Why do all lariat RNA introns have adenosine as the branch-point nucleotide? Conformational study of naturally-occurring branched trinucleotides and its eleven analogs by proton, phosphorus-31 NMR and CD spectroscopy

H-NMR conformational studies of six branched triribonucleotides where the branch-point nucleotide was either U, C or G (4–9) have been carried out by assigning 1H resonances through 2D NMR and then observing the temperature-dependent (i) chemical shifts of the aromatic and the anomeric protons, and (ii) shifts of the equilibrium of N and S pseudorometer popolations of each sugar moiety. The data have been compared with those of 2' ¿ 5' dimers (1–3) and other branched trimers (10–16). It emerged that all the branched trimers (4–16) adopt a conformational state closer to the corresponding 2' ¿ 5' dimers than the corresponding 3' ¿ 5' dimers. A temperature-dependent 31P chemical shift study confirmed that the conformational constraint is mainly associated with the 2' ¿ 5' phosphate linkage. Although, it appeared with the CD data that when C or especially when U is at the branch-point the overall constraint is weak. This suggests that even if these trimers adopt a 2' ¿ 5' dimer geometry, there is a lack of stabilization of strong stackings within the molecule. This is in sharp contrast with the results found for A (10–16) and to a smaller extent for G (8–9) at the branch-point.