Structure of branched DNA molecules: Gel retardation and atomic force microscopy studies

DNA heteroduplexes as models for slipped strand DNA have been analyzed by p olyacrylamide gel migration and atomic force microscopy (AFM). All heterodu plexes containing one hairpin or loop have reduced electrophoretic mobiliti es compared with that expected for their molecular weights. The retarded ge l mobility correlates with the formation of a sharp kink detected by AFM. I ncreasing the hairpin length from 7 bp to 50 bp results in a monotonous dec rease in gel mobility of heteroduplexes. This secondary retardation effect appears to depend only on the hairpin size since the AFM data show no depen dence of the kink angle on the hairpin length. Heteroduplex isomers with a loop or hairpin in opposite strands migrate with distinct mobilities. Analy sis of gel migration of heteroduplexes with altered hairpin orientations as well as of truncated heteroduplexes indicates that the difference in mobil ity is due to an inherent curvature in one of the long arms. This is confir med by the end-to-end distance measurements from AFM images. In addition, s ignificant variation of the end-to-end distances is consistent with a dynam ic structure of heteroduplexes at the three-way junction. Double heterodupl exes containing one hairpin in each of the complementary strands also separ ate in a gel as two isomers. Their appearance in AFM showed a complicated p attern of flat representations of the three-dimensional structure and may i ndicate a certain degree of interaction between complementary parts of the hairpins that are several helical turns apart. (C) 1999 Academic Press.