Synthesis of photo-responsive acridine-modified DNA and its application to site-selective RNA scission

Article abstract of DOI:10.1080/15257770802400099

Photo-responsive phosphoramidite monomers, which bear an azobenzene between acridine and the phosphoramidite unit, were synthesized, and incorporated into oligonucleotides. Upon UV irradiation, the azobenzene in the modified DNA efficiently isomerized from the trans isomer into the cis isomer. Although the Tm values of their duplexes with complementary DNA were not much changed by the isomerization, site-selective RNA scission was significantly accelerated by the UV irradiation when Mn(II) ion was used as the catalyst for RNA scission. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770802400099

Nucleosides, Nucleotides and Nucleic Acids, 27:1175–1185, 2008
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770802400099
SYNTHESIS OF PHOTO-RESPONSIVE ACRIDINE-MODIFIED DNA
AND ITS APPLICATION TO SITE-SELECTIVE RNA SCISSION
Keita Tanaka, Yoji Yamamoto, Akinori Kuzuya, and Makoto Komiyama
Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan
Photo-responsive phosphoramidite monomers, which bear an azobenzene between acridine and
2
the phosphoramidite unit, were synthesized, and incorporated into oligonucleotides. Upon UV
irradiation, the azobenzene in the modified DNA efficiently isomerized from the trans isomer into
the cis isomer. Although the Tm values of their duplexes with complementary DNA were not much
changed by the isomerization, site-selective RNA scission was significantly accelerated by the UV
irradiation when Mn(II) ion was used as the catalyst for RNA scission.
Keywords Photo-responsive; acridine; modified DNA
INTRODUCTION
To date, various conjugates of oligonucleotides with functional
molecules such as intercalating agents, fluorescent dyes, and metal com-
plexes have been prepared because of their attractive potentials for
biological and biochemical applications.[1–4] Acridine is known as an
intercalating agent and has been widely used for fluorescent labelling,
duplex stabilization, and so on.[5–12] Recently, we incorporated an acridine
to oligonucleotides and used these conjugates for site-selective scission
of RNA.[13–15] There, the phosphodiester linkages of the RNA in front
of the acridine were activated by noncovalent interactions such as local
perturbation of RNA-backbone conformations and promptly hydrolyzed by
metal ions. One attractive functionalization of such biochemical tool is to
add photo-responsibility, because light can then trigger RNA scission at
Received 15 April 2008; accepted 30 July 2008.
The authors thank Professor Hiroyuki Asanuma of Nagoya University for kind suggestions. This
work was partially supported by a Grant-in-Aid for Scientific Research from the Ministry of Education,
Science, Sports, Culture and Technology, Japan.
Address correspondence to Makoto Komiyama, Research Center for Advanced Science and
Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8904, Japan. E-mail:
komiyama@mkomi.rcast.u-tokyo.ac.jp
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K. Tanaka et al.
FIGURE 1 The structures of azobenzene-bearing acridine monomer residues (X, Y) and the oligonu-
cleotides used in the present work.
desired timing and place without changing chemical or physical conditions.
In this study, we have synthesized two new phosphoramidite monomers
that bear an azobenzene between an acridine and the phosphoramidite
group (12a and 12b), and incorporated them into oligonucleotides (2, 3,
and 5 in Figure 1). Azobenzene, which is isomerized from its trans-isomer
into cis-isomer upon UV irradiation (λ ≈ 320 nm) and goes back to the
trans-isomer upon visible light irradiation (λ ≈ 450 nm), is one of the most
popular photo-responsive molecules used in biological and biochemical
studies, because of its large photo-isomerization yield, high stability of
the isomers, and satisfactory reversibility. Here, these modified oligonu-
cleotides, which involve both an acridine (site-selective RNA activator)
and a photo-responsive azobenzene, are combined with Mn(II) ion for
site-selective RNA scission. The RNA scission, which is based on site-selective
RNA activation strategy, has been photo-controlled.
RESULTS AND DISCUSSION
Synthesis of Phosphoramidite Monomers and DNA Conjugates
Two kinds of photo-responsive monomers bearing azobenzene be-
tween the acridine and the phosphoramidite unit (12a and 12b)
were synthesized. With 12a, an azobenzene of para-geometry is intro-
duced to oligonucleotides (X in Figure 1), whereas the azobenzene from
12b takes ortho-geometry (Y in Figure 1). This difference should give
rise to subtle but significant change in the position of the acridine

