Clear distinction of purine bases on the complementary strand by a fluorescence change of a novel fluorescent nucleoside

Article abstract of DOI:10.1021/ja034090u

A new fluorescent nucleoside, benzopyridopyrimidine (BPP), which can sharply distinguish between A and G bases opposite BPP has been devised. The base-pairing degeneracy of BPP strongly contributes to the sharp fluorescence change that is dependent on the

Full text of DOI:10.1021/ja034090u

Published on Web 04/04/2003
Clear Distinction of Purine Bases on the Complementary Strand by a
Fluorescence Change of a Novel Fluorescent Nucleoside
Akimitsu Okamoto, Kazuki Tainaka, and Isao Saito*
Department of Synthetic Chemistry and Biological Chemistry, Faculty of Engineering, Kyoto UniVersity, and
SORST, Japan Science and Technology Corporation, Kyoto 606-8501, Japan
Received January 9, 2003; E-mail: saito@sbchem.kyoto-u.ac.jp
Table 1. The Oligodeoxynucleotides (ODNs) Used in This Study
Many fluorescent nucleosides have been used as site-specific
probes for studying structures and dynamics of nucleic acids.1
Nucleosides possessing various fluorophores have been explored,
including fluorescent nucleoside analogues and fluorophore-linked
base conjugates, as exemplified by 2-aminopurine,2 1,N-etheno-
adenine,3 ethynyl-extended pyrimidines and deazapurines,4 and
nucleoside analogues replaced by flat aromatic fluorophores.5 Such
fluorescent nucleosides are valuable as sensitive reporter probes
for detecting the change in the microenvironment of DNA, such
as that which occurs in hybridization and the conformational change.
However, there is no effective method that provides a clear
distinction of the type of counterbase by using the fluorescence
change caused by the change in DNA microenvironment.
We herein report on the clear distinction of purine bases on the
complementary strand using the fluorescence from a novel nucleo-
side, benzopyridopyrimidine (BPP). BPP has a base-pairing de-
generacy, which strongly contributes to the clear distinction of
purine bases on the complementary strand by a sharp fluorescence
change. The BPP-containing oligodeoxynucleotide (ODN) is ap-
plicable as a very effective reporter probe for typing of the A/G
single-nucleotide polymorphism (SNP).
sequences
ODN(BPP)
ODN(BPP*)a
ODN(A)
5′-d(CGCAAT[BPP]TAACGC)-3′
5′-d(CGCAAT[BPP*]TAACGC)-3′
5′-d(GCGTTAAATTGCG)-3′
5′-d(GCGTTAGATTGCG)-3′
5′-d(GCGTTAmGATTGCG)-3′
5′-d(CCACAT[BPP]TTATGA)-3′
5′-d(TCATAAAATGTGG)-3′
5′-d(TCATAAGATGTGG)-3′
ODN(G)
ODN(mG)b
ODNIFNG(BPP)
ODNIFNG(A)
ODNIFNG(G)
a BPP* ) [3-15N]- or [4-15N]-BPP. b mG ) N1-methylguanine.
The synthesis of BPP is outlined in Scheme 1. The hydroxy
groups of 5-iodo-2′-deoxycytidine 1 were protected, and then two
benzoyl groups were incorporated into the amino group to give 2.
Photoirradiation with a high-pressure Hg lamp efficiently afforded
the cyclized product 3. The deprotection of 3 gave BPP 2′-
deoxynucleoside 4, which was converted to phosphoramidite 5 for
use in a DNA synthesizer. The ODNs synthesized are summarized
in Table 1.
Figure 1. Absorption and fluorescence spectra of 2.5 µM ODN(BPP)
hybridized with 2.5 µM ODN(G) and ODN(A) (50 mM sodium phosphate,
0.1 M sodium chloride, pH ) 7.0, room temperature). λex ) 347 nm.
ODN(BPP) exhibited an absorption band at 347 nm arising from
BPP, and the absorption maxima shifted slightly to longer
wavelengths by duplex formation with ODN(A) and ODN(G). In
contrast, the fluorescence behavior of ODN(BPP) was strongly
dependent on the purine bases opposite BPP. The fluorescence
spectra of ODN(BPP)/ODN(A) had a strong fluorescence peak at
390 nm, whereas the fluorescence of the ODN(BPP)/ODN(G)
duplex was almost completely quenched. The fluorescence quantum
yield of ODN(BPP)/ODN(G) (Φ ) 0.0018) was approximately
20 times less than that observed for ODN(BPP)/ODN(A) (Φ )
0.035).6
Scheme 1 a
The fluorescent nucleobase BPP can form stable base pairs with
both A and G. In melting temperature (Tm) measurements of the
duplex,7 very high duplex stabilities were observed for both ODN-
(BPP)/ODN(A) and ODN(BPP)/ODN(G) duplexes (Tm ) 54.7 and
57.1 °C, respectively), which were comparable to those of the
natural base pairs T/A (Tm ) 52.5 °C) and C/G (Tm ) 56.0 °C).
Next, hydrogen bonds between BPP and purines have been
investigated by the 15N NMR of ODNs containing 15N-enriched
BPP. [3-15N]- and [4-15N]-enriched BPP (>98% 15N) were prepared
from [3-15N]- and [4-15N]-2′-deoxycytidine,8 respectively, and
incorporated into ODN (ODN(BPP*)). As is apparent in chemical
shifts shown in Figure 2, BPP in ODN(BPP*) exists in the N3-
imino and N4-amide forms, regardless of the type of purine opposite
the BPP. The change in chemical shift on hybridization indicated
that the hydrogen bond at N3 was tight for the BPP/G base pair,
a Reagents and conditions: (a) TBDMSCl, imidazole, DMF, room
temperature, 5 h; (b) benzoyl chloride (2.5 equiv), pyridine, room temper-
ature, 14 h, 82% (two steps); (c) hν (high-pressure Hg lamp), benzene, 10
min, 57%; (d) concentrated ammonia-methanol (1:1), 50 °C, 1.5 h, 82%;
(e) TBAF, THF, room temperature, 2 h, 72%; (f) 4,4′-dimethoxytrityl
chloride, pyridine, room temperature, 6 h, 51%; (g) (iPr2N)2PO(CH2)2CN,
1H-tetrazole, acetonitrile, room temperature, 30 min, quant.
We measured the absorption and fluorescence spectra of ODN-
(BPP)/ODN(A) and ODN(BPP)/ODN(G) duplexes (Figure 1).
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J. AM. CHEM. SOC. 2003, 125, 4972-4973
10.1021/ja034090u CCC: $25.00 © 2003 American Chemical Society

