Fluorometric sensing of the salt-induced B-Z DNA transition by combination of two pyrene-labeled nucleobases

Article abstract of DOI:10.1039/b416965d

We have developed a new fluorescent DNA sensor containing two pyrene-labeled nucleobases, ^PetG and ^PyC, and the fluorescence color was altered by the salt-induced B-Z DNA transtion. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b416965d

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Fluorometric sensing of the salt-induced B–Z DNA transition by
combination of two pyrene-labeled nucleobases{
Akimitsu Okamoto,*a Yuji Ochia and Isao Saito*b
Received (in Cambridge, UK) 5th November 2004, Accepted 6th December 2004
First published as an Advance Article on the web 17th January 2005
DOI: 10.1039/b416965d
We have designed two fluorescent nucleobases for the detection
of the B–Z transition, a guanine derivative in which a pyrene
chromophore is tethered to the C8 position via an ethynyl linker,
PetG,5 and a cytosine derivative in which a pyrene is tethered to the
C5 position via a propargyl linker, PyC (Fig. 1a). PetG will be a
fluorescent nucleoside for inducing a unique structure of G-rich
We have developed a new fluorescent DNA sensor containing
two pyrene-labeled nucleobases, PetG and PyC, and the
fluorescence color was altered by the salt-induced B–Z DNA
transtion.
Fluorescence labeling is a powerful tool for probing the structures
and interactions of biomolecules.1 One of the most attractive
fluorophores for DNA probes is a pyrene chromophore.2 Pyrene
excimer formation is very sensitive to its environment because of
the spatial requirement, and makes pyrene fluorescence a powerful
tool for investigating nucleic acid interactions and structures.
G-rich DNA sequences are known to often adopt unique
conformations, such as Z-DNA duplex, which are biologically
significant and structurally interesting.3 Well-designed pyrene
probes could provide useful information on such a conformational
transition of G-rich DNA.4
Here, we report a new fluorescent DNA probe in which the
fluorescence color is altered by the salt-induced B–Z DNA
transition. We synthesized CG-alternative oligodeoxynucleotides
(ODN) containing two pyrene-labeled nucleobases, and examined
their fluorescence properties. Two conformational states of the
doubly pyrene-labeled ODN, B-DNA and Z-DNA, showed
different colors. This ODN is promising as a fluorescent DNA
sensor sensitive to salt concentrations.
{ Electronic supplementary information (ESI) available: Experimental
details. See http://www.rsc.org/suppdata/cc/b4/b416965d/
*okamoto@sbchem.kyoto-u.ac.jp (Akimitsu Okamoto)
saito@mech.ce.nihon-u.ac.jp (Isao Saito)
Fig. 1 (a) Structure of PetG and PyC. (b) Molecular modeling of B-DNA
containing PetG and PyC, and (c) Z-DNA containing PetG and PyC.
Scheme 1 Reagents and conditions: (a) 1-ethynylpyrene, Pd(PPh3)4, CuI, triethylamine, DMF, 55 uC, 5 h, 70%; (b) TBAF, THF, rt, 2.5 h, 90%;
(c) N,N-dimethylformamide diethylacetal, DMF, rt, 1 h; (d) 4,49-dimethoxytrityl chloride, pyridine, rt, 1 h, 60% in 2 steps; (e) (iPr2N)2PO(CH2)2CN,
1H-tetrazole, acetonitrile, rt, 1 h, quantitative.
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DNA that contains a syn conformation at the N-glycosyl bond,
because a bulky substituent at C8 of PetG will make a syn
conformation predominant.6 In contrast, PyC, which we developed
earlier,7 has an anti conformation when PyC forms a base pair with
G, and thus the pyrene chromophore of PyC was exposed to the
outside of the major grove. Therefore, by using the combination of
two pyrene-labeled nucleosides, we have designed a new system for
fluorometrically monitoring the transition of B- and Z-DNA. In
B-DNA, as shown in Fig. 1b, the two pyrenes of PetG and PyC in a
59-d(PetGPyC)-39/59-d(GC)-39 duplex are far apart, because the
pyrene group of PyC is located at the major groove and the pyrene
group of PetG at the minor groove of the duplex as a result of the
predominant syn conformation. On the other hand, in Z-DNA,
where the bases alternate from G with a syn conformation to C
with an anti conformation, both pyrenes of a 59-d(PetGPyC)-39/
59-d(GC)-39 duplex would be located at the same groove, and
stacked together very closely (Fig. 1c). Thus, this pyrene-stacked
structure is expected to give the emission of an excited complex.
