Analysis of fluorescence energy transfer in duplex and branched DNA molecules.

Nonradiative fluorescence energy transfer (FET) is thought to be a highly sensitive measure of distance, occurring through a dipole coupling (Forster) mechanism in which the efficiency of FET depends on the inverse sixth power of the distance between fluorophores. The current work assesses the utility of FET for measuring distances in duplex and branched DNA molecules. The apparent efficiencies of FET between donor (fluorescein) and acceptor (eosin) fluorophores attached to opposite ends of oligonucleotide duplexes of varying length were determined; the results suggest that FET is a useful qualitative indicator of distance in DNA molecules. However, the apparent FET efficiency values cannot be fit to the Forster equation without the specification of highly extended DNA-to-fluorophore tethers and motionally restricted fluorophores, conditions that are unlikely to coexist. Three other lines of evidence further suggest that factors in addition to Forster transfer contribute to apparent FET in DNA: (1) The efficiency of FET appears to depend on the base sequence in some instances. (2) Donor fluorescence changes with the extent of thermally induced DNA melting in a sequence-dependent fashion, indicating dye-DNA interactions. (3) The distances between the ends of various pairwise combinations of arms of a DNA four-way junction do not vary as much as expected from previous work. Thus, the occurrence of any nondipolar effects on energy transfer in oligonucleotide systems must be defined before distances in DNA molecules can be quantified by using FET.

[1]  D. Söll,et al.  Studies of transfer RNA tertiary structure of singlet-singlet energy transfer. , 1974, Proceedings of the National Academy of Sciences of the United States of America.

[2]  J. Lakowicz,et al.  Resolution of end-to-end distance distributions of flexible molecules using quenching-induced variations of the Forster distance for fluorescence energy transfer. , 1988, Biophysical Journal.

[3]  J. Eisinger,et al.  The orientational freedom of molecular probes. The orientation factor in intramolecular energy transfer. , 1979, Biophysical journal.

[4]  P. Taylor,et al.  Fluorescence energy transfer on acetylcholinesterase: spatial relationship between peripheral site and active center. , 1980, Biochemistry.

[5]  P. Hagerman,et al.  Gel electrophoretic analysis of the geometry of a DNA four-way junction. , 1987, Journal of molecular biology.

[6]  C. Cantor,et al.  Studies of transfer RNA tertiary structure by singlet-singlet energy transfer. , 1970, Proceedings of the National Academy of Sciences of the United States of America.

[7]  Th. Förster Zwischenmolekulare Energiewanderung und Fluoreszenz , 1948 .

[8]  G. Weber,et al.  Determination of the absolute quantum yield of fluorescent solutions , 1957 .

[9]  J M Beechem,et al.  Simultaneous determination of intramolecular distance distributions and conformational dynamics by global analysis of energy transfer measurements. , 1989, Biophysical journal.

[10]  L. Hood,et al.  The synthesis of oligonucleotides containing an aliphatic amino group at the 5' terminus: synthesis of fluorescent DNA primers for use in DNA sequence analysis. , 1985, Nucleic acids research.

[11]  D. Lilley,et al.  Fluorescence energy transfer shows that the four-way DNA junction is a right-handed cross of antiparallel molecules , 1989, Nature.

[12]  E. S. Pearson,et al.  TABLES OF PERCENTAGE POINTS OF THE INVERTED BETA (F) DISTRIBUTION , 1943 .

[13]  L. Brand,et al.  Intramolecular transfer of excitation from tryptophan to 1-dimethylaminonaphthalene 5-sulfonamide in a series of model compounds. , 1968, Biochemistry.

[14]  P. Hagerman,et al.  Geometry of a branched DNA structure in solution. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[15]  C. Cantor,et al.  The use of singlet-singlet energy transfer to study macromolecular assemblies. , 1978, Methods in enzymology.

[16]  M. Caruthers,et al.  Chemical synthesis and biological studies on mutated gene-control regions. , 1983, Cold Spring Harbor Symposia on Quantitative Biology.

[17]  O. W. Odom,et al.  Position of transfer ribonucleic acid on Escherichia coli ribosomes. Distance from the 3' end of 16S ribonucleic acid to three points on phenylalanine-accepting transfer ribonucleic acid in the donor site of 70S ribosomes. , 1981, Biochemistry.

[18]  J. E. Mueller,et al.  T4 endonuclease VII cleaves the crossover strands of Holliday junction analogs. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[19]  L. Stryer,et al.  Detection of actin assembly by fluorescence energy transfer , 1981, The Journal of cell biology.

[20]  B. Baird,et al.  Structural studies on the membrane-bound immunoglobulin E (IgE)-receptor complex. 2. Mapping of distances between sites on IgE and the membrane surface , 1983 .

[21]  N. Seeman,et al.  An immobile nucleic acid junction constructed from oligonucleotides , 1983, Nature.

[22]  J. Lakowicz,et al.  Distance distributions in native and random‐coil troponin I from frequency‐domain measurements of fluorescence energy transfer , 1988, Biopolymers.

[23]  E. Haas,et al.  Determination of intramolecular distance distributions in a globular protein by nonradiative excitation energy transfer measurements. , 1986, Biopolymers.

[24]  B. Baird,et al.  Structural studies on the membrane-bound immunoglobulin E-receptor complex. 1. Characterization of large plasma membrane vesicles from rat basophilic leukemia cells and insertion of amphipathic fluorescent probes. , 1983, Biochemistry.

[25]  L. Stryer,et al.  Energy transfer: a spectroscopic ruler. , 1967, Proceedings of the National Academy of Sciences of the United States of America.

[26]  D. E. Wolf,et al.  Detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[27]  O. W. Odom,et al.  Distances between 3' ends of ribosomal ribonucleic acids reassembled into Escherichia coli ribosomes. , 1980, Biochemistry.