Photocontrolled binding and binding-controlled photochromism within anthracene-modified DNA

Article abstract of DOI:10.1021/ja304205m

Modified DNA strands undergo a reversible light-induced reaction involving the intramolecular photodimerization of two appended anthracene tags. The photodimers exhibit markedly different binding behavior toward a complementary strand that depends on the number of bases between the modified positions. By preforming the duplex, photochromism can be suppressed, illustrating dual-mode gated behavior.

Full text of DOI:10.1021/ja304205m

Communication
pubs.acs.org/JACS
Photocontrolled Binding and Binding-Controlled Photochromism
within Anthracene-Modified DNA
Jack Manchester,§ Dario M. Bassani,† Jean-Louis H. A. Duprey,§ Luciana Giordano,§ Joseph S. Vyle,‡
Zheng-yun Zhao,§ and James H. R. Tucker*,§
§School of Chemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K.
†Universite
́ ́
de Bordeaux, CNRS, ISM UMR 5255 351, Cours de la Liberation, 33400 Talence, France
‡School of Chemistry and Chemical Engineering, David Keir Building, Queen’s University Belfast, Stranmillis Road, Belfast BT9 5AG,
U.K.
S
* Supporting Information
Scheme 1. The S3-A↔S3-AP Photochromic System, in
Which the Anthracene Photodimer Consists of the Head-to-
Tail Isomer
ABSTRACT: Modified DNA strands undergo a reversible
light-induced reaction involving the intramolecular photo-
dimerization of two appended anthracene tags. The
photodimers exhibit markedly different binding behavior
toward a complementary strand that depends on the
number of bases between the modified positions. By
preforming the duplex, photochromism can be suppressed,
illustrating dual-mode gated behavior.
ithin the field of light-activated molecular and supra-
W
molecular systems, the photocontrol of biomolecular
binding processes in chemical or biological environments is
particularly appealing, due to the prospect of light-triggered
uptake or release of agents for various therapeutic and
nanodevice applications.1 An effective way of achieving
reversible control over such events is to attach photochromic
groups to biomolecular components in a way that allows a
structural change at the photochrome to impart a change in
binding affinity. As far as nucleic acids are concerned, trans−cis
isomerism (in particular that within azobenzene) has been
utilized extensively, allowing photocontrol over factors such as
secondary structure, binding, and catalysis.2,3 Until now,
anthracene photochromism has been used considerably less
in this capacity,4 but it is quite unusual among photochromic
systems in that it involves a photodimerization reaction, which
can lead to a profound change in structure when two
anthracenes are appended to one binding motif. We have
used this approach effectively in the past to demonstrate the
photoswitched binding of cations5a and also neutral mole-
cules5b,c using supramolecular receptor systems. Here we show
how the incorporation of two anthracene groups into one
oligonucleotide strand6 leads to an unusual example of a system
in which DNA duplex formation can either control, or
alternatively be controlled by, a photochromic process
(Scheme1).
a
Table 1. Sequences of the 14 DNA Strands Synthesized
S1-A
S2-A
S3-A
S1-B
S2-B
S3-B
S1-C
S2-C
S3-C
S1-D
S2-D
S3-D
T0
5′-TGGACTXTXTCAATG-3′
5′-TGGACXCTCXCAATG-3′
5′-TGGAXTCTCTXAATG-3′
5′-TGGACTXTYTCAATG-3′
5′-TGGACXCTCYCAATG-3′
5′-TGGAXTCTCTYAATG-3′
5′-TGGACTYTXTCAATG-3′
5′-TGGACYCTCXCAATG-3′
5′-TGGAYTCTCTXAATG-3′
5′-TGGACTYTYTCAATG-3′
5′-TGGACYCTCYCAATG-3′
5′-TGGAYTCTCTYAATG-3′
3′-ACCTGAGAGAGTTAC-5′
5′-TGGACTCTCTCAATG-3′
S0
a
Each strand composition is identified by the letter codes in Figure 1,
so that, for example, S2-B is the strand where anthracene tag X and
propyl linker Y are separated by three bases.
