Rational design of a photoswitchable DNA glue enabling high regulatory function and supramolecular chirality transfer

Article abstract of DOI:10.1039/d1sc02194j

Short, complementary DNA single strands with mismatched base pairs cannot undergo spontaneous formation of duplex DNA (dsDNA). Mismatch binding ligands (MBLs) can compensate this effect, inducing the formation of the double helix and thereby acting as a molecular glue. Here, we present the rational design of photoswitchable MBLs that allow for reversible dsDNA assembly by light. Careful choice of the azobenzene core structure results in excellent band separation of theEandZisomers of the involved chromophores. This effect allows for efficient use of light as an external control element for duplex DNA formation and for an in-depth study of the DNA-ligand interaction by UV-Vis, SPR, and CD spectroscopy, revealing a tight mutual interaction and complementarity between the photoswitchable ligand and the mismatched DNA. We also show that the configuration of the switch reversibly dictates the conformation of the DNA strands, while the dsDNA serves as a chiral clamp and translates its chiral information onto the ligand inducing a preference in helical chirality of theZisomer of the MBLs.

Full text of DOI:10.1039/d1sc02194j

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Rational design of a photoswitchable DNA glue
enabling high regulatory function and
supramolecular chirality transfer†
Cite this: Chem. Sci., 2021, 12, 9207
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
Nadja A. Simeth, a Shotaro Kobayashi,b Piermichele Kobauri, a Stefano Crespi,
a
ac
b
b
*
*
Wiktor Szymanski,
and Ben L. Feringa
Kazuhiko Nakatani,
Chikara Dohno
a
*
Short, complementary DNA single strands with mismatched base pairs cannot undergo spontaneous
formation of duplex DNA (dsDNA). Mismatch binding ligands (MBLs) can compensate this effect,
inducing the formation of the double helix and thereby acting as a molecular glue. Here, we present the
rational design of photoswitchable MBLs that allow for reversible dsDNA assembly by light. Careful
choice of the azobenzene core structure results in excellent band separation of the E and Z isomers of
the involved chromophores. This effect allows for efficient use of light as an external control element for
duplex DNA formation and for an in-depth study of the DNA–ligand interaction by UV-Vis, SPR, and CD
spectroscopy, revealing a tight mutual interaction and complementarity between the photoswitchable
ligand and the mismatched DNA. We also show that the configuration of the switch reversibly dictates
the conformation of the DNA strands, while the dsDNA serves as a chiral clamp and translates its chiral
information onto the ligand inducing a preference in helical chirality of the Z isomer of the MBLs.
Received 20th April 2021
Accepted 22nd May 2021
DOI: 10.1039/d1sc02194j
rsc.li/chemical-science
photoresponsive unit is addressed from those potentially pho-
toactive elements or structures in its surroundings.15–17
Introduction
Light is a convenient control element in biology,1–6 supramo-
lecular chemistry,7,8 and material science.9–11 At the molecular
level, such non-invasive control is enabled by photoresponsive
elements, i.e. optical molecular switches.9,10,12–14 These photo-
switches, when integrated into a system of choice, can affect its
properties dynamically and reversibly.7,9 A crucial requirement
in the photochemical control of a functional entity being part of
a biological system or a material is the ability to address the
photoactuator selectively in the complex spectral context of the
framework that it is supposed to regulate. In other words, the
key challenge in designing light-responsive systems is to ensure
the separation of the spectral region in which the
Molecular photoswitches have emerged as the addressable
unit of choice due to the reversible nature of their light-
stimulated isomerization.9,13,14 They have been employed to
control, for instance, the shape of polymer lms,18 surface
properties,19 nano architectures,20 probes in chemical biology,21,22
and drug activity.2 DNA binders based on various photo-
switches,23,24 such as overcrowded alkenes,25–27 diarylethenes,28,29
spiropyrans,30–32 hemiindigos,33,34 and azobenzenes,35–42 among
others, have been investigated. The photoisomerization needs to
be highly efficient to dynamically regulate such systems with
light.15,16 In biological and biohybrid systems, photons are oen
ltered by other chromophores, like amino acids, nucleotides,
and ligands interfering with the biomolecules (Fig. 1A, top) due to
spectral overlap, compromising effective switching.43 The chal-
lenge is to spectrally separate the absorption band of the switch
from those of all other components of the system (Fig. 1A,
bottom). Here, we show how careful design of azobenzene pho-
toswitches enables not only efficient photochemical intercon-
version, but also light-induced control of supramolecular
assemblies. Specically, light-induced isomerization between the
non-binding E and the stabilizing Z isomer allows for reversible
DNA ligand binding and duplex formation (Fig. 1B).
aCentre for Systems Chemistry, Stratingh Institute for Chemistry, Faculty for Science
and Engineering, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The
Netherlands. E-mail: b.l.feringa@rug.nl
bDepartment of Regulatory Bioorganic Chemistry, The Institute of Scientic and
Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki 567-0047, Japan.
