Oligomeric fluorescent labels for DNA.

In an effort to find fluorescent labels that have large Stokes shifts and increased emission intensity, a strategy for fluorescence labeling of DNA was explored in which multiple individual fluorophores are incorporated at adjacent positions at the end of a DNA probe. To encourage close interactions, hydrocarbon and heterocycle fluorophores were substituted at C-1 of deoxyribose, replacing the DNA base. The C-glycosides studied contained the well-known fluorophores terphenyl, pyrene, and terthiophene. For comparison, a commercial fluorescein-dU nucleotide was examined. Oligomeric labels containing up to five fluorophores were tested. Interestingly, all four dyes behaved differently on multiple substitution. Fluorescein displayed strong self-quenching properties, with the quantum yield dropping severalfold with each additional substitution and with a constant, small Stokes shift. In contrast, pyrene showed increases in quantum yield on addition of more than one fluorophore and yielded efficient long-wavelength emission on multiple substitution, with Stokes shifts of >130 nm. Oligomeric terphenyl labels gave a small progressive red shift in absorption and a marked red shift in emission wavelength and showed a strong increase in brightness with more monomers. Finally, terthiophene oligomers showed self-quenching combined with increasing Stokes shifts. Overall, the results suggest that some oligomeric fluorescent labels exhibit properties not available in common fluorescein class (or other commercial) labels, such as large Stokes shifts and increasing brightness with increasing substitution.

Libraries of composite polyfluors built from fluorescent deoxyribosides.

We report on a new class of water-soluble fluorescent molecules (polyfluors) that are composed of multiple individual fluorophores assembled on a DNA-like backbone. Four fluorophore deoxyribosides were synthesized, and these individual molecules were assembled into oligofluor strings on a DNA synthesizer. A library of 256 tetrafluors was generated by split and pool methods on polystyrene beads. Images of the library under a fluorescence microscope revealed at least 40-50 different hues and intensities. Selected tetrafluors were resynthesized in pure form in solution and displayed properties, such as large Stokes shifts, that individual fluorophores do not have.

Novel Nucleoside Analogues with Fluorophores Replacing the DNA Base.

We describe the preparation and fluorescence properties of a set of new nucleosides in which a known hydrocarbon or oligothiophene fluorophore replaces the DNA base at C(1) of the deoxyribose moiety (see 3a - f). These compounds are potentially useful as probes in the study of the structure and dynamics of nucleic acids and their complexes with proteins. In addition, they may find use as fluorescent labels for nucleic-acid-based biomedical diagnostics methods. The fluorophores conjugated to deoxyribose at C(1) in the α-d-form include terphenyl, stilbene, terthiophene, benzoterthiophene, and pyrene. Also included is a non-fluorescent spacer in which cyclohexene replaces the DNA base. The nucleosides are derived from brominated fluorophore precursors and Hoffer's 2-deoxy-3,5-di-O-(p-toluoyl)-d-ribofuranosyl chloride. The emission maxima of the free nucleosides range from 345 to 536 nm. Also described are the 5'-(dimethoxytrityl) 3'-O-phosphoramidite derivatives 5a - f, suitable for incorporation into oligonucleotides by automated synthesizers.