Photo-Responsive Acridine-Modified DNA
1177
SCHEME 1
when the oligonucleotides bind with the RNA substrate for site-selective
scission. First, the 6-chloro-9-(p-chlorophenoxy)-2-methoxyacridine (9)
was reacted with 4-aminobenzylamine to give 9-(p-aminobenzylamino)-
6-chloro-2-methoxyacridine (10). Then 10 was coupled with 4- or 3-
nitrosobenzyl alcohol (8a or b), which was obtained from Scheme 1, to
give acridine-azobenzene conjugate (11a and b). Finally, a 2-cyanoethyl-N,N-
diisopropylphosphoramidite group was introduced to 11a and b to provide
the phosphoramidite monomers 12a and 12b.
SCHEME 2
The photo-responsive acridine-modified oligonucleotides (2, 3, and
5 in Figure 1) were synthesized on an automated DNA synthesizer. In
the final step involving 12a or b, the coupling time was extended to
30 minutes. These modified oligonucleotides were purified as usual and
characterized with MALDI-TOFMS (Table 1). The short oligonucleotide 5
was also analyzed with electronic spray ionization (ESI) mass spectroscopy.
In site-selective RNA scission, 2 (or 3) is hybridized to substrate RNA 1 in
combination with another unmodified DNA (4). As the result, the acridine-
p-azobenzene residue (X) from 12a (or the acridine-o-azobenzene residue
Y from 12b) is placed just in front of the target U-19 residue in the RNA 1
(see Figure 1).
TABLE 1 Mass spectral data of the photo-responsive acridine-modified oligonucleotides.
calcd (M+H)
found
2
3
5
5980.5
5980.5
1393.5
5978.2
5986.4
1396.3

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K. Tanaka et al.
FIGURE 2 HPLC patterns of 5 before (front) and after (back) UV irradiation. Irradiation time is 3
minutes (details are presented in Experimental section).
Efficiency of the Photo-Isomerization of the Azobenzene
in the Modified Oligonucleotides
In order to evaluate the yield of the photo-isomerization of the azoben-
zene unit, the short oligonucleotide 5 was used and analyzed by RP-HPLC
before and after the UV irradiation (3 minutes). As seen in Figure 2, clearly
separated two peaks were observed. The faster peak (retention time = 21
minutes), whose area significantly increased after the UV irradiation,
is assignable to 5 involving cis-form azobenzene, while the slower one
(27 minutes) to the trans-isomer. The ratio of mole absorption coefficient of
the cis-isomer at the detecting wavelength (λ = 332 nm) to that of the trans-
isomer was independently determined to be 1:5.3. Before the UV irradia-
tion, the azobenzene in 5 exists mostly in the trans-form (the cis:trans ratio
= 1:10). After the UV irradiation for 3 minutes, 55% of the trans-azobenzene
was isomerized into the cis-form. The population of cis-azobenzene
in the photo-stationary state was 59% under the conditions employed
(the cis:trans ratio = 3:2).
The thermal isomerization rate of the reverse isomerization was deter-
mined from the time dependence of UV-VIS spectra of 2 (data not shown).
The pseudo-first order rate constant of cis- to trans-isomerization is 0.013 h−1
(t1/2 = 53 hours).