E-mail: cdohno@sanken.osaka-u.ac.jp; nakatani@sanken.osaka-u.ac.jp
cDepartment of Radiology, Medical Imaging Center, University of Groningen,
University Medical Centre Groningen, Hanzeplein 1, 9713 GZ Groningen, The
Netherlands
Deoxyribonucleic acid (DNA) is a biomacromolecule with
a dened supramolecular architecture, determined i.e. by selec-
tive hydrogen bond interactions and p stacking.44 Formation of
a three-dimensional helical structure is crucial for its biological
† Electronic supplementary information (ESI) available: Experimental procedures
and characterization data for all new compounds, photophysical and chemical
studies, CD experiments, Tm and SPR measurements, detailed protocols of the
computational calculation. See DOI: 10.1039/d1sc02194j
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Fig. 1 (A) Schematic representation of the different contributions to the UV-Vis spectrum in biological molecules or biohybrid systems with small
chromophores as ligands and azobenzene as photoactuator. (Top) Typical scenario where the pp* and np* transitions of both photoisomers,
and the spectrum of the ligands are extensively overlapping. (Bottom) Idealized scenario with high band separation of the respective electronic
transitions of ligands, and E, Z photoisomers. (B) E–Z photoisomerization of an azobenzene-based mismatch binding ligand (MBL) facilitates
reversible DNA hybridization.
function, frequently involved in disease-related malfunctions, hydrogen-bonding groups complementary to the mismatched
and represents a key organization element in DNA-based nano- bases. A molecule with this property is 2-carbonylamino-1,8-
materials.45–47 Small molecules, decorated with suitable substit- naphthyridine (Fig. 2A), a small chromophore that recognizes
uents, can interact with DNA,36 and consequently affect its guanine (G), and its covalent dimer was the rst ligand reported
properties, such as sequence-specic hybridization, without for GG mismatch binding.49 A naphthyridine carbamate (NC,
introducing chemical modications onto DNA. For example, Fig. 2A) was found to be a better G recognition unit in terms of
mismatch-binding ligands (MBLs) inuence DNA hybridization binding affinity to a GG homo mismatch and chemical stability of
and its secondary structure by enhancing the stability of the the molecule. Fig. 2B displays a few examples of MBLs based on
duplex DNA.24 Several selective MBLs have been developed that NC units introduced by Nakatani and coworkers. Specically,
discriminate, for instance, AA48 or GG homo mismatches.49,50 a naphthyridine carbamate dimer (NCD) was the rst MBL re-
Such MBLs are typically DNA intercalators, which possess ported as a molecular glue for DNA.51 A 2 : 1 ratio of NCD to
Fig. 2 (A) Naphthyridine carbamate (NC) units selectively bind to GG homo-mismatches. (B) Structures of diverse (activatable) MBLs. (C)
Overview of the current work.
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dsDNA stabilized a GG mismatch by DTm (difference in melting superb variants, we simulated the spectra of individual
temperature of DNA) of more than 30 ꢀC in an appropriate 11-mer components in water using time-dependent density function
duplex DNA.51 Hence, it can function as molecular glue between theory (TD-DFT) at the PBE0/6-311+G(2d,p) level of theory,
DNA strands that do not hybridize spontaneously in a certain a method proven to be reliable for azoswitches (with SMD
range of temperatures.51 Subsequently, both TD (a thermally implicit model used for water as a solvent, for details cf. Section
degradable NCD analog) and NCDA3 (a photoswitchable deriva- 6 in the ESI†).63 The calculated spectra of naphthyridine
tive of NCD) were developed (Fig. 2B).52,53 In NCDA3, an azo- methylcarbamate and the azobenzene core structure in NCDA3
benzene scaffold was employed as the photochromic core are depicted in Fig. 3 (black and red solid lines, respectively).
structure.53,54
The rst allowed transition of the naphthyridine methyl-
Azobenzenes are light-responsive molecules that have a ther- carbamate at 294 nm overlaps substantially with the main band
modynamically stable E and a photochemically generated, of E-CH2NHCH3, namely its pp* transition, at 355 nm. Due to
metastable Z isomer. While the E isomer is apolar and planar, the the spectral overlap, selective irradiation of the pp* transition
Z isomer has a dipole moment of ca. 3 D and a strongly twisted, of azobenzene might not be possible and photons needed to
helical shape.12,55,56 E–Z isomerization can be triggered photo- trigger the E–Z isomerization might be ltered by the NC
chemically in both directions in a reversible manner. Moreover, Z moiety. With this result in hand, we focused on nding azo-
/ E interconversion occurs spontaneously in a thermal process benzenes in which the pp* transitions are more bath-
and is associated with a dened thermal lifetime s of the Z ochromically shied and the separation between isomer bands
form.12,55 Azobenzenes are frequently used as photoswitchable is larger. Anticipating that 4-alkyloxy substituted azobenzenes
motif to design light-responsive DNA–ligands37–42 or nucleobase would absorb at longer wavelengths than alkyl-substituted
surrogates57–61 due to their facile synthesis, tunability, and analogs and show good photostationary distributions
pronounced geometrical change upon photoisomerization.12 (PSDs),64–66 we focused on 4,40-di-methoxy azobenzene E-OCH3.
Indeed, in MBLs, the helical conformation of the Z isomer has Using TD-DFT we established that the pp* transition of the E
been shown to have the ideal geometry to bind to DNA isomer is bathochromically shied (lmax ¼ 382 nm, cyan solid
mismatches while the E form had lower affinity.24
line, Fig. 3) and consequently, the overlap with the naphthyr-
In NCDA3, the E / Z photoisomerization was inefficient and idine carbamate absorption band is signicantly reduced. We
several minutes of irradiation with UV light were required to also found that in 4,40-di-methoxy azobenzene the absorption
induce sufficient isomerization. Moreover, extensive spectral
overlap of the various chromophores involved prevented
detailed spectroscopic studies and effective isomerization.53,54
These drawbacks impair the use of NCDA3 in rapidly respond-
ing DNA-based systems. Development of photoswitchable MBLs
with precisely tuned photoisomerization properties would pave
the way toward regulation of biological processes by targeting
endogenous DNAs and RNAs.62
Here, we focus on tuning the photochemical properties of the
azobenzene core structure to adjust them to the requirements of
the system (cf. Fig. 1A and B), which enabled fast dynamic control
of DNA double helix formation and real-time studies. Careful
design of a suitable photoactuator furnished a highly sensitive
ligand (Fig. 2C) featuring (i) excellent band separation, (ii)
quantitative photoisomerization, (iii) high quantum yield (QY),
(iv) fast response, and (v) excellent dsDNA assembly ability.