We synthesized the ODNs containing PetG and PyC bases. PyC
was prepared according to the protocol reported earlier.7 The
synthetic route for PetG-containing ODNs is outlined in Scheme 1.
O,O9-Bis(tert-butyldimethylsilyl)-8-bromo-29-deoxyguanosine (1)8
was converted by Sonogashira coupling with 1-ethynylpyrene to
give 2 (70%), which was then deprotected with tetrabutylammo-
nium fluoride to produce PetG (3) (90%, lmax = 386 and 408 nm,
e386 = 39 800, e408 = 38 900, lfl = 480 nm, WF = 0.058 in methanol).
The NOESY spectrum of PetG shows a correlation between the
1-imino proton and the 59-hydroxy proton, strongly indicating that
PetG prefers a syn conformation at the N-glycosyl bond. The
2-amino and 59-hydroxy groups of 8PyG were protected to give 5
(60% in two steps), and then quantitatively converted to
phosphoramidite 6 for DNA synthesis. The ODNs containing
PetG and PyC were efficiently synthesized via a conventional
protocol, and purified by reverse phase HPLC. The composition
of the ODN was determined by MALDI-TOF mass spectrometry.
A CG-alternative DNA is known to form a stable Z-DNA at
high salt concentration.9 We incorporated PetG and PyC into the
CG-repetitive sequence. The resulting ODN, ODN(PetGPyC)
59-d(CGCGCGCGCPetGPyCGCG)-39 (MALDI-TOF [M–H]2
calcd 4771.31, found 4770.92), formed a self-complementary
Z-DNA structure at high salt concentration. The CD spectrum
of ODN(PetGPyC) in an aqueous medium of 50 mM sodium
phosphate (pH = 7.0) and 4.5 M sodium chloride shows a positive
peak at 269 nm and a negative peak at 295 nm, indicative of a
typical Z-DNA (Fig. 2a).10 At a low salt concentration (50 mM
sodium phosphate, pH = 7.0, and 0.1 M sodium chloride), the CD
spectrum changed to a negative peak at 244 nm and a positive
peak at 282 nm, suggesting the formation of the self-complemen-
tary B-DNA.11
Fig. 2 (a) CD spectra of 5 mM ODN(PetGPyC) in 50 mM sodium
phosphate (pH = 7.0). Blue, with 0.1 M sodium chloride; red, with 4.5 M
sodium chloride. (b) Fluorescence spectra of 5 mM each of ODN(PetG) and
ODN(PyC) in 50 mM sodium phosphate (pH = 7.0) and 0.1 M sodium
chloride. Excitation wavelength was 350 nm for ODN(PyC) (green) and
350 (blue) and 420 nm (light blue) for ODN(PetG). (c) and (d) Absorption
and fluorescence spectra of 5 mM ODN(PetGPyC) in 50 mM sodium
phosphate (pH = 7.0), respectively. Blue, with 0.1 M sodium chloride; red,
with 4.5 M sodium chloride. (e) Fluorescence from a solution of 5 mM
ODN(PetGPyC) in 50 mM sodium phosphate (pH = 7.0). The fluorescence
was observed using a transilluminator at 366 nm. Sample 1, with 0.1 M
sodium chloride; sample 2, with 4.5 M sodium chloride.
of ODN(PyC), the fluorescence intensity of ODN(PetG) at
438 nm was ca. 3.4 times lower than that obtained on 420 nm
excitation.