Photo-Responsive Acridine-Modified DNA
1179
TABLE 2 The melting temperatures of the duplexes between acridine-modified DNA conjugate and
complementary DNA.
Tm(◦C)
Before UV irradiation
After UV irradiation
2/6
3/6
46.9
43.4
46.1
43.5
Duplex Forming Activity of the Modified Oligonucleotides
The Tm values of the duplexes of the photo-responsive acridine-
modified oligonucleotides 2 and 3 with their complementary oligonu-
cleotide (6) are shown in Table 2. The 3/6 duplex involving ortho-
azobenzene has similar Tm values with that (43.9◦C) of the unmodified
duplex without azobenzene unit, whereas the 2/6 duplex, in which the
azobenzene takes para-geometry, has significantly higher Tm. It is presumed
that the acridine successfully stacks on the adjacent base pairs when the
para-azobenzene unit is introduced, but the acridine attached to the ortho-
azobenzene does not. These Tm values hardly changed upon photo-induced
cis-trans isomerization of the azobenzenes, thus the isomerization does not
much alter the stacking modes of the acridines.
Effects of Photo-Isomerization of the Azobenzene on
Site-Selective RNA Scission Using the Photo-Responsive
Acridine-Modified Oligonucleotides
Figure 3 shows a typical polyacrylamide gel electrophoresis pattern of
site-selective RNA scission using the 2/4 or the 3/4 combinations. Mn(II)
ion was used as the catalyst for RNA hydrolysis. In the experiments, the mix-
tures of these oligonucleotides and the substrate RNA 1 were divided into
two portions and one portion was directly used for RNA scission by adding
MnCl2. Another portion was first irradiated with UV (λ = 300–400 nm)
for 3 minutes, and then used for the RNA scission. In the presence of either
the 2/4 combination or the 3/4 combination (without UV irradiation), 1
was cleaved site-selectively at the 3ꢁ-side of U-19 (lanes 4 and 6, respectively).
These results are completely consistent with previous results in which
modified oligonucleotides bearing only acridine (without the azobenzene
between the acridine and the main chain) were used in place of 2 and 3.[13]
Even with the incorporation of the azobenzene, the phosphodiester linkage
of RNA which is located in front of the acridine is also selectively activated
and hydrolyzed by the metal ion.
With the use of the 2/4 combination, the scission efficiency was notably
increased upon the UV irradiation (compare lane 4 with lane 5). As shown

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K. Tanaka et al.
FIGURE 3 Gel electrophoresis patterns for site-selective scission of 1 by combining 2/4 or 3/4
with Mn(II) ion. Lane 1, control; lane 2, treatment with Mn(II) alone; lane 3, RNase T1 digestion;
lane 4, 2/4 and Mn(II); lane 5, 2/4 and Mn(II) with UV irradiation; lane 6, 3/4 and Mn(II);
lane 7, 3/4 and Mn(II) with UV irradiation. Reaction conditions; [RNA] = 5 µM, [DNA] = 10
µM, [MnCl2] = 1 mM, [Tris] = 10 mM, [NaCl] = 200 mM, pH 8.0, 37◦C, 18 hours.
in Table 3, the conversion of scission at the 3’-side of U-19 was 2.7%
before the UV irradiation, but increased to 3.9% after the UV irradiation.
Thus, 1.4-fold acceleration was achieved simply by irradiating UV for 3
minutes. The reproducibility of this acceleration has been confirmed by
several separate reactions. With the 3/4 combination, however, the scission
efficiency was not much changed by UV irradiation (lanes 6 versus 7). In
lanes 4–7, a minor site-selective scission simultaneously occurred at the
5ꢁ-side of U-19 (the bands of larger mobility in the gel). This minor scission
by the 2/4 combination was also promoted by 1.4 fold by the photo-induced
isomerization of the azobenzene, whereas the corresponding scission by the
3/4 combination was little affected by the isomerization (see also Table 3).
Although the results in Table 2 indicate that the interactions between the
acridine and adjacent base pairs seem to be roughly the same before and
after the UV irradiation, a slight difference of the orientation of the acridine
in the DNA/RNA duplex probably causes notable change in the efficiency
of site-selective RNA scission. As shown in a previous work,[15] the scission
activities of DNAs bearing modified acridines were quite different even