Additionally, we provide in-depth insight into mutual interaction
of the photoswitchable MBLs and the dsDNA, based on extensive
spectroscopic and computational studies. In particular, we
present the discovery of a highly effective photoswitchable DNA
glue. This work highlights major steps towards the development
of photoresponsive, fully reversible MBLs and important
advances towards the rational design of light-responsive systems
for future non-invasive control of biological function with
potential for nanomaterial assembly.
Fig. 3 (A) Computed UV-Vis spectra of the photoactive core struc-
tures involved employing TD-DFT at the PBE0/6-311+G(2d,p) level of
theory using the SMD model for water. Vertical lines indicating the
position of the pp* transition bands allow to better appreciate the
Results and discussion
Computationally supported molecular design
To analyze the various contributions to the UV-Vis absorption
spectral separation of E-CH3NHCH3 and E-OCH3 with their respective
spectrum of our existing photoswitchable MBLs, and to design Z isomers. (B) Studied core structures.
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Scheme 1 Synthesis of target compounds 2 and 3 via alkylation as key step.
maximum of the E isomer corresponds to an absorption Subsequent deprotection using thiophenol gave target
minimum of the Z form at 388 nm (Fig. 3A). In contrast, the compounds 2 and 3 in 62% and 79% yield, respectively. MBL 4
maximum of the E-CH2NHCH3 (lmax ¼ 355 nm) and the was obtained using reductive amination as the key step for the
minimum of the Z form (lmin ¼ 388 nm) are more than 30 nm introduction of the NC groups onto the azobenzene core (see
apart, leading to a substantial overlap of the spectra. Hence, the Scheme S2, ESI, Sections 1.2 and 7,† together with a full char-
bands of both photoisomers in the alkoxy-substituted model are acterization and spectral data of 2–4).
predicted to be better separated than in the alkyl-substituted
core of NCDA3, which will facilitate higher PSDs and most
likely highly selective switching.
Photophysical and photochemical studies
We investigated the photophysical and photochemical proper-
ties of compounds 2–4 and compared them to the bis-alkyl
analogue NCDA3, as well as to the simulated spectra (Fig. 3A).
Synthesis
The synthesis of 4,40-bisalkoxy based MBLs started from the azo- An overview of the characteristics in the presence and absence
coupling between 4-hydroxy aniline (5) and phenol to result in of DNA is given in Table 1 and representative measurements are
4,40-di-hydroxy-azobenzene 6 (Scheme 1).67 Alkylation of 6 with displayed in Fig. 4 (see ESI† for the full set of spectra and
1,2-dibromoethane and 1,3-dibromopropane, provided 7 and 8, measurements). The parent alkyl substituted NCDA3 has an
respectively (59% and 56% yield). Treatment with Ns-protected absorption maximum at 320 nm in buffer, which lies between
amine 9, under basic conditions, yielded compounds 10 and 11. the predicted transition maxima of naphthyridine
Table 1 Overview of photophysical and photochemical properties of NCDA3 and compounds 2–4 in buffer in the presence and absence of
dsDNA (50-d(CTAACGGAATG)-30/30-d(GATTGGCTTAC)-50)
PSD430nm (PSD460nm
)
lpp*(E) (lNC
[nm]
)
3 (lpp*E)
FE/Z
{PSD470nm
}
Compound
[Mꢁ1‧cmꢁ1
]
lnp*(Z) [nm]
[%]
s at 25 ꢀCh
PSD365nm, [E : Z]
[E : Z]
Without DNA
NCDA3a
320d
28 850e
12 830
11 900
11 540
n.d.f
432
439
438
437
432
440
440
443
0.7
42
53
27
5
15
11
14
5.6
3.2
86 : 14h, 50 : 50i
21 : 79h, 17 : 83j
15 : 85h, 6 : 94g
12 : 88h, 3 : 97j
25 : 75i
88 : 12h
2a
365 (332)
365 (332)
367 (332)
321d
365 (334)
365 (337)
363 (333)
80 : 20h, (87 : 13)j
65 : 35h, (80 : 20)j
49 : 51h, (71 : 29)j
n.d.h
3b
7.4 (23)c
18.5
4c
With DNA
NCDA3b
9.4g
2b
3b
4b
n.d.f
12.2
32 : 68j
79 : 21j, {91 : 9}j
62 : 38j, {88 : 12}j
57 : 43j, {82 : 18}j
n.d.f
15.2 (13)g
13g
38 : 62j
n.d.f
31 : 69j
a
b
c
d
Aq. tris buffer (50 mM, 150 mM NaCl, pH ¼ 7.5). Aq. phosphate buffer (0.01 M, 100 mM NaCl, pH ¼ 7.0). Water. Absorption maximum
e
f
g
consists of contributions from both the azobenzene core and the NC moiety. At l ¼ 320 nm. Not determined. Aq. phosphate buffer (5 mM,
50 mM NaCl, pH ¼ 7.0). The photostationary state distribution (PSD) differs between. Assigned by 1H-NMR using a 365 nm lamp or a 430 nm
h
i
j
LED, respectively, 1 mM in D2O, see ESI. Data from ref. 53. Assigned by peak area integration at the isosbestic point in HPLC analysis aer
irradiation with a 365 nm lamp, or a 460 nm LED (values in “( )”), or a 470 nm LED (values in “{ }”), respectively, see ESI.
