On the base-stacking in the 5′-terminal cap structure of mRNA: a fluorescence study

Abstract

The fluorescence at 370 nm of the 7-methylguanosine residue (m⁷G) is found to be quenched when the base residue is involved in a stacking interaction with the adenosine residue in the cap structure m⁷G^5′pppA of an eukaryotic mRNA. On the basis of the observed degree of quenching, the amounts of the stacked and unstacked forms in the cap structure have been determined at various temperatures and pH's. It has been found that at pH 6.2 effective enthalpy and entropy in the unstacked → stacked change are ΔH° = −4.4 ± 0.1 kcal/mole and ΔS° = − 14.3 ± 0.2 e.u., respectively. The pk_a value for the m⁷G residue is found to be 7.7 at 10°C and 7.3 at 30°C. The stacked structure seems to be less favourable in the deprotonated form that occurs in the higher pH solution. A similar analysis of some other cap structures indicates that the stacked form in m⁷G^5′ pppN structure is favourable if N is a purine nucleoside or a 2′-O-methylpyrimidine nucleoside but not for an unmethylated pyrimidine nucleoside.