Fluorescence resonance energy transfer (FRET) has been used to demonstrate the bending of DNA and RNA bhelis for three series of double-stranded molecules containing bulge loops of unopposed nucleotides (A., n = 0-9). Fluorescein and rhodamine were covalently attahed to the 5' termini of the two component strands. Three diferent methods were applied to measure the FRET efficiencies. (26) was utilized to protect the 2' position of RNA (MilliGen). Fluorescein was introduced into the 5' end of the 18-base DNA oligonucleotides in the final coupling step using a mixture of 5-and 6-fluorescein phosphoramidites with C6 linkers (Pharmacia). For RNA only the 6-isomer was used. C6 linkers with amino groups were introduced on the 5' ends of all other oligonucleotides as described (21). The tertbutyldimethylsilyl protecting group was removed by a 16-hr treatment with 1 M tetrabutylammonium fluoride in tetrahydrofuran (Aldrich) (26). Fluorescein phosphoramiditelabeled strands were purified by reverse-phase HPLC. 5'-Amino oligonucleotides were purified by anion-exchange and reverse-phase HPLC prior to conjugation with either rhodamine N-hydroxysuccinimide ester (Molecular Probes) or 5-fluorescein isothiocyanate (for 27-base DNA oligonucleotides) (21). All dye-conjugated species were further purified by electrophoresis in 20% polyacrylamide/7 M urea gels. Base sequences of the three series of bulged duplexes (only the fluorescein-labeled top strands, containing n = 0, 1, 3, 5, 7, or 9 additional adenosine nucleotides An, are shown) are 5'-C3TAG2AnTCG2ATCTCG2-3' (18-bp DNA duplexes), 5'-C3UAG2AnUCG2AUCUCG2-3' (18-bp RNA duplexes), and 5'-C3TGTG2ATC2AG2AnTCG2ATC2TCG2-3' duplexes). Combinations of dye-labeled oligonucleotides were hybridized, purified by electrophoresis in nondenaturing 15% polyacrylamide gels, extracted into fluorescence buffer (90 mM Tris borate, pH 8.3/100 mM NaCl), and finally dialyzed against this buffer. For the AO, Al, and A3 bulged 18-bp DNA duplexes, the molecules containing 5-and 6-fluorescein isomers are well separated in the final electrophoresis step.The ratio of fluorescein to rhodamine absorbance is constant within each series of molecules while the ratio of dye to nucleic acid absorbance decreases (data not shown); the values indicate 100% labeling. All measurements were taken at 40C to ensure that the molecules were in the duplex form.This was verified by measuring the melting temperatures. The absorbances of all solutions were below 0.015 at the excitation wavelength.Abbreviation: FRET, fluorescence resonance energy transfer.tPresent address:

For the understanding of the catalytic function of the RNA hammerhead ribozyme, a three-dimensional model is essential but neither a crystal nor a solution structure has been available. Fluorescence resonance energy transfer (FRET) was used to study the structure of the ribozyme in solution in order to establish the relative spatial orientation of the three constituent Watson-Crick base-paired helical segments. Synthetic constructs were labeled with the fluorescence donor (5-carboxyfluorescein) and acceptor (5-carboxytetramethylrhodamine) located at the ends of the strands constituting the ribozyme molecule. The acceptor helix in helix pairs I and III and in II and III was varied in length from 5 to 11 and 5 to 9 base pairs, respectively, and the FRET efficiencies were determined and correlated with a reference set of labeled RNA duplexes. The FRET efficiencies were predicted on the basis of vector algebra analysis, as a function of the relative helical orientations in the ribozyme constructs, and compared with experimental values. The data were consistent with a Y-shaped arrangement of the ribozyme with helices I and II in close proximity and helix III pointing away. These orientational constraints were used for molecular modeling of a three-dimensional structure of the complete ribozyme.

Fluorescence steady-state and lifetime experiments have been carried out on duplex and single-stranded DNA molecules labeled at the 5' ends with 5-carboxytetramethylrhodamine (TMRh). The temperature and ionic strength of the solutions were varied over large ranges. The results reveal at least three well-defined states of the TMRh-DNA molecules for the single-stranded as well as for the double-stranded DNA molecules. Two states are fluorescent, with lifetimes in the range of 0.5-1 ns and 2.5-3 ns. A third state of TMRh-DNA does not fluoresce (a dark species of TMRh-DNA). The distribution of the TMRh-DNA molecules among these three states is strongly temperature and ionic strength dependent. Estimates are made of some reaction parameters of the multistate model. The results are discussed in terms of the photophysics of TMRh, and consequences of the multiple conformers of TMRh-DNA for studies involving fluorescence studies with TMRh-labeled DNA are considered.