Fluorescence resonance energy transfer analysis of the structure of the four-way DNA junction

Abstract

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.

Self-assembly of a nanoscale DNA box with a controllable lid

“…The kinetic studies were performed with a 10-nm slit centred at 665 nm, and recorded each 30 s with high shutter speeds to minimize bleaching. The FRET efficiency was estimated by the RatioA method 30 .…”

Observing the helical geometry of double-stranded DNA in solution by fluorescence resonance energy transfer.

“…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.…”

“…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 molecules other than nucleic acids (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26), to determine molecular distributions in condensed media (27)(28)(29)(30)(31), and to estimate distances and distributions in a large variety of other polymer problems (26,32).…”

Torsional Dynamics and Orientation of DNA−DAPI Complexes

“…In this article we report a time-resolved fluorescence depolarization study of DAPI bound to DNA to determine the potential use of such a minor groove binding ligand for probing nucleic acid structure and dynamics (Cooper & Hagerman, 1990;Clegg et al, 1992). DAPI is a minor groove probe; it binds with high affinity to A-T-A-T sequences, and it is strongly fluorescent when bound to DNA.…”