Triple fluorescence energy transfer in covalently trichromophore-labeled DNA.

DNA is a unique molecule that can be used to separate donor and acceptor for fluorescence energy transfer (ET)1 and longrange photoinduced electron-transfer studies.2 ET has been used extensively as a spectroscopic ruler for biological structures,3a-c and ET primers and terminators are markedly superior to single dye-labeled reagents in DNA sequencing and analysis.4a-b Molecular beacons using both organic dyes5 and metal complexes6 for genetic analysis were also developed. Most of the reported ET systems using DNA as a backbone are based on one donoracceptor pair.7a-b An ET system involving three chromophores that are located on separate strands of oligonucleotides has also been reported.8 Recently, we developed a novel approach for constructing a large number of combinatorial fluorescence energy transfer tags from a small number of chromophores for multiplex biological assays.9 We report here the systematic study of the photophysical properties of an ET system consisting of three different fluorophores that are covalently linked to a singlestranded (ss) DNA molecule. The trichromophore-labeled DNA that has a scaffold of 26 nucleotides (Chart 1) was constructed by solid-phase phosphoramidite synthesis and selective solution-coupling chemistry. The structure of the compound was confirmed by MALDI-TOF mass spectrometry.10 The 5′-end of the DNA was linked to 6-carboxyfluorescein (F). N,N,N′,N′-tetramethyl-6-carboxyrhodamine (R) was positioned four nucleotides away from F. A cyanine-5 monofunctional dye (Cy) was attached to the DNA six nucleotides from R. Twelve thymidine nucleotides followed the triple ET moiety with a cytidine at the 3′-end for DNA sequencing evaluations. The trichromophore-labeled DNA is designated as F-4-R-6-Cy-13 (number refers to the nucleotide residues) where F acts as the donor for R and Cy, R acts as an acceptor for F and a donor for Cy, while Cy acts as a final acceptor for both F and R. Figure 1 shows the spectra of F-4-R-6-Cy-13 that exhibits the characteristic absorption and emission of F (λabs: 496 nm, λem: 525 nm), R (λabs: 555 nm, λem: 585 nm), and Cy (λabs: 643 nm, λ em: 670 nm). The unique interaction of the three chromophores that are separated by defined number of nucleotides allows efficient ET to take place with dominant fluorescence emission from Cy at 670 nm with 488 nm excitation, leading to a “Stokes shift” of 182 nm. The quenching efficiency for F (QF) is 99%, and the overall fluorescence quantum yield (φ) for F-4-R-6-Cy-13 is 0.13,10 while φ for the Cy monomer is 0.27. 11 To evaluate the ET property of F-4-R-6-Cy-13 in ssDNA with different lengths, we used it as a primer in Sanger sequencing method12 to produce DNA extension fragments terminated by biotinylated dideoxycytidine triphosphate (ddCTP-biotin) on a template generated from human genomic DNA. The solid-phase sequencing chemistry using ddCTP-biotin and streptavidin-coated magnetic beads allows the isolation of pure DNA extension fragments which are free from false terminations.13 These ssDNA fragments, analyzed by a three-color capillary array electrophoresis (CAE) system and resolved at single base pair (bp) resolution, produced an electropherogram as shown in Figure 2. All the peaks for each of the DNA fragments (92 to 110 bp are shown) extended by F-4-R-6-Cy-13 display a constant fluorescence emission signature of 2:3:7 (blue:green:red) that is defined by the ratio of the fluorescence intensity from each of the three detection channels (F, blue, 520 ( 20 nm; R, green, 585 ( 20 nm; Cy, * Corresponding authors: E-mail: dj222@columbia.edu (J.J.) and turro@ chem.columbia.edu (N.J.T.). † Columbia Genome Center, Columbia University. ‡ Department of Chemical Engineering, Columbia University. § Department of Chemistry, Columbia University. | City University of New York. (1) Clegg, R. M. Methods Enzymol. 1992, 211, 353. (2) Murphy, C. J. et al. Science 1993, 262, 1025. (3) (a) Stryer, L. Annu. ReV. Biochem. 1978, 47, 819. (b) Cha, A.; Snyder, G. E.; Selvin, P. R.; Bezanilla, F. Nature 1999, 402, 809. (c) Fairclough, R. H.; Cantor, C. R. Methods Enzymol. 1978, 48, 347. (4) (a) Ju, J.; Ruan, C.; Fuller, C. W.; Glazer, A. N.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 4347. (b) Rosenblum, B. B. et al. Nucleic Acids Res. 1997, 25, 4500. (5) Tyagi, S.; Kramer, F. R. Nat. Biotechnol. 1996, 14, 303. (6) Joshi, H. S.; Tor, Y. Chem. Commun. 2001, 549. (7) (a) Selvin, P. R.; Hearst, J. E. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 10024. (b) Ha, T.; Enderle, T.; Ogletree, D. F.; Chemla, D. S.; Selvin, P. R.; Weiss, S. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 6264. (8) Kawahara, S.; Uchimaru, T.; Murata, S. Chem. Commun. 1999, 563. (9) Tong, A. K.; Li, Z.; Jones, G. S.; Russo, J. J.; Ju, J. Nat. Biotechnol. 2001, 19, 756. (10) See the Supporting Information for experimental details. (11) Mujumdar, R. B.; Ernst, L. A.; Mujumdar, S. R.; Lewis, C. J.; Waggoner, A. S. Bioconj. Chem. 1993, 4, 105. (12) Sanger, F.; Nicklen, S.; Coulson, A. R. Proc. Natl. Acad. Sci. U. S.A. 1977, 74, 5463. (13) Ju, J. U.S. Patent 5,876,936, 1999. Figure 1. Absorption (‚‚‚) and emission (-) spectra of F-4-R-6-Cy-13.