STRUCTURAL-ANALYSIS OF DNA BENDING INDUCED BY TETHERED TRIPLE-HELIX FORMING OLIGONUCLEOTIDES

In order to monitor DNA flexibility, we have recently reported the des ign of an artificial DNA bending system consisting of two triple helix forming oligonucleotides (TFOs) connected by a flexible linker [Akiya ma, T., & Hogan, M. E. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 12122- 12127], which spans a single turn of DNA helix. Those data suggested t hat up to 60 degrees of bending deformation could be induced with an e xpenditure of energy which is much smaller than predicted from bulk fl exibility parameters. In this report, the detailed structure of the be nd has been investigated by three different methods: circular permutat ion analysis, phasing analysis, and ring closure. Circular permutation and phasing analysis suggest that the magnitude of the bend is depend ent on linker length. The apparent location of the bend was estimated from circular permutation analysis to be at the duplex region interven ing the two sites of triple helix formation. The electrophoretic mobil ity of the bent complex appears to vary with the sequence of the inter vening duplex region of the binding site complex, in the order of AT-r ich >random greater than or equal to CC-rich sequence. Detailed fittin g of the phasing data has shown that bending is not accompanied by sig nificant twisting deformation. Ring closure analysis with T-4 DNA liga se has confirmed the general magnitude of the TFO-induced bend and has additionally suggested that formation of the simple linear antiparall el triple helix does not enhance DNA flexibility.