Monitoring of microenvironmental changes in the major and minor grooves of DNA by dan-modified oligonucleotides

Article abstract of DOI:10.1021/ol052473m

(Graph Presented) We describe the synthesis of new environmentally sensitive fluorescence probes to elucidate DNA structures. DNA oligonucleotides containing fluorophore dan (6-(dimethylamino)-2-acylnaphthalene)-modified dC or dG were able to monitor the

Full text of DOI:10.1021/ol052473m

ORGANIC
LETTERS
2005
Vol. 7, No. 26
5829-5832
Monitoring of Microenvironmental
Changes in the Major and Minor
Grooves of DNA by Dan-Modified
Oligonucleotides
Takumi Kimura, Kiyohiko Kawai, and Tetsuro Majima*
The Institute of Scientific and Industrial Research, Osaka UniVersity,
8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan
majima@sanken.osaka-u.ac.jp
Received October 12, 2005
ABSTRACT
We describe the synthesis of new environmentally sensitive fluorescence probes to elucidate DNA structures. DNA oligonucleotides containing
fluorophore dan (6-(dimethylamino)-2-acylnaphthalene)-modified dC or dG were able to monitor the microenvironmental changes in both the
major and minor grooves of DNA with a B- to A-DNA conformational transition and RNA hybridization.
The structure and dynamics of the grooves of DNA are of
significant importance for the recognition of DNA by
proteins, drugs, and metal complexes.1-3 Such DNA com-
plexes are induced in the major or minor grooves of DNA
via hydrogen bonding and electrostatic or hydrophobic
interactions.4,5 Therefore, it is important to understand the
microenvironment of the major and minor grooves of DNA.
In this study, we selected a dan fluorophore as a probe
for the hydration in the grooves of DNA duplexes. It is
known that the fluorophore 6-(dimethylamino)-2-acylnaph-
thalene (dan) undergoes a large charge redistribution upon
excitation and has nearly ideal environmental sensor
properties.6-8 Hence, the polarity-sensitive dan fluorophore
has been used to understand the environment of the binding
site in proteins or DNA.9,10 We designed novel groove
environmentally sensitive fluorescence probes and demon-
strated their ability to probe the microenvironmental changes
in the major and minor grooves by DNA-drug interactions
and conformational transition. It is known that the nonbase-
(6) Pierce, D. W.; Boxer, S. G. J. Phys. Chem. 1992, 96, 5560-5566.
(7) Yamana, K.; Mitsui, T.; Nakano, H. Tetrahedron 1999, 55, 9143-
9150.
(1) Steitz, T. A. Q. ReV. Biophys. 1990, 23, 205-280.
(2) Zimmer, C.; Wahnert, C. U. Prog. Biophys. Mol. Biol. 1986, 47,
31-112.
(3) Neidle, S.; Pearl, L.; Skelly, J. L. Biochem. J. 1987, 243, 1-13.
(4) Dickerson, R. E.; Drew, H. R. J. Mol. Biol. 1981, 149, 761-786.
(5) Sugiyama, H.; Lian, C.; Isomura, M.; Saito, I.; Wang, A. H. Proc.
Natl. Acad. Sci. U.S.A. 1996, 93, 14405-14410.
(8) Svensson, R.; Greno¨, C.; Johansson, A. S.; Mannervik, B.; Morgen-
stern, R. Anal. Biochem. 2002, 311, 171-178.
(9) Cohen, B. E.; McAnaney, T. B.; Park, E. S.; Jan, Y. N.; Boxer, S.
G.; Jan, L. Y. Science 2002, 296, 1700-1703.
(10) Kamal, J. K.; Zhao, L.; Zewail, A. H. Proc. Natl. Acad. Sci. U.S.A.
2004, 101, 13411-13416.
10.1021/ol052473m CCC: $30.25
© 2005 American Chemical Society
Published on Web 11/18/2005