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methylcarbamate (294 nm) and E-CH2NHCH3 (355 nm). Due to (Fig. 4C and ESI, Section 2.2†). 1H-NMR measurements and the
the overlap with an absorption band of the NC moiety, the exact presence of isosbestic points in the UV-Vis spectra indicate
position of the pp* transition of the azobenzene moiety in E- a clean two-component isomerization process (cf. Fig. 4D and
NCDA3 could not be assigned (Fig. 4A).53 In contrast, the E ESI, Section 2.6†). The QY for this photoreaction is around 0.7%
isomers of the 4,40-alkoxy azobenzene derivatives 2–4 exhibit for NCDA3, a quite low value for classical azobenzene scaffolds,
a
well-dened pp* transition that is signicantly bath- which show usually QYs FE/Z of around 10–20% in polar protic
ochromically shied to ca. 365 nm. This shi leads to a clear solvents.55 We hypothesize that, in this case, overlap with the
separation from the absorption band of the NC moiety (lNC NC unit diminishes the isomerization efficiency of the azo-
,
Fig. 4C and ESI, Section 2.2†) and is in agreement with the TD- benzene moiety in the MBL, which is supported by the TD-DFT
DFT calculations; the calculations predict the position of the calculations (vide supra). Much to our delight, the spectral
transition maximum ca. 20 nm bathochromically shied separation as well as the isomerization was found to be drasti-
compared to measurements (Fig. 3). Gratifyingly, the addition cally improved in compounds 2–4 with QYs between 27% and
of dsDNA containing a d(CGG)/d(CGG) mismatch does not 53%. These high photoisomerization efficiencies allowed for
signicantly inuence the positions of the pp* transition bands rapid E / Z isomerization in the seconds range at spectro-
of the azobenzene core structures (see Table 1 and ESI, Section scopic concentrations using a standard UV-LED (cf. ESI, Section
2.2†). However, lNC experiences a slight bathochromic shi of 2.3†). The presence of duplex DNA inuences the photo-
1–5 nm in compounds 2, 3, and 4, an effect that can be attrib- isomerization efficiencies (ca. 5% for NCDA3 and 11–15% for 2–
uted to the intercalation of the corresponding chromophore,68 4, cf. Table 1) suggesting a tight interaction of the MBL motif
here the NC.
with the DNA strands.
Irradiation of the thermally equilibrated samples with
The photostationary state distribution (PSD) differs between
365 nm light induced photochemical E / Z isomerization in all the various azobenzene-based MBLs. Notably, in the parent
investigated compounds. The photochemical process is NCDA3 only ca. 50% of Z isomer could be observed at the
accompanied by characteristic changes in the UV-Vis absorp- PSS365nm
,
while in compounds 2–4 up to 97% of the meta-
53
tion spectra: upon irradiation, the pp* transition of the E stable Z was detected (HPLC analysis, cf. Table 1 and ESI,
disappears leaving an absorption minimum and the np* band Sections 2.6 and 2.7†). The relatively low PSD365nm in NCDA3
of the Z form around 440 nm increases as predicted by TD-DFT can be rationalized by an overlap of the UV-Vis absorption
Fig. 4 (A) The UV-Vis spectrum of compound NCDA3 (25 mM in tris buffer) is shown for comparison at the thermal equilibrium (cyan), the
PSS365nm (red), and the PSS420nm (grey). (B) Photoinduced E–Z isomerization of compounds 2–4. (C) UV-Vis spectrum of compound 3 (50 mM in
phosphate buffer) at the thermal equilibrium (cyan), the PSS365nm (red), and the PSS445nm (grey). (D) Excerpt of the 1H-NMR spectrum showing the
protons of the azo-core moiety of compound 3 at the thermal equilibrium (cyan), the PSS365nm (red), and the PSS430nm (grey). (E) Repeated
irradiation of compound 3 with 365 nm and 445 nm light in the presence (red) and absence (cyan) of duplex DNA (50-d(CTAACGGAATG)-30/30-
d(GATTGGCTTAC)-50).
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spectra of the E and Z photoisomer according to TD-DFT found that the isolated switches suffered fatigue aer multiple
calculations (vide supra). The alkoxy-substituted derivatives repetitions. In the presence of DNA, though, photochemical
show signicantly higher PSD365nm, most likely due to a better isomerization appeared to be very robust as exemplied for 3 in
separation of the transition bands of E and Z form, as predicted Fig. 4E (see also ESI, Section 2.4†). This behavior, together with
by computational analysis (Fig. 3). Photochemical Z / E the prolonged thermal stability (increased lifetimes of the Z
isomerization was facilitated by irradiation with blue light isomers, vide supra), indicates a tight interaction of the photo-
(430 nm, 460 nm, or 470 nm depending on the setup used, see switchable MBLs and the dsDNA, albeit without compromising
ESI, Section 2†) for NCDA3 and compounds 2–4, respectively. In the photoswitching, which offered intriguing opportunities for
NCDA3, 88% of the E form could be regenerated,53 while for 2–4 a more detailed investigation, as outlined below.
between 71 and 87% E isomer was observed (HPLC analysis, cf.
Table 1, Fig. 4D, and ESI, Sections 2.6 and 2.7†).