Photo-Responsive Acridine-Modified DNA
1181
TABLE 3 Conversions of the site-selective scissions of 1 at U-19 before and after UV irradiation.
3ꢁ-side of U-19 (%).
5ꢁ-side of U19 (%)
2/4
2.7
3.9
3.1
3.4
1.0
1.4
1.3
1.4
2/4 + UV Irradiation
3/4
3/4 + UV Irradiation
though they have almost the same Tm values with the complementary RNA.
It was accordingly suggested that subtle difference in the orientations of the
acridine residues notably alters the scission activity.
The trans-2 and the cis-2 species never exist in 100% in the mixture, and
thus the difference in the intrinsic activities of trans-2/4 and cis-2/4 should
be still larger than observed above in the RNA hydrolysis. As estimated by
the HPLC analysis using 5, the ratio of the trans-isomer and the cis-isomer
in the reaction mixture is 10:1 before the UV irradiation and 2:3 after
the irradiation (the population of the cis-isomer was 59%). By analyzing
the 1.4 fold difference of RNA hydrolysis rate using these two ratios, the
intrinsic activity of the cis-2/4 combination is at least twice as high as that
of the trans-2/4 combination. This ratio may be still greater, since the
isomerization of azobenzene is suppressed in DNA-RNA duplexes because
of steric hindrance and thus the population of the cis-2 isomer could be still
smaller even after the UV irradiation.
CONCLUSION
Acridine-modified oligonucleotides which bear photo responsive
azobenzene between the acridine and the main chain have been synthe-
sized. By combining one of them with Mn(II) ion, site-selective RNA scission
has been photo-controlled. Intrinsic activity of the cis-isomer is at least
twice as high as that of the trans-isomer. Except for previous reports on
photo-responsive ribozymes or deoxyribozymes,[16–19] this is the first photo-
responsive site-selective RNA scission system. In order to accomplish more
clear-cut switching, still more efficient isomerization of the azobenzene is
necessary. Such attempt is now under way in our laboratory.
EXPERIMENTAL
Materials
Water was deionized by Millipore Water Purification System (Billerica,
1
MA, USA), and sterilized immediately before use. H NMR spectra were
obtained on a 500 MHz NMR spectrometer (Bruker Avance600). For