control strands containing the propyl linker Y were synthesized,
with letter codes B, C, and D identifying the particular
combination of X and Y used. The anucleosidic threoninol unit
in X (in this study used in its D-configuration) has previously
been shown to be readily incorporated into oligonucleotides via
The sequences of the DNA strands made for this study are
presented in Table 1, and the structures of the anucleosidic
groups X and Y incorporated into the modified strands are
depicted in Figure 1. Sequences S1-A, S2-A, and S3-A were
designed to monitor the effect of increasing the number of
bases between the two anthracene tags X. In addition, various
Received: May 4, 2012
Published: June 13, 2012
© 2012 American Chemical Society
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Communication
target strand T0, as evidenced by melting temperatures from
variable-temperature UV/vis spectroscopy (phosphate buffer,
250 mM NaCl, strand concentration = 5 μM). The Tm values
are presented in Table 2. The duplex between strands S2-A and
Table 2. Tm Data (°C) for Duplexes Formed by Various
a
Strands with the Complementary Target Strand T0
modification
S1
S2
45
S3
35
Figure 1. Structures of the anucleosidic groups X and Y when
incorporated into DNA (phosphodiester groups toward 3′ end).
A
37.5
37
b
AP
B
25
<5
38
40
28.5
27
C
37.5
30
40
the corresponding phosphoramidite monomer.2a,b,7 The
anthracene tag was connected to the threoninol unit according
to a procedure described previously for related systems,7 prior
to DNA incorporation via standard automated synthesis. All the
strands, including the target strand T0 and its unmodified
complementary S0, were purified by reversed-phase HPLC and
characterized by ESI mass spectrometry (see the Supporting
Information (SI)).
D
31.5
19.5
a
Conditions: 5 μM, pH 7.0, 10 mM sodium phosphate buffer, 250
b
mM NaCl. No duplex formation observed down to 5 °C, the lowest
temperature the conditions allowed for in water.
T0 is the most stable, which is consistent with it containing two
more GC base pairs. Significantly, the values for the A duplexes
are all higher than those for most of the control duplexes
involving strands B, C, and D that contain the propyl linker Y
instead of the anthracene tag X at one or both positions.9 This
indicates that the anthracene groups stabilize their respective
duplexes through an intercalative interaction with the base-pair
stack, in agreement with our previous findings7b on the same
15-mer sequence. A striking trend is apparent when comparing
the Tm data for the three photoproducts with those for the
corresponding starting materials. For the S1-A system, there is
essentially no change in duplex stability upon photocyclization.
However, the ΔTm value is 20 °C for the S2-A system, and for
the five-base-separated system, no inflection was observed at all
(Figure 2), indicating no duplex formation whatsoever between
Upon photoirradiation with filtered light from a high-
pressure Hg-Xe lamp (365 5 nm) of Ar-degassed solutions of
each of the doubly tagged strands S1-A, S2-A, and S3-A (ca. 20
μM, 10 mM phosphate buffer, pH 7.0, 100 mM NaCl), the
characteristic anthracene band centered at ca. 360 nm was
observed to decrease significantly over a period of 40 min.
HPLC runs of these irradiated solutions indicated a clean
photoreaction with generally high conversion (see the SI), with
the appearance in each case of one new major peak and one
new minor peak in addition to the residual starting material.
Mass spectrometry analysis of the isolated major photoproduct
from each reaction (designated S1-AP, S2-AP, and S3-AP,
respectively) revealed a mass identical to that of the
corresponding starting material in each case, in agreement
with the formation of an intramolecular photodimer. The
absence of any photoreactivity in the singly tagged B and C
control strands for S1, S2, and S3 excludes the occurrence of
other significant intermolecular (e.g., between anthracenes on
separate strands) or intramolecular (e.g., with DNA bases)
photoinduced processes. At room temperature, each major
photoproduct was found to be quite stable, whereas the minor
product readily converted back to the starting material. In line
with previous work on related anthracene systems,5b,c,8 this
trend indicates a head-to-tail (see Scheme 1) and a head-to-
head orientation for the major and minor photoadducts,
respectively.
A series of thermal reversion studies were then undertaken
on buffered solutions of the three major photoproducts (ca. 5
μM). In each case, no significant changes were noted below 55
°C, but upon continued heating at 80 °C for 16−20 h, each
compound reverted back cleanly to its respective starting
material, as indicated by HPLC. The opening rate constants at
80 °C were determined to be 2 × 10−3, 1.9 × 10−3, and 2.6 ×
10−3 s−1, respectively for S1-AP, S2-AP, and S3-AP. The data
indicate that the base separation between the photoligated units
influences the reversion kinetics to some extent, with the five-
base separation giving the fastest rate, presumably due to
greater destabilization of the cyclodimer by the longer
oligonucleotide spacer.
