Intramolecular stacking association and conformation properties of a 'cap' structure, m7G5'pppUm, and the related model compounds.

The stacking equilibrium quotient of the m7G5'pppUm unit, which occurs as the 5'-terminal "cap" of certain eukaryotic mRNA's, was determined by temperature-dependent difference spectrophotometry as Kstack = 1.82 at 25 degrees and pH 5. In order to evaluate the contribution of different structural modifications to the net stabilization of the cap structures of mRNA, a variety of compounds related to m7G5'pppUm were synthesized and their stacking properties were studied by the same method and compared. The results are summarized as: (1) Introduction of a methyl group into N-7 of G residue results in an increase in base stacking. (2) Methylation at 2'-OH of U residue also stabilizes the stacked structure of G-containing dimers, but it does not influence stacking interaction in m7G-containing dimers. (3) The effect of different types of internucleotide linkages on the order of stacking tendencies is: N5'ppN' greater than N5'pppN' greater than NpN'. UV hypochromicity and CD spectral measurements of the relevant dimers were also conducted, and the hypochromicity values and CD spectra of dimers in their stacked conformation were estimated by making use of the determined Kstack values. The results indicate that, while 2'-O-methylation exerts very little effect on the stacked conformation of the dimers, methylation at N-7 and the nature of the internucleotide linkage strongly influence the stacked conformation, thereby forming unusual left-handed conformations in m7G5'pppU(m), m7G5'ppU(m), and G5'ppU(m).