1182
K. Tanaka et al.
ESI-MS, a Hitachi M-8000 LC/3DQMS mass spectrometer was used. MALDI-
TOF mass spectra were measured on an AutoFLEX (Bruker, Billerica,
MA, USA). Photo-irradiation was achieved by using UV Spot Light Source
(Hamamatsu Photonics, Hamamatsu, Japan; 200 W) through a UV-D36C
filter (Asahi Technoglass, Tokyo, Japan). Accordingly, the light of 300–400
nm wavelength was irradiated onto the samples.
4-Hydroxyaminobenzyl alcohol (7a). To a solution of 4-nitrobenzyl
alcohol (3.06 g, 19.7 mmol) and NH4Cl (1.57 g, 29.4 mmol) in 2-
methoxyethanol/H2O (30 ml/10 ml), Zn powder (3.84 g, 58.8 mmol) was
added by portions, and the mixture was stirred for 15 minutes at room
temperature. After filtration of Zn powder, the solution was concentrated
under reduced pressure. The product was purified by silica gel column
chromatography (10:1 CHCl3/MeOH) to afford 0.80 g of 7a (29% yield)
1
as a white-yellow solid. TLC: Rf = 0.20, 10:1 CHCl3/MeOH, H NMR
(DMSO-d6) δ 8.27 (d, 1H), 8.18 (s, 1H), 7.12 (d, 2H), 6.80 (d, 2H), 4.96
(t, 1H), 4.39 (d, 2H), MS (ESI): m/z 138 (M−H).
2-Hydroxyaminobenzyl alcohol (7b). The synthetic procedure was the
same as described above for 7a. 1H NMR (DMSO-d6) δ 8.28 (d, 1H), 7.90 (s,
1H), 7.18–7.09 (m, 3H), 6.95 (t, 1H), 5.13 (t, 1H), 4.34 (d, 2H), MS (ESI):
m/z 138 (M–H).
4-Nitrosobenzyl alcohol (8a). In ice cold H2O (60 ml), FeCl3 (3.21 g,
19.8 mmol) was dissolved. To the stirring FeCl3 solution was added dropwise
ethanol solution (2 ml) of 7a (0.50 g, 3.6 mmol), and the mixture was stirred
for additional 20 minutes. After the solution was removed, the residue was
re-dissolved in CHCl3, and the organic layer was washed with H2O. The
organic layer was dried over anhydrous Na2SO4 and concentrated under
reduced pressure to afford 0.30 g of 8a as a crude product. TLC: Rf = 0.61,
10:1 CH2Cl2/MeOH, 1H NMR (DMSO-d6) δ 7.94 (d, 2H), 7.68 (d, 2H), 5.58
(t, 1H), 4.78 (d, 2H).
2-Nitrosobenzyl alcohol (8b). 1H NMR (DMSO-d6) δ 7.99 (d, 1H), 7.91
(t, 1H), 7.43 (t, 1H), 6.60 (d, 1H), 5.66 (s, 3H)
9-(p-aminobenzylamino)-6-chloro-2-methoxyacridine (10). To a solution
of 6-chloro-9-(p-chlorophenoxy)-2-methoxyacridine 9 (2.0 g, 5.2 mmol)
in DMF (10 ml) was added 4-aminobenzylamine (1.3 ml) and N,N-
diisopropylethylamine (1 ml), and the mixture was stirred for 16 hours at
90◦C. After the solvent was removed, CHCl3 and saturated aqueous NH4Cl
were poured to make an emulsion. After filtration, washing the solids with
methanol, DMF, aqueous sodium hydroxide (1 N), and H2O gave 1.5 g of 10
(77% yield) as yellow solid. TLC: Rf = 0.25, 10:1 CH2Cl2/MeOH, 1H NMR
(DMSO-d6) δ 8.34 (d, 1H), 7.86 (d, 1H), 7.82 (d, 1H), 7.62 (d, 1H), 7.48
(t, 1H), 7.40 (q, 1H), 7.28 (q, 1H), 7.07 (d, 2H), 6.52 (d, 2H), 4.99 (s, 2H),
4.80 (d, 2H), 3.79 (s, 3H), MS (ESI): m/z 364 (M + H).
Acridine-azobenzene conjugate (p-isomer; 11a). To a solution of 10 (0.25
g, 0.69 mmol) in acetic acid (10 ml) was added dropwise 8a (0.19 g, 1.4

