Solution structure of a five-adenine bulge loop within a DNA duplex

The three-dimensional solution structure of a DNA molecule of the sequence 5'-d(GCATCGAAAAAGCTACG)-3' paired with 5'-d(CGTAGCCGATGC)-3' containing a f ive-adenine bulge loop (dA(5)-bulge) between two double helical stems was d etermined by 2D H-1 and P-31 NMR, infrared, and Raman spectroscopy. The DNA in both stems adopt a classical B-form double helical structure with Watso n-Crick base pairing and C2'-endo sugar conformation. In addition, the two dG/dC base pairs framing the dA(5)-bulge loop are formed and are stable at least up to 30 degrees C. The five adenine bases of the bulge loop are loca lized at intrahelical positions within the double helical stems. Stacking o n the double helical stem is continued for the first four 5'-adenines in th e bulge loop. The total rise (the height) of these four stacked adenines ro ughly equals the diameter of the double helical stem. The stacking interact ions are broken between the last of these four 5'-adenines and the fifth lo op adenine at the 3'-end. This 3'-adenine partially stacks on the other ste m. The angle between the base planes of the two nonstacking adenines (A10 a nd A11) in the bulge loop reflects the kinking angle of the global DNA stru cture. The neighboring cytosines opposite the dA(5)-bulge (being parts of t he bulge flanking base pairs) do not stack on one another. This disruption of stacking is characterized by a partial shearing of these bases, such tha t certain sequential NOEs for this base step are preserved. In the base ste p opposite the loop, an extraordinary hydrogen bond is observed between the phosphate backbone of the 5'-dC and the amino proton of the 3'-dC in about two-thirds of the conformers. This hydrogen bond probably contributes to s tabilizing the global DNA structure. The da(5)-bulge induces a local kink i nto the DNA molecule of about 73 degrees (+/-11 degrees). This kinking angl e and the mutual orientation of the two double helical stems agree well wit h results from fluorescence resonance energy transfer measurements of singl e- and double-bulge DNA molecules.