C O M M U N I C A T I O N S
Figure 3. A/G SNP typing using the change of BPP fluorescence. The
fluorescence from a solution containing 5 µM ODNIFNG(BPP)/ODNIFNG(G)
(left) or ODNIFNG(BPP)/ODNIFNG(A) (right) in 50 mM sodium phosphate,
0.1 M sodium chloride, pH ) 7.0, was measured using a transilluminator
at 366 nm.
Figure 2. 15N NMR chemical shifts of 15N-enriched BPP in ODN(BPP*)
and the base-pairing mode of BPP and purines. (a) BPP in ODN(BPP*),
(b) BPP/G base pair in ODN(BPP*)/ODN(G), (c) BPP/A base pair in ODN-
(BPP*)/ODN(A). The 15N-enriched nitrogen atoms are shown in bold.
Table 2. Fluorescence Lifetime Analysis for ODN(BPP)a
(Figure 3). The hybridization of the BPP-containing ODN with a
target DNA facilitates the judgment with the naked eye of the type
of purine located at a specific site on the target DNA, although the
general utility of our method is limited by the flanking base pair
of BPP.12
In summary, we have devised a new fluorescent nucleoside, BPP,
which can sharply distinguish between A and G bases opposite
BPP. The base-pairing degeneracy of BPP may play a key role in
the fluorescence emission and quenching of BPP. The hybridization
of an ODN probe containing BPP with a target DNA facilitates
judgment with the naked eye of the type of purine base located at
a specific site on the target DNA. BPP-containing ODN is a very
effective probe for A/G SNP typing.
strands hybridized
with ODN(BPP)
Φ
τ1 (ps)
(R1)
τ2 (ns)
(R2)
τ3 (ns)
(R3)
(10-2
)
ør2
single strand
ODN(A)
4.09
3.52
0.18
1.26
158 (48)
113 (54)
37b (81)
122 (73)
0.91 (31)
0.44 (43)
1.03 (12)
0.91 (16)
2.40 (21)
2.43 (3)
3.28 (7)
2.67 (11)
1.00
1.13
1.07
1.01
ODN(G)
ODN(mG)c
a ODN(BPP) hybridized with ODN(G) and ODN(A) was monitored (2.5
µM in 50 mM sodium phosphate, 0.1 M sodium chloride, pH ) 7.0, room
temperature). λex ) 295 nm, λem ) 380 nm. The fluorescence lifetime profile
is shown in the Supporting Information. b This value is shorter than the
measurement time scale (41.2 ps/channel). c mG ) N1-methylguanine.
whereas N4 was preferentially used for the BPP/A base pair
hydrogen bond. Thus, BPP is an effective degenerate base, forming
stable base pairs in the Watson-Crick pairing mode for BPP/G
and in the wobble mode for BPP/A.
Acknowledgment. We thank Prof. S. Ito and Dr. H. Ohkita
(Department of Polymer Chemistry, Kyoto University) for valuable
assistance and discussions on the fluorescence decay experiments.
The base-pairing degeneracy of BPP plays an important role in
the sharp fluorescence change, in particular, the quenching in BPP/
G. Accordingly, we measured the quenching of the fluorescence
of BPP by N1-methylguanine (mG),9 which does not form a stable
base pair with BPP,10 to investigate the effect of the stable BPP/G
base pair on the quenching of BPP. The fluorescence of ODN-
(BPP) obtained by hybridization with ODN(mG) was considerably
quenched (Φ ) 0.0126), but the extent of the quenching was not