DNA melting experiments
The Z / E isomerization could also be achieved thermally.
The thermal lifetimes of all compounds were mostly found to be
prolonged in the presence of dsDNA, being an indication that
the Z isomer is energetically stabilized in the presence of DNA
(cf. Table 1 and ESI, Section 2.5†).
The photochemically induced E–Z isomerization was
reversible for all switchable ligands 2–4 and could be repeated
over several cycles both in the presence and absence of DNA. We
Thermal melting curves were measured for an 11-mer dsDNA (5
mM) containing a d(CGG)/d(CGG) mismatch in the presence of
compounds 2, 3, and 4 (20 mM) with and without photo-
irradiation (365 nm for 20 s). The absorbance of the sample was
monitored at 260 nm inꢁt1he temperature range 2–80 ꢀC with
a heating rate of 1 ꢀC min . Melting temperatures (Tm, ꢀC) were
calculated by a median method except for Tm*, where the
Fig. 5 DNA melting experiment: the absorbance of the sample was monitored at 260 nm from 2 to 80 ꢀC with a heating rate of 1 ꢀC minꢁ1
.
Conditions: dsDNA (50-d(CTAACGGAATG)-30/30-d(GATTGGCTTAC)-50, 5 mM) in phosphate buffer (10 mM, 100 mM NaCl, pH 7.0), in absence
(black open circle) and presence of each ligand (compounds 2–4, 20 mM). (A) Compound 2; (B) 3; (C). 4. Ligands were either pretreated with heat
at 60 ꢀC in the dark to reach thermally equilibrated state (blue filled circle) or photo-irradiated at 365 nm for 20 s (red filled diamond). (D)
Repeated irradiation of 3 with dsDNA. The sample before irradiation (cyan half-filled circle) was irradiated at 365 nm (red filled diamond),
subsequent irradiation at 460 nm (blue filled circle), followed by irradiation at 365 nm (red half-filled diamond).
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differential method was applied. Ligands were preincubated at in the presence of 3 aer repeated irradiations with 365 nm and
60 ꢀC before the thermal melting experiments to reach the 460 nm light, are shown in Fig. 5D. Tm without irradiation was
thermally equilibrium where the E isomers were exclusive. The around 30 ꢀC (cyan half-lled circle), which is slightly higher
presence of the E isomers had neglectable effect on the Tm than that in Fig. 5B because of residual Z isomer existing under
values under the investigated conditions; the increase in Tms ambient light. Irradiation at 365 nm increased Tm (red lled
ꢀ
was less than 1 C compared to the ones in the absence of the diamond), blue-light irradiation decreased Tm back to 30 ꢀC
ligand (Tm1, Fig. 5A–C, blue lled circles). In contrast, all (blue lled circle), and subsequent irradiation at 365 nm again
ligands showed a stabilization effect on the dsDNA aer irra- increased Tm (red half-lled diamond) exhibiting a prole
diation with 365 nm light indicated by increasing Tm (Tm2
,
almost identical to the one aer the rst UV-light irradiation.
Fig. 5A–C, red lled diamond). The use of compound 3, which The reversibility of Tm demonstrates that the light-mediated
contains a C3 linker, resulted in the highest DTm value. Aer photoisomerization of MBL 3 (Fig. 5D) can be fully translated
photoirradiation, Z-3 stabilized the target DNA duplex by up to to govern the stability of the mismatch containing dsDNA.
ca. 16 ꢀC (from 27.5 to 43.3 ꢀC, Fig. 5A), while the Z isomers of 2
and 4 showed smaller DTm values thaꢀn for 3, raising the
ꢀ
ꢀ
Surface plasmon resonance
temperature by 11 C (from 27.6 to 38.4 C, Fig. 5A) and 9 C
(from 27.8 to 37.0 ꢀC, Fig. 5C), respectively. The observations are
supported by molecular dynamics (MD) simulations which are
in agreement indicating that the DNA double helix in the Z-3-
dsDNA complex is consistently the most stable one over 300 ns
in temperature-dependent simulations (cf. Section 6.3 in the
ESI†). In previous studies NCDA3 was found to exhibit a DTm of
Binding of the ligands to DNA was further evaluated by surface
plasmon resonance (SPR). Hairpin forming dsDNA (50-CTAA
CNG AATG TTTT CATT CMG TTAG-30) containing a d(CNG)/
d(CMG) site was immobilized onto a sensor chip surface. The
dsDNA containing a GG mismatch (N ¼ M ¼ G), CC mismatch
(N ¼ M ¼ C), and GC match (N ¼ C, M ¼ G) were used to
compare the binding selectivity. Solutions of each ligand at
concentrations ranging from 0.031 to 0.5 mM, with and without
irradiation, were injected to the surface. Fig. 6 shows SPR curves
for the GG mismatch dsDNA. Increasing concentrations of
ligands at the PSS365 (main isomers Z-2–4) showed an increas-
ingly strong response signal (Fig. 6D–F), while ligands at the
thermal equilibrium (main isomer E-2–4) exhibited response of
much smaller intensities (Fig. 6A–C). It is evident that at similar
concentrations, the Z isomer shows drastically enhanced
ca. 15 C (from 32.7 to 48.0 ꢀC).53 However, E-NCDA3 showed
ꢀ
background activity and stabilized the mismatched DNA to
some extent (Tm1 ¼ 32.7 ꢀC),53 while E-3 shows hardly any effect
on Tm1. This does not only make 3 the MBL providing the largest
Tm change before and aer irradiation (DTm ¼ 16 ꢀC), but also
the photoswitch with the clearest difference between the inac-
tive E and the stabilizing Z isomer. The stabilization effect is
fully reversible by photoswitching upon irradiation with
different wavelengths as expected from Fig. 4E. Melting proles
Fig. 6 SPR analysis of the binding of ligands 2 (A and D), 3 (B and E), and 4 (C and F) to hairpin dsDNA containing a CGG/CGG site. Ligands at
a series of concentrations (0.031, 0.063, 0.13, 0.25, 0.5 mM) were injected during association steps (from 57 to 117 s) followed by dissociation step
where running buffer was injected. Either ligands at thermal equilibrium (A–C) or ligands after irradiation at 365 nm (D–F) were injected to surface
immobilized with the dsDNA containing a GG mismatch.
