dinucleoside phosphates, ApA, UpU, GpA, and UpA, and reasonable agreement was obtained for ApG and ApU. The effect of changing conformation on the CD spectrum was studied for ApA, UpU, UpA, and ApU; the spectra of UpU, UpA, and ApU were sensitive to small change in conformation, whereas ApA was insensitive over the range of conformation studied. Further studies await detailed knowledge of the structure of dinucleoside phosphates in solution.     

Synthesis and properties of photolabile caged phosphotriester derivatives of dinucleoside phosphates

Dinucleoside phosphates that harbor phosphate groups transiently blocked (caged) by o-nitrobenzyl or o-nitroveratryl residues were synthesized. It was shown that the conditions of the UV-induced deprotection largely depend on the nature of the protective group. The phosphotriesters obtained were resistant toward snake venom phosphodiesterase and nucleases of the cellular extract. The synthesis of the dinucleoside phosphates containing a photolabile group preceded the incorporation of the modified blocks into extended oligonucleotides by the phosphoramidite method.     

Sequence specific fragments in the field desorption mass spectra of dinucleoside phosphates

In field desorption mass spectrometry of ribodinucleoside phosphates the formation of nucleoside cyclophosphates can be used to determine the base sequence.