Table 1. Stokes Shift (∆ν)a and Dielectric Constants (ꢀ)b of Dan Fluoroprobe and Melting Temperatures (Tm)c of Duplexes
No. dioxane (%)d
ꢀ
sequencese
λex (nm) λem (nm) ∆ν (cm-1
)
Tm (°C)
0
15
30
45
78.5 danC monomer
63.3 danC monomer
50.5 danC monomer
37.3 danC monomer
24.0 danC monomer
321
322
325
326
328
323
323
325
323
322
459
457
454
450
444
457
457
443
437
452
9366
9168
8743
8452
7965
9078
9078
8196
8076
8932
60
1
2
3
4
5
6
7
61
61
30
27
56
5′CGCTTTTdanCAAAACGC3′/3′GCGAAAAGTTTTGCG5′
5′CGCCAGGdanCCGCACGC3′/3′GCGGTCCGGCGTGCG5′
5′CGCTTTTdanGAAAACGC3′/3′GCGAAAACTTTTGCG5′
5′CGCTTTTdanCAAAACGC3′/3′r(GCGAAAAGUUUUGCG)5′f
5′CGCTTTTdanGAAAACGC3′/3′r(GCGAAAACUUUUGCG)5′f
5′CGCTTTTCAAAACGC3′/3′GCGAAAAGTTTTGCG5′
5′CGCTTTTGAAAACGC3′/3′GCGAAAACTTTTGCG5′
50
72
54
59
55
50
49
a All samples were excited at 330 nm, and the emission was monitored at 460 nm using a spectral bandwidth of 2.5 nm. b ꢀ values were calculated from
ref 13. c Thermal denaturation profiles were recorded on an Jasco V-530 UV/vis spectrophotometer. Absorbance of the samples was monitored at 260 nm
from 10 to 80 °C. d Mixed solvents were prepared by stirring distilled water with appropriate volume percent of 1,4-dioxane (spectroscopic grade). e DNA
samples are duplex concentration of 80 µM (base concentrated), sodium chloride of 100 mM, and pH 7, buffered by 5 mM sodium phosphate solution, at
7 °C in the following conditions. f The complementary strand is 2′-O-Me substituted RNA oligomer.
paired substituent on the N4- or N2-exocyclic amino groups
of dC and dG in B-DNA extends into the center of the major
and minor grooves, respectively.11,12 Therefore, the dan
fluoroprobe modifications to the amino group of dC or dG
are expected to show only a modest steric effect during the
duplex formation compared with rigid spacer arms in
previous studies (Figure 1).13,14
linkers of nucleobases were conjugated with 6-dimethyamino-
2-naphthoic acid by using activating reagents for peptide
synthesis, N-hydroxybenzotriazole and N,N′-dicyclohexyl-
carbodiimide.
We synthesized a series of dan-labeled dC (danC) or dan-
labeled dG (danG) modified 15 mer oligodeoxynucleotides
(ODNs). The danC containing ODN1 exhibited a melting
temperature (Tm) up to about 1 °C higher than the unmodified
ODN6 (Table 1). On the other hand, the Tm of the danG-
modified ODN3 notably increased (up to 5 °C) more than
the unmodified ODN7. The stabilization of the dan-modified
duplexes seems to reflect the location of the dan moiety in
the major and minor grooves.
ODN1,2 showed a 2-nm blue shift of the dan emission
compared with the dan-modified single strand ODNs
(λmax ) 459 nm). It is known that a subtle difference exists
between the hydration of the G/C and A/T base pairs in
DNA.17 However, no sequence-dependent change in the
fluorescence property was observed for ODN1 and ODN2.
The difference between the hydration of the G/C- and A/T-
rich sequence may be too small for detection in the dan-
modified nucleobase. A dramatic blue shift of the dan
emission was observed for the ODN3 in which the dan
moiety is located in the minor groove. It is known that the
blue shift in the fluorescence emission is dependent upon a
large decrease in the dipole moment in the fluorescent state
compared with that in the ground state.18 Therefore, the
microenvironment of dan fluorophore in the minor groove
of B-DNA shows a higher hydrophobicity compared with
that of the major groove. These results unambiguously
indicated that the dan fluorophore is excellent as a groove
microenvironment-sensitive probe of DNA, and the major
and minor grooves of DNA have significant differences in
polarity.
Figure 1. Dan-modified dC (danC) and dG (danG).
As the flexible linker between dan fluorophore and
nucleobase, a ethylaminolinker was attached to the N4-
position of deoxycytidine and the N2-position of deoxygua-
nosine according to reported procedures.15,16 Ethylamino
(11) Nakatani, K.; Dohno, C.; Saito, I. J. Am. Chem. Soc. 2002, 124,
6802-6803.
(12) Tang, X.; Dmochowski, I. J. Org. Lett. 2005, 7, 279-282.
(13) Barawkar, D. A.; Ganesh, K. N. Nucleic Acids Res. 1995, 23, 159-
164.
(14) Jadhav, V. R.; Barawkar, D. A.; Ganesh, K. N. J. Phys. Chem. B
1999, 103, 7383-7385.
(15) Noll, D. M.; Noronha, A. M.; Miller, P. S. J. Am. Chem. Soc. 2001,
123, 3405-3411.
(17) Barsky, D.; Colvin, M. E.; Zon, C. G.; Gryaznov, S. M. Nucleic
Acids Res. 1997, 25, 830-835.
(18) Weber, G.; Farris, F. J. Biochemistry 1979, 14, 3075-3078.
(16) Adib, A.; Potier, P. F.; Doronina, S.; Huc, I.; Jean, P. Tetrahedron
Lett. 1997, 38, 2989-2992.
5830
Org. Lett., Vol. 7, No. 26, 2005

