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(ethylphospahte) (17), and adenosine 3′,5′-bis(ethylphospahte) (19) is reported. These compounds, along with methyl β-D-ribofuranosyl-bis-2′,3′-ethylphosphate (9), were used as reference systems for 31P and1H-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 temperture 31P-NMR experiments. The data indicate that the contribution of g,g around the P-O3′ (ζ) and P-05′ (α) boonds is significantly greater for the 2′-phosphate group than for the 3′-phosphate grooup. 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′ stacking 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 of31P-NMR experiments. The results obtained with the series of compounds 20 (2′p5′G3′p5′U), 26a (UA2′p5′G3'o5′U, 27 (A2′p5′GU3′p5′UC), 28 (CUA2′p5′GU3′p5′UC), 29 (CUA2′p5′GUG3′p5′UCA), and 30 (CCUA2′p5′GUG3′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 (A2′p5′g3′p5′U) (31 P 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),???(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.