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response and as a consequence the SPR curves are convex different chromophores in the MBLs. While the compounds in
upward in the association step beginning from 57 to 117 s. This their E form did not show any Cotton effect in the absence of
feature suggests the contribution of aggregation/dissociation of DNA, an increasing CD signal is observed by titrating MBLs to
ligands and/or conformational changes of the dsDNA–ligand the GG mismatch-containing DNA 11-mer for compounds E-2
complexes on the surface. Although it is difficult to estimate the and E-4 (Fig. 7A, and ESI, Section 5†). In particular, the positive
binding constants because of the uncommon shape of the induced CD (ICD) signal at ca. 340 nm corresponds well with the
curves, ligand Z-3 (Fig. 6E) provided the highest response, NC absorption band and the one at 365 nm with the pp*
which is well-correlated with stabilization effects on dsDNA as transition band of the azobenzene core indicating an interac-
observed in Tm (Fig. 5).54 Compounds with linkers that are one tion of the MBL with the DNA (Fig. 7A). In contrast, E-3 hardly
methylene unit shorter (2) or longer (4) than in 3 resulted not showed any ICD (Fig. 7C). While addition of increasing
only in weaker binding affinities but also in smaller changes amounts of E-3 and E-4 led to a decrease in intensity of the
before and aer irradiation. We also compared the binding of signal in the DNA region of the CD spectrum (Fig. 7B and S31†),
the different photoirradiated ligands towards GG and CC homo a larger change was found for E-2 with the formation of an
mismatches and a GC match. The results conrm the marked additional negative signal at 228 nm (Fig. 7A). We mainly
selectivity towards the GG mismatch, as expected from the observed this characteristic structural change for Z forms of the
recognition moieties in MBLs consisting of dimeric NC (Fig. S25 MBLs (vide infra).
and S26†).24
Irradiation of the samples with 365 nm light induces E / Z
isomerization and results in higher Tms (vide supra), greater
response in SPR (vide supra), and more pronounced changes in
the CD spectrum (exemplarily shown for MBL 3 in Fig. 7C, for
further measurements see ESI, Section 5†). The latter shows
a signicant change of the Cotton effect below 300 nm, which
indicates a structural change of the dsDNA and additional
contribution originated from NC embedded in the helix.75 As
already described for E-2, formation of a new signal at 228 nm is
observed for Z-2–4 with Z-3 inducing the most prominent
change. In the MBL region, photoisomerization is accompanied
CD spectroscopy
CD spectroscopy is frequently used to probe ligand–DNA
interactions37,38,40,69–72 and was previously employed to study the
affinity of MBLs and their effect on DNA hybridization.48,73,74
Due to the excellent separation of the absorption bands of DNA,
NC, and azobenzene our system allows for clear identication of
the different transitions contributing to the CD spectrum. The
part of the spectrum below 300 nm is mainly attributed to the
DNA, while the one above 300 nm can be assigned to the
Fig. 7 Binding studies using CD spectroscopy by titrating increasing amounts of MBLs 2 (A), and 3 (B) to dsDNA (50-d(CTAACGGAATG)-30/30-
d(GATTGGCTTAC)-50, 5 mM) in phosphate buffer at their thermal equilibrium and of 3 at the PSS365nm (C). (D) CD spectra of compound 3 (10 mM)
in the presence of dsDNA (5 mM) in phosphate buffer. Altering irradiation with 365 nm and 470 nm light and reversible structural change in DNA
and of the ICD of 3. (E) Excerpt showing the reversible structural change in the DNA-region of the CD spectra. (F) Irradiation of MBL 3 with
different colors of light: CD spectrum at the thermal equilibrium (black), the PSS365nm (red), the PSS430nm (grey), and the PSS470nm (cyan) con-
taining varying amounts of Z isomer (a wider spectral range can be found in Fig. S30†).
9214 | Chem. Sci., 2021, 12, 9207–9220
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by a decrease of the pp* absorption band of the E isomer in the decreased the affinity to DNA by loss of the p–p-interaction and
UV-Vis absorption spectrum (vide supra) and the corresponding thus allowed for photocontrol of the system's architec-
signal in the CD spectrum practically disappears. A pronounced ture.39,41,61,76 These reports highlight the possibility of using CD
negative signal around 440 nm is observed (Fig. 7C and ESI, spectroscopy both to follow the process, usually showing
Section 5†), while the positive signal at ca. 320 nm intensies. a decrease of the Cotton effect for the Z isomer38,58,76 to assign
The position of l ¼ 440 nm the signal is in good accordance the binding mode of both photoisomers, if they are used as
with the lmax values of the np* transition band of the Z isomer ligands, by the pattern of the ICD signal.38,39
in 4,40-di-alkoxy azobenzene (cf. TD-DFT spectrum in Fig. 3).