To estimate the dielectric constants (ꢀ) of the major and
minor grooves in DNA, the fluorescence parameters (λex,
λem) of the danC monomer were measured in media of
different dielectric constants generated from varying the
ratios of dioxane/water (Table 1). We followed the earlier
method of measuring the Stoke’s shift (1/λex - 1/λem) of a
related polarity-sensitive fluorophore in various media of
known ꢀ values.14 The Stoke’s shift (∆ν) value of the major
groove-modified ODN1,2 is 9078 cm-1. These values
correspond to a dielectric constant of 61 D. On the other
hand, the ∆ν of the minor groove-modified ODN3 was 8196
cm-1, which corresponds to the ꢀ value of 30D. The results
showed that the major groove (61 D) of DNA is more polar
than the minor groove (30 D). Therefore, the dan probe is
effective in monitoring the microenvironment of DNA. Jin
and Breslauer measured the minor groove environment with
bisbenzimide as a probe molecule, which was added to the
poly[d(AT)]-poly[d(AT)] duplex without covalent bonding,
and reported an ꢀ value (ꢀ ) 20 D) similar to that (ꢀ ) 30
D) measured in the present study.19 Moreover, many studies
have focused on the hydration process of biomaterials such
as DNA, RNA, or protein.20-22 Therefore, DNA has different
hydration conditions in its two grooves. These results
correspond to the fact that the major groove is about 2 times
wider than the minor groove.
was more polar than that of the major groove. Moreover,
the estimated polarity of the minor groove in the A-like
(ꢀ ) 56 D) was lower than the major groove in the B-like
(ꢀ ) 61 D). It should be noted that the former is more polar
than that in the minor groove of the B-like (ꢀ ) 30 D).
3+
Recently, it has been suggested that Co(NH3)6 induces
the A-DNA structure in DNA with GC-rich sequences, as
evidenced, among other data, by the characteristic changes
in their circular dichroism (CD) spectra. It is known that
3+
Co(NH3)6 can adhere to guanine bases in the deep major
groove of the A-DNA helix, as is evident from the significant
3+
direct NOE cross-peaks from the protons of Co(NH3)6 to
GH8.25 Therefore, we performed the titration of Co(NH3)6
3+
with the major groove-modified ODN1 and 2. As shown in
Figure 2B, the fluorescence spectra of the GC-rich sequence
DNA/RNA hybrids are important intermediates in tran-
scription, the normal replication of double-stranded DNA,23
and the reverse transcription by retroviruses. To compare
the microenvironment of the DNA/RNA hybrid duplexes (A-
like) with that of the DNA duplexes (B-like), the 2′-OMe-
RNA complementary strand such as the RNA-like strand was
hybridized to the dan-modified ODN (ODN4,5). In the major
groove-modified A-like ODN4, a remarkable blue shift of
the dan emission was observed when compared with the
B-like (ODN1,2). However, in the minor groove (ODN5)
of the A-like, an interesting red shift was observed. The
estimated ꢀ values of the major and minor grooves in the
A-form are 27 D and 56 D, respectively. It is known that
the DNA/RNA hybrid is globally closer to the A-form than
the B-form. The X-ray data revealed that the minor and major
grooves of the A-form DNA become progressively wider
and shallower and deeper and narrower than those of the
B-form, respectively. Therefore, it is suggested that the
increase in polarity at the minor groove of the DNA/RNA
hybrid reflects the enlargement of the groove width in the
minor groove. These results suggest that the DNA/RNA
hybrid is more similar to the crystal structure of A-form
DNA.24 The 2′-OMe substitution of the RNA strand may
lose flexibility of the duplex compared with the native RNA.
The microenvironment in the minor groove of the A-like
3+
Figure 2. Fluorescence titration of Co(NH3)6 with the solution
of dan-modified duplexes. (A) Free ODN1 duplex and the 0.1:1,
0.2:1, 0.5:1, 1:1, 2:1, 4:1, and 8:1 Co3+/duplex complexes. (B) Free
ODN2 duplex and the 0.1:1, 0.2:1, 0.5:1, 1:1, 2:1, 4:1, and 8:1
Co3+/duplex complexes.
ODN2 showed a dramatic change in the fluorescence
property of the dan fluorophore. The fluorescence maxima
of ODN2 shifted from 457 to 435 nm by adding Co(NH3)6
It was noted that this tendency is similar to the A-like DNA/
RNA hybrid. It is known that A-DNA is conformationally
similar to the DNA/RNA hybrid.26,27 On the other hand, the
3+
.
3+
AT-rich sequence ODN1, in which Co(NH3)6 does not
induce A-DNA, had only a 6-nm blue shift under the same
conditions. We also observed the intensity decrease of the
dan fluorescence in the presence of Co(NH3)63+. We observed
fluorescence quenching of danC monomer with various ratios
(19) Jin, R.; Breslauer, K. J. Proc. Natl. Acad. Sci. U.S.A. 1988, 85,
8939-8942.
(20) Kochoyan, M.; Leroy, J. L. Curr. Opin. Struct. Biol. 1995, 5, 329-
333.
(21) Lamm, G.; Pack, G. R J. Phys. Chem. B 1997, 101, 959-965.
(22) Young, M. A.; Jayaram, B.; Beveridge, D. L. J. Phys. Chem. B
1998, 102, 7666-7669.
(25) Robinson, H.; Wang, A. H. J. Nucleic Acids Res. 1996, 24, 676-
682.
(26) Egli, M.; Usman, N.; Zhang, S.; Rich, A. Proc. Natl. Acad. Sci.
U.S.A. 1992, 89, 534-538.
(27) Egli, M.; Usman, N.; Rich, A. Biochemistry 1993, 32, 3221-3237.
(23) Hansen, U. M.; McClure, W. R. J. Biol. Chem. 1980, 255, 9564-
9570.
(24) Conn, G. L.; Brown, T.; Leonard, G. A. Nucleic Acids Res. 1999,
27, 555-561.
Org. Lett., Vol. 7, No. 26, 2005
5831