Figure 2. Normalized graphs showing the change in absorbance as a
function of temperature for T0 in the presence of S3-A (red) and S3-
AP (dashed blue).
S3-AP and T0 under the conditions used. These differences in
duplex stability are comparable with the best results obtained in
other photoswitchable systems2b,3b where normally more than
one photochromic unit is required to generate large ΔTm
values. These studies indicate that the greater the base
separation between the reacting anthracene units, the greater
the structural change upon photodimerization, which then
hinders or even prevents duplex formation with the
complementary strand.
To further probe these dramatic differences in duplex
stability, two other independent sets of experiments were
undertaken. First, CD spectroscopy was performed at 20 °C
under the same conditions as the melting curves. For both S1-
The extent to which DNA duplex formation could affect, or
be affected by, anthracene photochromism was then inves-
tigated. Each of the doubly tagged A strands was found to form
a stable duplex at room temperature with the complementary
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Journal of the American Chemical Society
Communication
AP and S2-AP in the presence of T0, the characteristic negative
and positive bands associated with duplex B-DNA were
observed. However, in the corresponding scan for S3-AP, the
negative band at ca. 245 nm correlating to duplex helicity10 was
absent, with the observed spectrum essentially the same as that
for the two strands measured independently and then
mathematically added together (Figure 3).
This system therefore represents an example of gated
photochromism,11 where a separate external input (in this case,
a binding process via the addition of DNA strand) can control a
photochromic process by switching it ON or OFF. The
addition of T0 restricts photochromism, but by then adding the
competing strand S0, the stronger duplex S0/T0 is formed,
thereby unlocking the system and allowing photochromism to
recommence (Scheme 2). Therefore, in this particular system,
it is possible to demonstrate both photocontrolled duplex
formation and binding-controlled photochromism.
Scheme 2. Representation of Photocontrolled Binding of T0
and Binding-Controlled Photoreactivity within the S3-A↔
S3-AP Photochromic System
Figure 3. CD spectra of T0 with 1 molar equivalent of S3-A (red),
with 1 molar equivalent of S3-AP (dashed blue), and measured alone
and then mathematically added to an independent spectrum of S3-AP
(black). Conditions: 5 μM, pH 7.0, 10 mM sodium phosphate buffer,
250 mM NaCl, 293 K.
Second, a series of native gel electrophoresis experiments was
undertaken. Under the conditions used, which required a lower
NaCl concentration of 25 mM, neither S2-AP nor S3-AP could
form a stable duplex with T0 at 20 °C, as illustrated for the
three-base-separated system in Figure 4. Ethidium bromide
To conclude, these studies further demonstrate the scope
and potential of photochromism in the design of functional and
controllable nanodevices comprising biomolecular components.
Further studies are now underway to explore and exploit these
findings further in related nucleic acid and peptide systems.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures for new compounds, oligomer
characterization (HPLC, MS), selected UV/vis and fluores-
cence spectra, CD spectra, electrophoresis. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
j.tucker@bham.ac.uk
■
Notes
The authors declare no competing financial interest.
Figure 4. 20% Native-PAGE experiment illustrating differing duplex-
forming abilities with T0 between strands S0, S2-A, and S2-AP: lane 1,
S0; lane 2, T0; lane 3, S0 + T0; lane 4, S2-AP; lane 5, S2-AP + T0;
lane 6, S2-A; lane 7, S2-A + T0. Conditions: 25 mM NaCl, 1× TB, 24
h at 100 V, 293 K.
ACKNOWLEDGMENTS
■
This research was funded by an EPSRC Leadership Fellowship
to J.H.R.T. (EP/G007578/1). Advantage West Midlands and
staff within the School of Chemistry Analytical Facility are
gratefully acknowledged. J.H.R.T. thanks Prof. Alison Rodger
(Warwick) for helpful suggestions in the preparation of this
manuscript.
staining experiments (ethidium binds preferentially to duplex
DNA, see the SI) confirmed unambiguously that only the
undimerized strands S2-A and S3-A formed a duplex under
these conditions.
Finally, whereas the photoreaction of the single strands S1-A,
S2-A, and S3-A was straightforward, little photoreactivity was
observed upon photoirradiation of their corresponding
duplexes (again formed with the complementary strand T0)
under the same conditions (see SI). This supports the notion of
the anthracene units interacting with the base-pair stack (vide
supra), which precludes their availability for photodimerization.
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Communication
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