Triple fluorescence energy transfer in covalently trichromophore-labeled DNA.
Triple fluorescence energy transfer in covalently trichromophore-labeled DNA.
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DOI：
10.1021/ja016904h
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发表时间：
2001-11
影响因子：
15
通讯作者：
A. Tong;S. Jockusch;Zengmin Li;Han-Ru Zhu;D. Akins;N. Turro;J. Ju
中科院分区：
文献类型：
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作者：
A. Tong;S. Jockusch;Zengmin Li;Han-Ru Zhu;D. Akins;N. Turro;J. Ju
DNA是一种独特的分子，可以用来分离供体和受体，用于荧光能量转移（ET）和远程光致电子转移研究ET已被广泛用作生物结构的光谱标尺，在DNA测序和分析中，3a-c和ET引物和终止物明显优于单一染料标记试剂。还开发了利用有机染料和金属配合物进行遗传分析的分子信标。大多数使用DNA作为主干的ET系统都是基于一个供体-受体对。还报道了一个涉及位于不同寡核苷酸链上的三个发色团的ET系统最近，我们开发了一种从少量发色团构建大量组合荧光能量转移标签的新方法，用于多种生物检测我们在这里报告了由三个不同的荧光团组成的ET系统的光物理性质的系统研究，这些荧光团共价连接到单链（ss） DNA分子。通过固相磷酸酰胺合成和选择性溶液偶联化学构建了具有26个核苷酸支架的三色细胞标记DNA（图1）。用MALDI-TOF质谱法证实了化合物的结构DNA的5 '端与6-羧基荧光素(F)相连。N，N,N ',N ‘ -四甲基-6-羧基罗丹明(R)被定位在离f 4个核苷酸的位置，一个花青素-5单功能染料（Cy）附着在离R 6个核苷酸的DNA上，12个胸腺嘧啶核苷酸与3 ’端胞苷相连，用于DNA测序评估。trichromophore-labeled DNA被指定为F-4-R-6-Cy-13(数量是指核苷酸残留物)F R和Cy作为供体,为F R作为受体和捐赠者Cy,虽然Cy作为最终受体对F和R .图1显示了光谱的F-4-R-6-Cy-13展品的特征吸收和发射F(λabs: 496 nm,λem: 525海里)、R(λabs: 555 nm,λem: 585海里),和Cy(λabs: 643 nm,λem: 670海里)。三种发色团的独特相互作用被确定数量的核苷酸分开，使得有效的ET发生在670 nm和488 nm激发下，Cy的优势荧光发射，导致182 nm的“斯托克斯位移”。F （QF）的猝灭效率为99%，F-4- r -6-Cy-13的总荧光量子产率（φ）为0.13,10，而Cy单体的φ为0.27。11为了评估F-4-R-6-Cy-13在不同长度的ssDNA中的ET特性，我们将其作为Sanger测序方法的引物12，在由人类基因组DNA生成的模板上产生以生物素化二脱氧胞苷三磷酸（ddCTP-biotin）终止的DNA延伸片段。使用ddctp -生物素和链霉亲和素包被磁珠的固相测序化学可以分离出没有错误终止的纯DNA延伸片段这些ssDNA片段通过三色毛细管阵列电泳（CAE）系统进行分析，并以单碱基对（bp）分辨率进行解析，产生如图2所示的电泳图。由F-4-R-6-Cy-13延伸的每个DNA片段（92至110 bp）的所有峰显示出恒定的荧光发射特征为2:3:7（蓝：绿：红），这是由三个检测通道(F，蓝色，520 (20 nm)； R，绿色，585 （20 nm); Cy， *通讯作者：E-mail: dj222@columbia.edu （J.J.）和turo @ chem.columbia.edu (N.J.T.)）的荧光强度之比定义的。†哥伦比亚大学哥伦比亚基因组中心。哥伦比亚大学化学工程系。§哥伦比亚大学化学系。b|纽约城市大学。(1)李建平，李建平，李建平，等。(2) Murphy， C. J.等。科学1993,262,1025。(3) (a) Stryer， L. Annu。生物化学学报，1987,47(8):819。(b) Cha, a；施耐德，g.e.；Selvin, p.r.；刘志强，陈志强。自然科学，1999,42,809。(c)费尔克劳，r.h.；李建军，张建军。生物化学学报，1997,18(3):349 - 347。(4) (a) Ju, j；阮,c;富勒，c.w.；格雷泽，a.n.；马蒂斯，r.a.。学会科学。美国1995,92,4347。(b) Rosenblum, b.b.等。核酸学报，1997,25,4500。(5) Tyagi, s；陈晓明。生物技术学报。1997,14(3):393。(6)乔希，h.s.；化学。common . 2001,549。(7) (a) Selvin, p.r.；赫斯特，j.e.。学会科学。美国1994,91,10024。(b)哈，T.；恩德勒、t;Ogletree, d.f.；Chemla博士；Selvin, p.r.；Weiss， S. Proc. Natl。学会科学。美国1996,93,6264。(8)河原，S.；Uchimaru t;Murata， S. Chem。common . 1999,563。(9)唐亚奎；李,z;琼斯，g.s.；罗素，j.j.；朱俊杰。生物技术。2001,19(1):56。（10）实验细节见配套资料。（11）穆琼达尔，r.b.；恩斯特，洛杉矶；穆琼达尔，s.r.；刘易斯，c.j.；瓦格纳，A. S. Bioconj。化学，1993,4,105。（12） Sanger， F.；Nicklen,美国;科尔森，a.r.。学会科学。美国，1977,74,5463。（13）朱，J.美国专利5,876,936,1999。图1所示。F-4-R-6-Cy-13的吸收光谱和发射光谱。
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.