NMR-STUDIES AND CONFORMATIONAL-ANALYSIS OF A DNA 4-WAY JUNCTION FORMED IN A LINEAR SYNTHETIC OLIGONUCLEOTIDE

A linear DNA oligomer (M(r) 14 000, 46 nucleotides) was especially des igned, chemically synthesized, and studied by means of H-1 NMR spectro scopy. The design of the oligomer was guided by the idea that incorpor ation of three short palindromic sequences, each interspersed by 5'-CT TG-3' motifs at predetermined positions in the oligomer, would give ri se to the formation of three stable minihairpin loops [Ippel, J. H., e t al. (1992) J. Biomol. Struct. Dyn. 9, 1-16], which in turn were expe cted to encourage further folding of the strand into a stable four-way junction containing three ''hairpin'' arms and an open-ended duplex s tem as the fourth arm. Linear DNA four-way junctions constructed accor ding to this concept can be more compact and are therefore expected to be more suitable as model compounds for conformational studies compar ed to junctions that are built from two or more separate strands. A st able cruciform conformation was substantiated for the 46-mer in aqueou s solution in the presence of Mg2+ Complete sequential H-1 NMR assignm ents of the nonexchangeable protons (except H4', H5', and H5'') were o btained with the aid of NOESY and HOHAHA experiments. The NMR data gav e evidence for the expected existence of minihairpin-loop structures a t the three 5'-CTTG-3' motifs in the sequence. The complementary stem domains adopt a regular B-DNA form. Watson-Crick type base pairing is preserved for all residues in the stem domains, including the residues at the center of the junction. A systematic investigation of the inte rresidual NOEs observed between the protons of the eight central resid ues revealed the complete stacking pattern of the residues at the bran ch point.