The efficiency of fluorescence resonance energy transfer (FRET) between fluorescein and rhodamine covalently attached to both 5' termini of a series of doublestranded DNA species (ranging from 8 to 20 bp) was measured. FRET efficiency varied with a dependence compatible with dye-to-dye distances (R) calculated on the basis of doublestranded B-DNA structure; the helical geometry of doublestranded DNA in solution is clearly evident. The experimental data were consistent with a 1/[1 + (RIRo)6] dependence of FRET efficiency characteristic for the Forster dipole-dipole mechanism. The thermal dissociation of the strands of the duplex DNA species can be followed by using FRET, and from these data we have been able to obtain enthalpies of duplex formation in good agreement with earlier measurements using alternative techniques. FRET measurements at very different salt concentrations can be accurately compared. We conclude that FRET is a reliable and valuable method for studying structure and conformational transitions in nucleic acids.Nucleic acids may adopt specific and sometimes complex folded structures that are critical for their biological function. Full determination of these structures requires the measurement of distances up to 80 A or more, but there are few techniques that allow such distances to be determined in solution. This is particularly important for nucleic acids in view of the extended helical structures involved, which can lead to underdetermination of structures by methods that can only yield short distances. Fluorescence resonance energy transfer (FRET) is sensitive to distances in the longer size range and has recently proved to be very useful in the study of nucleic acid structures, such as the solution structure of the four-way DNA junction (1, 2). FRET-derived distance information in DNA and RNA could be complementary to the shorter distances determined by NMR. However, the application of FRET to fluorescence probes covalently linked to nucleic acid structures has been relatively infrequent (1,(3)(4)(5)(6)(7)(8)(9)(10)(11).The rate of nonradiative energy transfer from an excited donor molecule (D) to a nearby acceptor molecule (A) depends in a characteristic manner on the distance between the two chromophores and their relative angular disposition (12)(13)(14). Depending on the D-A molecular pairs, the efficiency of transfer responds sensitively to relatively small changes of distances in the range of 10 to 80 A. Careful evaluations of FRET experiments can yield quantitative estimates of distances between labeled positions in macromolecules, or in molecular aggregates, provided that certain spectroscopic parameters are known. Even if sufficient information is not available to calculate exact distances, relative dimensions of molecular structures can often be deduced (1, 2, 11), and in principle FRET is applicable to very complex molecular structures. The method has been applied successfully to estimate intra(inter)molecular distances between donor and acceptor molecules in biological mo...

We have carried out fluorescence resonance energy transfer (FRET) measurements on four-way DNA junctions in order to analyze the global structure and its dependence on the concentration of several types of ions. A knowledge of the structure and its sensitivity to the solution environment is important for a full understanding of recombination events in DNA. The stereochemical arrangement of the four DNA helices that make up the four-way junction was established by a global comparison of the efficiency of FRET between donor and acceptor molecules attached pairwise in all possible permutations to the 5' termini of the duplex arms of the four-way structure. The conclusions are based upon a comparison between a series of many identical DNA molecules which have been labeled on different positions, rather than a determination of a few absolute distances. Details of the FRET analysis are presented; features of the analysis with particular relevance to DNA structures are emphasized. Three methods were employed to determine the efficiency of FRET: (1) enhancement of the acceptor fluorescence, (2) decrease of the donor quantum yield, and (3) shortening of the donor fluorescence lifetime. The FRET results indicate that the arms of the four-way junction are arranged in an antiparallel stacked X-structure when salt is added to the solution. The ion-related conformational change upon addition of salt to a solution originally at low ionic strength progresses in a continuous noncooperative manner as the ionic strength of the solution increases. The mode of ion interaction at the strand exchange site of the junction is discussed.

Equilibrium binding experiments using fluorescence and absorption techniques have been performed throughout a wide concentration range (1 nM to 30 microM) of the dye Hoechst 33258 and several DNAs. The most stable complexes found with calf thymus DNA, poly[d(A-T)], d(CCGGAATTCCGG), and d(CGCGAATTCGCG) all have dissociation constants in the range (1-3) X 10(-9) M-1. Such complexes on calf thymus DNA occur with a frequency of about 1 binding site per 100 base pairs, and evidence is presented indicating a spectrum of sequence-dependent affinities with dissociation constants extending into the micromolar range. In addition to these sequence-specific binding sites on the DNA, the continuous-variation method of Job reveals distinct stoichiometries of dye-poly[d(A-T)] complexes corresponding to 1, 2, 3, 4, and 6 dyes per 5 A-T base pairs and even up to 1 and 2 (and possibly more) dyes per backbone phosphate. Models are suggested to account for these stoichiometries. With poly[d(G-C)] the stoichiometries are 1-2 dyes per 5 G-C pairs in addition to 1 and 2 dyes per backbone phosphate. Thermodynamic parameters for formation of the tightest binding complex between Hoechst 33258 and poly[d(A-T)] or d-(CCGGAATTCCGG) are determined. Hoechst 33258 binding to calf thymus DNA, chicken erythrocyte DNA, and poly[d(A-T)] exhibits an ionic strength dependence similar to that expected for a singly-charged positive ion. This ionic strength dependence remains unchanged in the presence of 25% ethanol, which decreases the affinity by 2 orders of magnitude. In addition, due to its strong binding, Hoechst 33258 easily displaces several intercalators from DNA.