Furthermore, we and others have reported that the chiral
Also, in this case, the strongest signals originate from MBL Z-3. environment provided by DNA can inuence the chiroptical
Both the structural change in the DNA and the CD signal in the properties of organic chromophores77–80 as well as the stereo-
MBL region can be reverted by irradiation with blue light chemical outcome of a photoisomerization reaction as shown, for
(Fig. 7D and E and ESI, Section 5†). Specically, the ratio of the instance, for DNA-binding diarylethenes.29 The same experiment,
two photoisomers can be adjusted using different wavelengths however, cannot easily be repeated for azobenzenes, as the E
of light and the composition in the dark and at the PSS365nm
,
isomer is achiral and the Z(P) and Z(M) enantiomers (inter-
PSS430nm, and the PSS470nm directly correlates with the observed converting helical conformers, Fig. 8A) undergo fast isomeriza-
CD response and the degree of stabilization of the dsDNA tion (racemization) in solution81 i.e. the dynamical helicity of Z-
(Fig. 7F, a wider spectral range can be seen in Fig. S30†).
azobenzene as shown in Fig. 8A. As a comparison, the photoin-
Repeated irradiation with 365 nm and 470 nm light of the duced closure reaction in diarylethenes forms a new covalent
same sample showed reversible stabilization and destabilization bond and hence, the enantiomers are congurationally stable
of the mismatched DNA as exemplarily by MBL 3 shown in and can be isolated.29 For this reason, a chirality transfer onto the
Fig. 7D and E. This behavior demonstrates a reversible mutual Z-azobenzene unit has been reported for azobenzenes aer
change in DNA structure and binding of the ligands. Analyzing covalently attaching a stereogenic element,82,83 oen in cyclic
the titration experiments by plotting the CD signal at 341 nm of systems to induce additional strain,81,84,85 but not for supramo-
the ligand versus the ligand concentration allowed for a qualita- lecular systems based on DNA as described here.
tive description of the binding affinities of the photochromic
Conversely, in our system the functionalized azobenzenes do
MBLs (ESI, Section 5.5†). Using a single set of independent not serve as a mere intercalators or groove-binding ligands
binding sites model and assuming a 2 : 1 ligand : dsDNA stoi- towards a canonical DNA, but as photocontrollable glue mole-
chiometry as reported previously,53,54 we found an apparent KD of cule to promote double helix formation in a mismatched
0.7 mM for compound 3 as the strongest binder, followed by dsDNA. Due to the tight binding of the NC moieties, which was
compound 2 with 15.2 mM and compound 4 with 23.6 mM. This is previously demonstrated by the necessity to use a thermally
in agreement with the order observed from DNA melting exper- degradable variant (TD in Fig. 2B) to make the system revers-
iments (vide supra). Also, in SPR compound 3 showed the stron- ible,52 our system is closer to a macrocyclic structure, where the
gest response while compound 4 and 2 gave weaker signals (vide dsDNA serves as a chiral clamp, than to a classical DNA–ligand
supra). Though the response of MBL 4 is stronger than the one of interaction. Hence, we focused on studying the origin of the
2 in SPR, this could be also due to unspecic binding to DNA and observed Cotton effect around 440 nm in the Z isomers (Fig. 8D)
is consequently not necessarily contradictory to the Tm and CD in more detail aiming to elucidate whether the signal is derived
studies. Altogether, compound 3, featuring a C3 linkage, emerged from the excitonic coupling between the chromophores or from
throughout our studies to be both the strongest binder and DNA imprinting its chiral information in the Z azobenzenes.
simultaneously the photoswitch with the largest differences in The transfer of chirality works through energetical discrimina-
affinity between the two isomers. A pronounced inuence of tion of the enantiomers of the Z-azobenzene inducing a bias in
linker length on the binding to DNA was already observed for the equilibrium between Z(M)and Z(P) helical conguration
photoswitchable MBL in our earlier studies.54 We hypothesize (Fig. 8B) and can be approximated by theoretical calcula-
that a combination of factors contribute to the observed behavior, tions.81,83,84 Consequently, we performed a thorough computa-
such as relative stabilities of conformations of MBL in both tional investigation models of MBLs 2–4 in a 2 : 1 complex with
bound and unbound states, and the entropic costs as well as the the DNA 11-mer. The DNA complexes were built by modifying
solvation changes upon binding.
a similar structure that was previously released (PDB ID:
1X26).48 To minimize any bias originated by the selection of the
initial structures, we performed a preliminary energy minimi-
zation with the OPLS3e force eld and subsequently we per-
Chirality transfer
Photoswitches such as azobenzenes have been studied previ- formed multiple 300 ns MD simulations with the parmbsc1
ously in combination with DNA, either as nucleobase surro- force eld. Randomly sampled structures were nally optimized
gates23,57–61 or as non-covalent ligands.23,37–40 Commonly, the E with Grimme's GFN force eld.86 Fig. 8C shows as an example
isomer showed the stronger interaction with DNA due to its p- the optimized geometry of 3 bound to dsDNA (note that two
stacking ability employing dsDNA38,39,60,61,76 or
a G-quad- molecules of 3 are bound to dsDNA). We analyzed the frequency
ruplex.41,42 In sharp contrast, in our system it is the Z isomer of and energy distributions of both Z(P) and Z(M) congurations
compound-3 that shows the stronger CD signal (Fig. 7B and C). present in the two MBLs. As two ligands are required to stabilize
In the more common designs, E / Z photoisomerization the mismatched DNA, the two azobenzenes can either both
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Fig. 8 (A) Unbiased equilibrium of interconverting helical conformers. (B) DNA-induced biased equilibrium favoring Z(P) configuration in the
alkoxy azobenzene-based MBLs 2–4. (C) Calculated structure of Z-3 in DNA (note two molecules of Z-3 bind to form stable dsDNA). (D)
Experimental CD spectrum of compound 3 (10 mM) in the presence dsDNA (50-d(CTAACGGAATG)-30/30-d(GATTGGCTTAC)-50, 5 mM) in
phosphate buffer at the thermal equilibrium (grey solid line) and after E / Z isomerization at the PSS365nm (cyan solid line), which corresponds
well with the computed CD spectrum of Z(P)-OCH3 (red dotted line, TD(25 states)-PBE0/6-311+G(2d,p) level of theory using the SMD implicit
model for water). (E) Violin plot of the energy and distribution of two molecules of Z-3 in a 2 : 1 complex to mismatched DNA adopting (PP)Z,
(PM/MP)Z, and (MM)Z configuration (for details see ESI, Section 6.3†).