3+
of Co(NH3)6 (data not shown), suggesting that electron
that a critical blue shift of the dan emission depends on the
B- to A-DNA conformational transition of the ODN.
3+
transfer occurs between dan and Co(NH3)6
.
We synthesized the novel dan-modified nucleosides (dan
C
The CD spectra are good indicators of the DNA confor-
mation. Figure 3B shows the CD spectra of the GC-rich
and danG). The ODNs containing a dan-modified nucleoside
exhibit interesting fluorescence properties upon binding to
DNA and RNA duplexes. We measured the local dielectric
constant of the environment in several different DNA
grooves. Dramatic fluorescence changes of the dan-modified
ODNs were observed upon binding to the minor groove of
DNA and the major groove of RNA. These probes can
discriminate between the microenvironments in both grooves
of the duplexes and also detect the B- to A-DNA confor-
mational transition. Therefore, these probes are applicable
to measuring the microenvironment in a variety of DNA
structures. The potential of such a probe can be exploited
by incorporating danC or danG into specific sequences for
studying the microenvironments in different DNA poly-
morphs such as Z-DNA,28,29 triplexes,30 or G-quadruplex31
and by observing hydration dynamics at specific sites in
DNA.
Acknowledgment. This work has been partly supported
by a Grant-in-Aid for Scientific Research (Project 17105005,
Priority Area (417), 21st Century COE Research, and others)
from the Ministry of Education, Culture, Sports, Science and
Technology (MEXT) of the Japanese government.
3+
Supporting Information Available: Experimental de-
tails, synthesis, and characterization of all oligonucleotides,
melting curves, and absorption spectra. This material is
available free of charge via the Internet at http://pubs.acs.org.
Figure 3. Titration of Co(NH3)6 with the solution of dan-
modified duplexes as monitored by their CD spectra. (A) CD spectra
of free ODN1 duplex and the 4:1 and 8:1 Co3+/duplex complexes.
(B) CD spectra of free ODN2 duplex and the 4:1 and 8:1 Co3+
/
duplex complexes.
OL052473M
(28) Kimura, T.; Kawai, K.; Tojo, S.; Majima, T. J. Org. Chem. 2004,
69, 1169-1173.
ODN2. The characteristic spectral shift toward the longer
wavelength associated with a B- to A-DNA transition is
evident.25 However, no change was observed in the CD
spectrum of ODN1 upon the addition of Co(NH3)6 as
compared with the GC-rich sequence. These results suggest
(29) Kimura, T.; Kawai, K.; Majima, T. Chem. Commun. 2004, 268-
269.
(30) Rajeev, K. G.; Sanjayan, G. J.; Ganesh, K. N. J. Org. Chem. 1997,
62, 5169-5173.
(31) Kimura, T.; Kawai, K.; Fujitsuka, M.; Majima, T. Chem. Commun.
2004, 1438-1439.
3+
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Org. Lett., Vol. 7, No. 26, 2005