Next, the absorption and fluorescence spectra of a doubly
pyrene-labeled ODN, ODN(PetGPyC) were measured. The absorp-
tion spectrum of ODN(PetGPyC) contained absorption peaks
arising from both PetG and PyC (Fig. 2c). For the ODN(PetGPyC)
at high salt concentration, a fluorescence was observed at 507 nm
on excitation at 350 nm (WF = 0.028) (Fig. 2d). The fluorescence
wavelength from ODN(PetGPyC) was much longer than those
from ODN(PetG) or ODN(PyC). This fluorescence wavelength was
very close to that of a typical pyrene excimer,12 suggesting that the
excited complex of PetG and PyC was formed in the duplex. On the
other hand, the B-form ODN(PetGPyC) at low salt concentration
showed a fluorescence at 440 nm (WF = 0.033). Although the
duplex was excited at 350 nm where PyC was effectively excited as
shown in Fig. 2b, the fluorescence at 400 nm originating from PyC
Prior to the investigation of the fluorescence property of
ODN(PetGPyC), we measured the absorption and fluorescence
spectra of single pyrene-labeled ODNs, ODN(PetG) 59-
d(CGCGCGCGCPetGCGCG)-39 and ODN(PyC) 59-d(CGCG-
CGCGCGPyCGCG)-39. The lmax of ODN(PyC) was 346 nm
(e = 17 200) in 50 mM sodium phosphate (pH = 7.0) and 0.1 M
sodium chloride, and the lfl was observed at 400 nm on 350 nm
excitation (Fig. 2b). The lfl of ODN(PetG) were 38 nm longer than
those of ODN(PyC) (lmax = 392, 402, and 423 nm, lfl = 438 nm
on 420 nm excitation). On excitation at 350 nm, which is the lmax
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was not observed, and only the fluorescence emission at 438 nm,
arising from PetG, was observed. This fluorescence wavelength
indicated that the fluorescent emission process via fluorescence
resonance energy transfer from PyC to PetG was predominant in
B-DNA. Therefore, B- and Z-form structures of ODN(PetGPyC)
induced by salt concentration are easily distinguishable, by
monitoring the change in the fluorescence wavelength.
Two conformations of duplex ODN(PetGPyC) induced by
changing the salt concentration showed different visible colors
(Fig. 2e). For B-DNA, a strong pale blue fluorescence was
observed, whereas a bluish-green fluorescence was emitted by
Z-DNA. The change in the fluorescence color facilitates the
monitoring of salt-induced conformational transition of
ODN(PetGPyC).
Notes and references
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In conclusion, we were able to monitor B–Z DNA transition
using the fluorescence from pyrene-labeled bases. Two pyrene-
labeled bases, PetG and PyC, were designed for monitoring the
structural change. The fluorescence emitted from the labeled ODN
was distinguishable for two conformational states, B- and Z-DNA.
This pyrene-labeled CG-alternative ODN is promising as a new
optical DNA sensor in which the fluorescence color is altered by
changing the salt concentration, and would also be applicable to
DNA–protein interaction assays in which CD spectroscopy is not
suitable.
Akimitsu Okamoto,*a Yuji Ochia and Isao Saito*b
aDepartment of Synthetic Chemistry and Biological Chemistry, Faculty
of Engineering, Kyoto University, Kyoto, 615-8510, Japan.
E-mail: okamoto@sbchem.kyoto-u.ac.jp; Fax: +81 75 383 2759;
Tel: +81 75 383 2757
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I. Tinoco, Jr., Nucleic Acids Res., 1985, 13, 4983.
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Structures, Properties, and Functions; University Science Books:
Sausalito, CA, 2000.
12 A. Okamoto, T. Ichiba and I. Saito, J. Am. Chem. Soc., 2004, 126, 8364;
K. Yamana, T. Iwai, Y. Ohtani, S. Sato, M. Nakamura and H. Nakano,
Bioconjugate Chem., 2002, 13, 1266.
bNEWCAT Institute, School of Engineering, Nihon University and
SORST, Japan Science and Technology Corporation, Tamura,
Koriyama, 963-8642, Japan. E-mail: saito@mech.ce.nihon-u.ac.jp;
Fax: +81 24 956 8924; Tel: +81 24 956 8911
1130 | Chem. Commun., 2005, 1128–1130
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