Polyfluorophore excimers and exciplexes as FRET donors in DNA

Abstract

We describe studies aimed at testing whether oligomeric exciplex and excimer fluorophores conjugated to DNA have the potential to act as donors for energy transfer by the Forster mechanism. Oligodeoxyfluorosides (ODFs) are composed of stacked, electronically interacting fluorophores replacing the bases on a DNA scaffold. The monomer chromophores in the twenty tetramer-length ODFs studied here include pyrene (Y), benzopyrene (B), perylene (E), dimethylaminostilbene (D), and a nonfluorescent spacer (S); these are conjugated in varied combinations at the 3' end of a 14mer DNA probe sequence. In the absence of an acceptor chromophore, many of the ODF-DNAs show broad, unstructured long-wavelength emission peaks characteristic of excimer and exciplex excited states, similar to what has been observed for unconjugated ODFs. Although such delocalized excited states have been widely studied, we know of no prior report of their use in FRET. We tested the ability of the twenty ODFs to donate energy to Cy5 and TAMRA dyes conjugated to a complementary strand of DNA, with these acceptors oriented either at the near or far end of the ODF-conjugated probes. Results showed that a number of the ODF fluorophores exhibited relatively efficient energy transfer characteristic of the Forster mechanism, as judged by drops in donor emission quantum yield and fluorescence lifetime, accompanied by increases in intensity of acceptor emission bands. Excimer/exciplex bands in the donors were selectively quenched while shorter-wavelength monomer emission stayed relatively constant, consistent with the notion that the delocalized excited states, rather than individual fluorophores, are the donors. Interestingly, only specific sequences of ODFs were able to act as donors, while others did not, even though their emission wavelengths were similar. The new FRET donors possess large Stokes shifts, which can be beneficial for multiple applications. In addition, all ODFs can be excited at a single wavelength; thus, ODFs may be candidates as "universal FRET donors", thus allowing multicolor FRET of multiple species to be carried out with one excitation.

Figure 1

Structures in this study. A. Monomeric 2’-deoxyfluorosides B, E, Y, D and S. B. Structures of two representative ODFs (BEBY and YSSY) that were studied as DNA conjugates. These were attached to the 3’ end of a DNA probe sequence (R). C. An example of a hybridization experiment: hybridization of BEYY-labeled probe (P2) with an acceptor-labeled complement (C3’).

Figure 1

Structures in this study. A. Monomeric 2’-deoxyfluorosides B, E, Y, D and S. B. Structures of two representative ODFs (BEBY and YSSY) that were studied as DNA conjugates. These were attached to the 3’ end of a DNA probe sequence (R). C. An example of a hybridization experiment: hybridization of BEYY-labeled probe (P2) with an acceptor-labeled complement (C3’).

Figure 2

Representative evidence for FRET between ODF fluorophores and TAMRA in a DNA duplex (see full data in Supporting Information). Fluorescence emission spectra (excitation 330 nm) are shown for hybridization of P1 and P6 with T3’ and T5’ (red lines) and individual emission spectra of P1, P6 (blue), T3’ and T5’ (green). Note spectral changes characteristic of FRET for the distal-substituted TAMRA (A, C).

Figure 3

Evidence for FRET between ODF donors and a Cy5 acceptor in duplex DNA. Fluorescence emission spectra are shown for hybridization of ODF conjugates P2 and P6 with C3’ and C5’ (red lines) (λ_ex = 330 nm) and individual emission spectra of P2, P6, C3’ and C5’ (λ_ex = 330 nm) (blue and green lines). Note quenching of excimer and exciplex bands (440–560 nm) concomitant with enhancement of Cy5 emission at ∼670 nm.

Figure 4

Testing possible FRET donation from pyrene excimers to TAMRA. Fluorescence emission spectra upon hybridization of A. P3; B. P4 and C. P5 with T3’ respectively (red lines) (λ_ex = 330 nm) and individual emission spectra of P3, P4, P5 and T3’ (λ_ex = 330 nm).

Figure 5

Evidence for strong FRET donation from pyrene excimers to Cy5 acceptor in DNA. Fluorescence emission spectra upon hybridization of P3, P4 and P5 with C3’ and C5’ respectively (red lines) (λ_ex = 330 nm) and individual emission spectra of P3, P4, P5, C3’ and C5’ (blue and green lines) (λ_ex = 330 nm).