Synthesis of tetrameric branched RNA-DNA conjugate & branched-RNA analogue & their comparative conformational studies by 500 MHz NMR spectroscopy

We report herein the unambiguous synthesis of pure tetrameric branched oligonucleotides A3'p5'G3'p5'C2'p5'[dC] (13) found naturally in gram-negative bacterium Stigmatella aurantiaca, and corresponding branched RNA analogue A3'p5'G3'p5'C2'p5'C (14). The conformational features of branched tetramers 13 and 14 have been elucidated and compared by assesing temperatue- and concentration-dependent H-1 and P-31 chemical shifts, (C2'-exo and C3'-endo) reversible (C2'-endo, C3'-exo) equilibrium, and equilibrium amongst staggered gamma and beta-rotamers using various 2D homo- and heteronuclear correlation, NOESY and ROESY experiments by 500 MHz NMR spectroscopy. Subsequently the conformational features of 13 and 14 have been compared with those of A3'p5'C2'p5'G (ref. 19) and U3'p5'A3'p5'C2'p5'G (ref. 24) found as the branch-point in the lariat formed in the pre-mRNA processing reaction (Splicing). These studies have clearly shown that (1) the intramolecular geometries of both 13 and 14 are dominated by stacking along the axis A3' --> 5'G2' --> 5'dC(C), but the RNA-DNA conjugate 13 has a more defined tertiary structure than that of 14, (2) these branched tetramers tend to associate intermolecularly above approximately 2 mM concentration producing an aggregate which is vertically stacked along the axis A3' --> 5'G2' --> 5'dC(C), (3) the G2' --> 5'dC(C) stacking and the predominant S conformation of branch-point G found in 13 and 14 suggest that their structures are quite different from the ones found for U3'p5'A3'p5'C2'p5'G (ref. 24). Note however that the structures found for 13 and 14 are reminiscent of A2' --> 5'G stacking found in the branched trimer A3'p5'C2'p5'G (ref. 19).