Determination of DNA helical handedness by fluorescence resonance energy transfer.

Fluorescence resonance energy transfer (FRET) has been used to determine the helical handedness, twist and rise of different DNA conformations. The approach is based on the construction of a set of molecules consisting of two fused helical segments, one of which is in a known reference helical form. The duplexes are covalently labeled at one end with a donor and at the other with an acceptor. By systematically shifting the position of the junction while maintaining constant the total length in base-pairs, the variation in the efficiency of energy transfer can be shown to depend primarily and sensitively on the differences in helical twist and rise of the two constituent segments. If the latter have the same helical sense, one predicts a FRET signal that is a monotonic function of the junctional position. In contrast, a periodic function arises when two segments are of opposite handedness. The formalism includes explicit consideration of dye orientation (the dipole-dipole orientation factor kappa) and an implementation valid for single helix molecules, and introduces new functions of measured fluorescence signals for establishing the FRET efficiency. The method has been applied to a family of oligonucleotides forming hairpin duplexes containing an antiparallel-stranded (aps) d(m5C.G)m segment labeled at the 5' end with fluorescein (donor) and a second parallel- stranded d(A.T)N-m segment (psAt-DNA) labeled at the hairpin loop with the sulfoindocyanine dye Cy3. The segment lengths were in the range 4 to 12, but the total length N was maintained constant at 16. The d(m5C.G) sequence was chosen due to its capacity for adopting a B or a Z conformation at low and high concentrations of salt, respectively. The parallel-stranded d(A.T) sequence served as the second segment in order to determine the helical rise and twist of psAT-DNA, presumed to be right-handed from molecular modeling and a prior study of topologically constrained DNA. A Z-DNA/ps-DNA junction was created between the two segments by inducing a B-Z transition in d(m5C.G)m with MgCl2. The range of required salt concentration was established by circular dichroism measurements. FRET efficiency values of 0.38 to 0.41 were obtained for the oligonucleotides with the d(m5C.G) segment in the B conformation. In contrast, upon induction of the B-Z transition the FRET efficiency was a decreasing function of the d(m5C.G) content (0.38 to 0.28 for m = 6 to 12). Helical parameters were estimated from functional fits of the data, and were consistent with the known properties of B- and Z-DNAs and with the conclusion that psAT-DNA has a helical rise and twist close to that of B-DNA. The approach outlined here is not restricted to DNA but can be applied to other helical structures, e.g. RNA, proteins, and protein-nucleic acid complexes.