Coumarin-fluorescein pair as a new donor-acceptor set for fluorescence energy transfer study of DNA

Article abstract of DOI:10.1016/S0040-4039(00)00215-X

A method for introduction of the 2'-coumarin labeled nucleoside as a fluorescence energy donor into DNA duplexes has been described. Efficient FRET occurs between the coumarin-fluorescein pair in DNA owing to the high quantum yield of the donor. The present donor-acceptor pair may be useful as FRET indicator of DNA structures in solution. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)00215-X

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 2605–2608
Coumarin–fluorescein pair as a new donor–acceptor set for
fluorescence energy transfer study of DNA
Tsuneo Mitsui, Hidehiko Nakano and Kazushige Yamana ∗
Department of Applied Chemistry, Himeji Institute of Technology, 2167 Shosha, Himeji, Hyogo 671-2201, Japan
Received 27 December 1999; revised 26 January 2000; accepted 28 January 2000
Abstract
A method for introduction of the 20-coumarin labeled nucleoside as a fluorescence energy donor into DNA
duplexes has been described. Efficient FRET occurs between the coumarin–fluorescein pair in DNA owing to
the high quantum yield of the donor. The present donor–acceptor pair may be useful as FRET indicator of DNA
structures in solution. © 2000 Elsevier Science Ltd. All rights reserved.
Keywords: coumarin–fluorescein pair; nucleoside; fluorescence energy transfer; DNA.
There have been several reports concerned with applications of fluorescence energy transfer (FRET)
in studies on nucleic acid structures such as helical four-way junctions in DNA topology,1 geometry of
bent DNA molecules,2 and relative orientation of the helical segments in RNA hammerhead ribozyme.3
In these applications, the 50- or 30-end or the phosphate backbone of oligonucleotides has been used for
the attachment of the donor and acceptor molecules. Although FRET has been successfully applied to
analysis of complex nucleic acid structures, there remained the difficulty in generating a donor–acceptor
pair at appropriate positions of nucleic acids. We have recently shown that the sugar 20-position of DNA
is a suitable site for covalent attachment of 6-dimethylamino-2-naphthamide (DAN)4 and fluorescein
(F) moieties as a fluorescence energy donor–acceptor pair. Our approach to the synthesis of fluorescent
labeled DNA is based on the combined use of pre- and post-labeling technique,5 thus providing great
flexibility for rapid construction of a donor–acceptor pair in DNA.
The DAN–F pair has been expected to be useful in FRET measurement of DNA.5 However, the
fluorescence of the donor (DAN) incorporated into DNA exhibited weak emission of ca. 1% quantum
yield even in the absence of the acceptor. This low quantum yield causes difficulty or inaccuracy in
determination of distance-dependent FRET by the usual fluorescence measurements. The donor quantum
efficiency also affects the critical distance at which FRET efficiency is 0.5.6 Higher quantum yield of the
donor leads to longer critical distance of FRET. Efficient FRET at long distance (several tenth Å) should
be necessary for analysis of complex nucleic acid structures in solution. We now describe the synthesis
of DNA duplexes containing a coumarin–fluorescein pair at the sugar residues. This new fluorescence
energy donor–acceptor set is promising as a FRET indicator of DNA.
∗
Corresponding author. Fax: +81-792-67-4895; e-mail: yamana@chem.eng.himeji-tech.ac.jp (K. Yamana)
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)00215-X
tetl 16495

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We have chosen the highly fluorescent coumarin derivative as the fluorescence energy donor, because
coumarin and fluorescein fluorophores show suitably overlapping spectra for FRET.7 For site-specific
incorporation of coumarin fluorophore into oligonucleotides, the nucleoside phosphoramidite 2 was
synthesized. As shown in Scheme 1, 20-amino-20-deoxyuridine was allowed to react with 7-hydroxy-
3-coumarin carboxylic acid8 using DCC (rt, overnight) giving modified uridine with coumarin at the
20-position [U(HCC) 1].9 After protection at 50-hydroxyl function by dimethoxytrityl and 7-OH of
the coumarin ring by a trimethylacetyl group, nucleoside 1 was converted by the usual method to the
phosphoramidite 2.
Scheme 1. (i) 7-Hydroxy-3-coumarin carboxylic acid (1 equiv.), DCC (1.2 equiv.), HOBT (1.2 equiv.) in DMF; 89% yield; (ii)
dimethoxytrityl chloride (1.2 equiv.) in pyridine; 80% yield; (iii) trimethylacetyl chloride (1 equiv.) in pyridine; 79% yield; (iv)
2-cyanoethyl-N,N0,N00,N000-tetraisopropylphosphorodiamidite (1.5 equiv.), tetrazole (1 equiv.) in CH2Cl2; 59% yield
Oligonucleotides containing one coumarin fluorophore at the different sites, 3–5, were synthesized by
using 2 according to a fully automated procedure described earlier.5 After deprotection, the oligomers
were purified with 20% denaturing polyacrylamide gel electrophoresis. The integrity of each oligomer
was verified by ion-spray MS.10 The binding and fluorescence properties of oligomers 3–5 are summari-
zed in Table 1. The oligonucleotide 3 that contains U(HCC) at the terminal fraying 50-end retains normal
binding affinity for DNA, whereas incorporation of U(HCC) into other sites as in oligonucleotides 4
and 5 causes destabilization of the modified duplexes. Similar duplex stability depending on the site
of incorporation has been observed for the duplexes containing dansyl modified uridine11 and U(DAN)
modified uridine.5 The fluorescence emission maximum of U(HCC) modified oligonucleotides appeared
at 450 nm which is well overlapped with the UV–vis absorption of fluorescein. Fluorescence quantum
yields of the modified duplexes were estimated to be ca. 10%. These observations indicate that the
U(HCC) modified oligonucleotides may be a suitable donor to fluorescein labels in FRET study.
Table 1
Tm values and fluorescence properties of U(HCC) modified oligonucleotide duplexes