as large as that with ODN(BPP)/ODN(G). This result suggests that
the formation of a stable BPP/G base pair is a key factor for the
effective quenching of the BPP fluorescence.
To further analyze the character of the fluorescence of BPP in
the duplex in detail, we examined the fluorescence lifetime of
duplexes containing ODN(BPP). The fluorescent decay profiles
of these duplexes were fitted to triexponential functions (Table 2).
The fluorescence decay was strongly influenced by the type of
purine base opposite BPP. An extremely rapid quenching process
that contributed to a strong quenching was only found in ODN-
(BPP)/ODN(G), as judged by the shortest lifetime and the
preexponential factor, R1. This observation strongly suggests that
tight hydrogen bonds between BPP as a fluorophore and G as a
quencher form a complex that undergoes an extremely rapid
quenching and result in an effective quenching of the BPP
fluorescence.
Supporting Information Available: Detailed experimental data
of BPP and the related ODNs (PDF). This material is available free of
charge via the Internet at http://pubs.acs.org.
References
(1) (a) Wojczewski, C.; Stolze, K.; Engels, J. W. Synlett 1999, 1667-1678.
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(6) The fluorescence quantum yields (Φ) of ODN(BPP)/ODN(T) and ODN-
(BPP)/ODN(C) were 0.026 and 0.025, respectively.
(7) All Tm’s of the duplexes (2.5 µM) were measured in 50 mM sodium
phosphate and 100 mM sodium chloride, pH ) 7.0. The absorbance of
the duplexes was monitored at 260 nm from 10 to 80 °C using a heating
rate of 1 °C/min.
(8) (a) Kupferschmitt, G.; Schmidt, J.; Schmidt, T.; Fera, B.; Buck, F.;
Ruterjaus, H. Nucleic Acids Res. 1987, 15, 6225-6241. (b) Ariza, X.;
Bou, V.; Vilarrasa, J. J. Am. Chem. Soc. 1995, 117, 3665-3673.
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639.
(10) The Tm of ODN(BPP)/ODN(mG) was 44.0 °C, indicating that the base
pair between BPP and N1-methylguanine is very loose.
This clear change in fluorescence that depends on the type of
purine base opposite BPP will be very useful for SNP typing of
genes. We tested the distinction of purine bases by BPP hybridiza-
tion using the A/G SNP sequence of the human interferon-γ gene
(IFNG).11 As a result, a strong visible emission was obtained with
the ODNIFNG(BPP)/ODNIFNG(A) duplex, whereas the emission
from the ODNIFNG(BPP)/ODNIFNG(G) duplex was negligible
(11) (a) Iwasaki, H.; Ota, N.; Nakajima, T.; Shinohara, Y.; Kodaira, M.;
Kajita, M.; Emi, M. J. Hum. Genet. 2001, 46, 32-34. (b) http://
snp.ims.u-tokyo.ac.jp/cgi-bin/SnpInfo.cgi?SNP_ID)IMS-JST037908.
(12) When the flanking base pair is a G/C base pair, the fluorescence of BPP
is considerably suppressed. Thus, the SNP typing method would be
inaccurate for the sequence containing a G/C base pair flanking to SNP
site.
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