exhibit P (PP), both M (MM), or a mixed conguration (PM or azobenzene core structure for the next generation of photo-
MP). In most cases, the two azobenzenes in the MBLs adopted switchable DNA mismatch binding ligands. TD-DFT calcula-
a PP or a mixed MP/PM helicity in the Z conguration over the tions suggested employing the 4,40-di-alkoxy substituted
MM one (see Fig. 8E and Section 6.3 in the ESI†). These results azobenzene core to improve the spectral band separation of the
suggest that the system biases the formation of PP helicity over involved chromophores. UV-Vis spectroscopy of the synthesized
MM helicity in the two azobenzenes (Fig. 8B). To support the compounds conrmed the predicted l-orthogonality between
interpretation of a preference in the helicity of the switches, we the absorption band of the naphthyridine carbamate moiety
calculated the CD spectrum of Z-OCH3 in its P form, that and the one corresponding to the pp* transition of the E
matched the Cotton effect observed experimentally (cf. Fig. 8D, isomers in compounds 2–4. Compared to the MBLs based on
red dotted line for the simulation, cyan line for the experiment). classical azobenzene, the present ligands exhibit improved
These results indicate that the dsDNA and the MBL show photophysical and photochemical properties, such as up to 75
a highly mutual supramolecular interaction: on the one hand, times higher E / Z photoisomerization quantum yields.
the Z form of the MBL can provide enhanced stabilization of the Moreover, the improved band separation in the UV-Vis
helical structure of DNA. On the other hand, the DNA is trans- absorption spectra between the various chromophores
lating its chirality onto the Z isomer of the azoswitch (inducing involved resulted in PSDs up to >95% for the new system we
right-handed (P) helicity in the dynamic helical system). To the developed (in contrast to ca. 50% in NCDAs). These benecial
best of our knowledge this has only been observed so far in properties allowed to induce close to quantitative E / Z pho-
azobenzenes with
a
covalently attached stereogenic toisomerization within a few seconds, a central premise for
element,9,81–85 and in supramolecular host–guest assemblies rapidly responding systems.
based on anion binding stilbenes.87 DNA was up to now only
Detailed studies using various complementary techniques
used to template the formation a preferred enantiomer during revealed that the Z isomer of the new MBLs stabilized dsDNA
an electrocyclic reaction in DAE photoswitches,29 but never to more than the E form. Among the tested ligands, compound 3
induce a biased equilibrium in rapidly interconverting helical with a C3 linker showed the best stabilization effect on dsDNA
conformers such as in Z(P)-3 discussed in this work.
containing a GG mismatch showing a light-induced DTm of
16 ꢀC. While in previous studies both photoisomers had affinity
to mismatched DNA,53 the E isomer in MBL 3 does hardly show
any stabilizing effect on the dsDNA. In stark contrast, the
photochemically generated Z form of the same compound
rapidly induces DNA duplex formation as seen CD spectroscopy.
Conclusions
In summary, we used rational design aided by computational
analysis to tune the photochemical properties of the
9216 | Chem. Sci., 2021, 12, 9207–9220
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A strong response in SPR and titration studies revealing an Education, Culture and Science of the Netherlands (Gravitation
apparent KD of 0.7 mM for Z-3 conrming its high affinity. Both Program no. 024.001.035 to BLF), and the Netherlands Orga-
the lack of activity of the thermodynamically stable E form and nization for Scientic Research (NWO, VIDI grant no.
the high activity of the rapidly light-generated meta-stable Z 723.014.001 for WS). This work was partially supported by
isomer make MBL 3 and excellent candidate for further studies Grants in Aid for Scientic Research (B) (18H02107) to CD from
in real time.
the Japan Society for the Promotion of Science (JSPS).
Additionally, binding of the photoswitchable MBLs to
dsDNA facilitates a unique supramolecular transfer of chirality
onto the helically shaped Z form of the azo-switch as demon-
strated by CD spectroscopy supported by an extensive compu-
tational analysis. Specically, the presence of the dsDNA
induced a bias in the dynamic equilibrium between Z(M) and
Z(P) conguration, favoring an overall P conguration. This
shows that photoswitchable MBL and mismatched DNA strands
have a distinctive mutual interaction in which the photo-
switches serve as DNA glue while the nucleic acid induces
a transfer of chirality onto the switch. These ndings present an
important step towards fully dynamic and external control in
reversibly photo-responsive DNA-based architectures, with
prospects for adaptive nano systems and genetic regulation,
and demonstrate the potential of rational design in the context
of photo-responsive systems.
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