Assessment of competing 2'→5' versus 3'→5' stackings in solution structure of branched-RNA by 1H- and 31P-NMR spectroscopy

Article abstract of DOI:10.1016/S0040-4020(01)88130-8

Preparation of five novel phosphorylated derivatives of adenosine, i.e. adenosine 2',3'-bis(ethylphosphate) (11), adenosine 2',3'-bis(phosphate) (13), adenosine 2',3',5'-tris(ethylphosphate) (15), adenosine 2',5'-bis(ethylphosphate) (17), and adenosine 3',5'-bis(ethylphosphate) (19) is reported. These compounds, along with methyl β-D-ribofuranosyl-bis-2',3'-ethylphosphate (9), were used as reference systems for ³¹P and ¹H-NMR conformational studies on the branched RNA structures 20-30. Compounds 11, 13, 15, 17, and 19 preserve the essential structural elements of the branch point adenosine, while the intramolecular base stacking interactions are removed. The ³¹P-NMR chemical shifts of 20-30, referenced against 9, 11, 13, or 15, show a pattern that is generally consistent with our previous results from variable temperature ³¹P-NMR experiments. The data indicate that the contribution of g,g around the P-O3' (ζ) and P-O5' (α) bonds is significantly greater for the 2'-phosphate group than for the 3'-phosphate group. These results point towards preferential 2'→5' rather than 3'→5' base stacked structures in all of these synthetic models of the lariat. This is especially the case for the branched trimer 20 and the pentamer 27 which are part of a naturally occurring lariat structure. Note that the strongest 2'→5' stackins is however found in the unnatural trimers 22 and 23 in which the 2'-linked residue is a pyrimidine nucleotide. Compounds 11, 13, and 15 were also used to calibrate the ¹H-NMR oligomerization shifts of the H2 protons of the branch-point adenosine. These data show a consistency with the results from variable temperature ¹H-NMR experiments, as well as with the results of ³¹P-NMR experiments. The results obtained with the series of compounds 20 (A(2'p5'G)/(3'p5'U)), 26a (UA(2'p5'G)/(3'p5'U)), 27 (A(2'p5'GU)/(3'p5'UC)), 28 (CUA(2'p5'GU)/(3'p5'UC)), 29 (CUA(2'p5'GUG)/(3'p5'UCA)), and 30 (CCUA(2'p5'GUG)/(3'p5'UCA)) are of special interest since these structures are constituents of the naturally occurring lariat in the excised intron in Group II splicing of bl 1 of Yeast mitochondria. Qualitatively, the present ¹H- and ³¹P-NMR data on 26a, 27, 28, 29, and 30 show 2'→5' base stacking of an intermediate strength: 2'→5' base stacking is substantially stronger for trimer 20 and pentamer 27. This difference is ascribed to the 5'-conformational transmission effect owing to the presence of at least one nucleotide upstream of the branch-point. 5'-Conformational transmission appears to weaken the 2'→5' stacking at the expense of some 3'→5' stacking. The experimental data on the conformation of 20 (A(2'p5'G)/(3'p5'U)) (³¹P and ¹H chemical shifts, vicinal ¹H-¹H, ¹H-³¹P, and ¹³C-³¹P coupling constants) formed the basis for a series of AMBER molecular mechanics calculations. These molecular modelling studies allowed us to conclude that g,g conformation in the 2'-phosphate group is primarily g^-(ζ),g^-(α). This is found to be the only conformation that gives 2'→5' base stacking as evident in the temperature dependent chemical shift and in the oligomerization shift studies. Modelling studies furthermore showed two energetically possible ζ and α torsions for the 3'-phosphate group (g^-(ζ),g^-(α) and g^-(ζ),t(α)). The present use of reference compounds 9, 11, 13, 15, 17, and 19 has led to a refined and partially revised concept for the conformational description of oligomeric branched RNAs.