Photo-Responsive Acridine-Modified DNA
1183
mmol) in acetic acid (10 ml), and the mixture was stirred for 32 hours at
room temperature. After the solvent was removed, the product was purified
by silica gel column chromatography (10:1 CH2Cl2/MeOH) to afford 30 mg
1
of 11a (9% yield) as a red solid. TLC: Rf = 0.14, 10:1 CH2Cl2/MeOH, H
NMR (DMSO-d6) δ 8.32 (s, 1H), 7.90–7.84 (7H), 7.70 (d, 2H), 7.58 (s, 1H),
7.54 (d, 2H), 7.41 (d, 1H), 7.32 (d, 1H), 5.41 (t, 1H), 5.07 (s, 2H), 4.61 (d,
2H), 3.76 (s, 3H), MS (ESI): m/z 483 (M + H), 481 (M–H)
1
Acridine-azobenzene conjugate (o-isomer; 11b). H NMR (DMSO-d6) δ
8.35 (d, 1H), 7.91–7.88 (7H), 7.73–7.65 (4H), 7.58 (m, 2H), 7.46–7.34 (3H),
5.29 (s, 1H), 5.23 (s, 2H), 5.14 (s, 2H), 3.79 (s, 3H), MS (ESI): m/z 483 (M
+ H)
Phosphoramidite monomer of 11a (12a). To a solution of 11a (30 mg,
0.062 mmol) and 2-cyanoethyl N,N,Nꢁ,Nꢁ-tetraisopropylphosphoramidite
(21 mg, 0.068 mmol) in anhydrous DMF (1 ml) on ice was slowly added
1H-tetrazole (6 mg, 0.085 mmol) in anhydrous DMF (1 ml) under nitrogen,
and the mixture was stirred for 1 hour at room temperature. After the
solvent was removed, ethyl acetate (50 ml) was poured, and the organic
layer was washed with saturated aqueous NaCl (50 ml × 2). This layer was
dried over anhydrous Na2SO4 and concentrated under reduced pressure to
afford 46 mg of 12a (quant). This product was dissolved in anhydrous DMF
(0.1 M) and used for automated DNA synthesis without further purification.
TLC: Rf = 0.57, 10:1 CH2Cl2/MeOH, MS (ESI): m/z 683 (M + H),
Phosphoramidite monomer of 11b (12b). This was prepared in a similar
manner as described for 12a. MS (ESI): m/z 683 (M + H)
Preparation of modified oligonucleotides. The substrate 1 is 36mer-RNA
(5ꢁ-end FAM-labeled) and the U19 was chosen as the target-site for the
scission. The complementary A was replaced with the unit from either 12a
or 12b. All the oligonucleotides were synthesized on an ABI 3400 DNA
Synthesizer in 1 µmole scale. An extended coupling time of 30 minutes
was adopted for 12a and 12b. The oligonucleotides were cleaved from
the resin, and deprotected by a treatment with 0.4 M methanolic NaOH
(methanol:water = 4:1, 1 ml) at room temperature for 16 hours.
Purification and characterization of the acridine-modified DNAs. The
oligonucleotides were purified by Poly-Pak II cartridges (Glen Research
Co., Sterling, VA, USA). The resulting crude product was then purified
by denaturing 20% PAGE. Final purification was carried out by a reversed
phased HPLC equipped with an RP-C18 column (Cica-Merck LiChroCART
125–4; a linear gradient of 0–25% acetonitrile in 50 mM ammonium formate
over 25 minutes; flow rate 0.5 ml/minutes). They were characterized by
MALDI-TOFMS (Bruker AutoFLEX) in the positive ion mode. 5 was also
characterized with ESI-MS in the negative ion mode.
HPLC assay for photo-isomerization of the azobenzene. Aqueous solu-
tion of 5 (100 µM) was divided into two portions, and both were heated
to 97◦C for 1 minute and slowly cooled down to room temperature. One

1184
K. Tanaka et al.
portion was irradiated with UV for 3 minutes to isomerize trans-azobenzene
into cis-form. 30 µl of each solution was analyzed by a reversed phased HPLC
(a linear gradient of 15–50% acetonitrile in 50 mM ammonium formate over
40 minutes; flow rate 0.5 ml/minute; wavelength 332 nm).
Measurement of melting temperatures (Tm) of the duplexes between
acridine-modified oligonucleotides and its complementary DNA. Melting
profiles were measured on a JASCO V-530 UV/Vis spectrophotometer. The
absorbance at 260 nm was measured from 5 to 60◦C in a quartz cell of 1
cm path length with a rate of 1.0◦C/minute. The specimens contained 2
or 3 (1 µM) and 6 (1 µM) in pH 8.0 Tris-HCl buffer. For the “cis-isomer”
experiments, the specimens were irradiated with UV for 30 minutes before
the Tm measurements, and soon used for the measurements.
RNA scission assay. The substrate RNA (5 µM), acridine modified DNA
(2 or 3; 10 µM) and unmodified DNA (6; 10 µM) were added to Tris buffer
(10 mM) containing NaCl (200 mM). The mixture was then divided, and
both portions were heated to 97◦C for 1 minute and slowly cooled down to
room temperature. One of them was directly used for the RNA hydrolysis,
while another was irradiated with UV light for 3 minutes to isomerize
trans-azobenzene into cis isomer and then used for RNA scission. Selective
cleavage reaction was initiated by adding aqueous solution of MnCl2 to the
mixture. After incubation for a predetermined reaction time at 37◦C, the
reaction was quenched by saturated EDTA·2Na solution and analyzed on
20% denaturing PAGE. All the reactions were carried out in shielded tubes
to avoid the isomerization of the cis-azobenzene into trans-form by ambient
light.
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