Triple Fluorescence Energy Transfer in Covalently Trichromophore-Labeled DNA Anthony K. Tong, †,‡ Steffen Jockusch, § Zengmin Li, †,‡ Han-Ru Zhu, | Daniel L. Akins, | Nicholas J. Turro,* ,§,‡ and Jingyue Ju* ,†,‡ Columbia Genome Center Departments of Chemical Engineering and Chemistry Columbia UniVersity, New York, New York 10027 Department of Chemistry, City College City UniVersity of New York, New York, New York 10031 ReceiVed August 21, 2001 DNA is a unique molecule that can be used to separate donor and acceptor for fluorescence energy transfer (ET) 1 and long- range 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 Mo- lecular beacons using both organic dyes 5 and metal complexes 6 for genetic analysis were also developed. Most of the reported ET systems using DNA as a backbone are based on one donor- acceptor 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 single- stranded (ss) DNA molecule. The trichromophore-labeled DNA that has a scaffold of 26 nucleotides (Chart 1) was constructed by solid-phase phosphora- midite 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-carboxy- fluorescein (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 (λ abs max : 496 nm, λ em max : 525 nm), R (λ abs max : 555 nm, λ em max : 585 nm), and Cy (λ abs max : 643 nm, λ em max : 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 (Q F ) 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 method 12 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 electrophore- sis (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. 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