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To test the validity of the coumarin–fluorescein pair in distance-dependent FRET measurement, we
prepared the DNA duplexes possessing different numbers of base-pairs between these fluorophores
(6+10, 7+11, 8+12, and 9+13).12 Fig. 1 represents the fluorescence spectra observed in the typical FRET
experiments. The duplex of oligomer 6 with unmodified oligonucleotide exhibited fluorescence at 450
nm whose quantum yield is 25%. When compared with this duplex, the fluorescence of the donor was
largely weakened by introduction of the acceptor. With this fluorescence quenching, enhancement of the
emission derived from the acceptor at 523 nm was observed. On the contrary, little or no fluorescence
changes were observed in the presence of non-complementary oligonucleotides with fluorescein label.
All the labeled duplexes displayed similar fluorescence properties. These observations indicate that the
intramolecular FRET occurs between coumarin and fluorescein labels along the DNA duplex. The FRET
efficiencies (Eapp) measured for the duplexes are shown graphically in Fig. 1 where the Eapp from the
duplexes labeled with DAN–fluorescein pair5 are also represented. It is seen that the Eapp is clearly
dependent on the number of nucleotides in the DNA. The relatively large Eapp was observed between
the coumarin–fluorescein pair when compared with the DAN–fluorescein pair in the DNA of the same
length.13 Since the spectral overlap integral is similar in DAN–fluorescein and coumarin–fluorescein
pairs, the highly fluorescent coumarin is mainly responsible for the large Eapp.6
Fig. 1. Left panel: Fluorescence spectra for typical FRET measurement of DNA duplex (10-mer) at a total strand concentration of
4.0×10−5 M. The measurements were carried out at 3°C in a buffer containing 1.0 M NaCl and 0.01 sodium phosphate, adjusted
to pH 7.0. Oligomer 6+50-TA9 (- - - -), 50-T9A+oligomer 10 (· · · · ·), oligomer 6+10 (solid line). Right panel: Apparent FRET
efficiency (Eapp) in DNA duplexes containing various numbers of base pairs in which the coumarin–fluorescein pair is shown
by black bars and the DAN–fluorescein pair by white bars. The sequences of oligomers used for FRET study are shown below.
For the study of the DAN–F pair, U(DAN) was incorporated into the donor strand in place of U(HCC)5
We have described a method for introduction of the 20-coumarin labeled nucleoside as a FRET donor
in DNA duplexes. Efficient FRET occurs between the coumarin–fluorescein pair in DNA owing to the
high quantum yield of the donor. The present FRET donor–acceptor pair may thus be useful for analysis
of DNA structures in solution.
Acknowledgements
We are very grateful to Professor Hiroshi Sugiyama for ion-spray mass spectral analysis.

2608
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6. For energy transfer occurring via the Forster mechanism, the distance in Å between donor and acceptor fluorophores are
deduced from the relationship, R=R0[(1−E)/E]1/6. R0 is the critical distance at which transfer efficiency E is 0.5, calculated
6
according to R0 =8.8×10−25×κ2 F J n−4, where κ2 is the orientation factor, J is the spectral overlap integral, n is the
refractive index of the medium, and F is the fluorescence quantum yield of the donor in the absence of the acceptor.
7. Zolkarnik, G.; Negulescu, P. A.; Knapp, T. E.; Mere, L.; Burres, N.; Feng, L.; Whitney, M.; Roemer, K.; Tsien, R. Y. Science
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9. Nucleoside 1, TLC (silica, CH2Cl2:MeOH=1:1, v/v) Rf 0.61; 1H NMR (500 MHz, DMSO-d6): δ: 3.62 (m, 2, H5 , 5 ), 4.00
0
00
0
0
0
0
0
(m, 1, H4 ), 4.17 (m, 1, H3 ), 4.58 (m, 1, H2 ), 5.22 (t, 1, 5 -OH, D2O exchange), 5.71 (d, 1, uracil H5), 5.96 (d, 1, H1 ),
6.15 (d, 1, 30-OH, D2O exchange), 6.79 (d, 1, coumarin H4), 6.86 (dd, 1, coumarin H5), 7.80 (d, 1, coumarin H6), 7.96 (d, 1,
uracil H6), 8.83 (s, 1, coumarin H8), 9.18 (d, 1, C20-amide), 11.22 (s, 1, uracil NH, D2O exchange).
10. Ion-spray mass data for oligomers 3–5 were 3849.5 (calcd 3848.6), 3848.9 (calcd 3848.6), and 3849.0 (calcd 3848.6),
respectively.
11. Yamana, K.; Ohashi, Y.; Nunota, K.; Nakano, H. Tetrahedron 1997, 53, 4265.
12. The fluorescein labeled oligonucleotides were synthesized by the published procedure (Ref. 5).
13. The distances between coumarin and fluorescein fluorophores estimated from the theory (Ref. 6) were in fairly good
agreement with the values based on the molecular model of DNA.