A DNA hairpin with a single residue loop closed by a strongly distorted Watson-Crick G center dot C base-pair

Our previous NMR and modeling studies have shown that the single-stranded 1 9mer oligonucleotides d(AGCTTATC-ATC-GATAA GCT) -ATC- and d(AGCTTATC-GAT-GA TAAGCT) -GAT- encompassing the strongest topoisomerase II cleavage site in pBR322 DNA could form stable hairpin structures. A new sheared base-pair, t he pyrimidine-purine C.A, was found to close the single base -ATC- loop, wh ile -GAT- displayed a flexible loop of three/five residues with no stabiliz ing interactions. Now we report a structural study on -GAC-, an analog of - GAT-, derived through the substitution of the loop residue T by C. The resu lts obtained from NMR, non-denaturing PAGE, UV-melting, circular dichroism experiments and restrained molecular dynamics indicate that -GAC- adopts a hairpin structure folded through a single residue loop. In the -GAC- hairpi n the direction of the G9 sugar is reversed relative to the C8 sugar, thus pushing the backbone of the loop into the major groove. The G9.C11 base-pai r closing the loop is thus neither a sheared base-pair nor a regular Watson -Crick one. Although G9 and C11 are paired through hydrogen bonds of Watson -Crick type, the base-pair is not planar but rather adopts a wedge-shaped g eometry with the two bases stacked on top of each other in the minor groove . The distortion decreases the sugar C1'-C1' distance between the paired G9 and C11, to 8 Angstrom versus 11 Angstrom in the standard B-DNA. The A10 r esidue at the center of the loop interacts with the G9 C11 base-pair, and s eems to contribute to the extra thermal stability displayed by -GAC- compar ed to -GAT-. Test calculations allowed us to identify the experimental NOEs critical for inducing the distorted G.C Watson-Crick base-pair. The prefer ence of -GAC- for a hairpin structure rather than a duplex is confirmed by the diffusion constant values obtained from pulse-field gradient NMR experi ments. All together, the results illustrate the high degree of plasticity o f single-stranded DNAs which can accommodate a variety of turn-loops to fol d up on themselves. (C) 1999 Academic Press.