In search of visible-light photoresponsive peptide nucleic acids (PNAs) for reversible control of DNA hybridization

Article abstract of DOI:10.3762/bjoc.15.243

Photoswitchable oligonucleotides can determine specific biological outcomes by light-induced conformational changes. In particular, artificial probes activated by visible-light irradiation are highly desired in biological applications. Here, we report two novel types of visible-light photoswitchable peptide nucleic acids (PNAs) based on the molecular transducers: hemithioindigo and tetra-ortho-fluoroazobenzene. Our study reveals that the tetra-ortho-fluoroazobenzene–PNA conjugates have promising properties (fast reversible isomerization, exceptional thermal stability, high isomer conversions and sensitivity to visible-light irradiation) as reversible modulators to control oligonucleotide hybridization in biological contexts. Furthermore, we verified that this switchable modification delivers a slightly different hybridization behavior in the PNA. Thus, both melting experiments and strand-displacement assays showed that in all the cases the trans-isomer is the one with superior binding affinities. Alternative versions, inspired by our first compounds here reported, may find applications in different fields such as chemical biology, nanotechnology and materials science.

Full text of DOI:10.3762/bjoc.15.243

In search of visible-light photoresponsive peptide nucleic
acids (PNAs) for reversible control of DNA hybridization
Lei Zhang, Greta Linden and Olalla Vázquez*
Letter
Open Access
Address:
Beilstein J. Org. Chem. 2019, 15, 2500–2508.
Fachbereich Chemie, Philipps-Universität Marburg, Hans-Meerwein
Straße 4, 35043 Marburg, Germany
doi:10.3762/bjoc.15.243
Received: 26 July 2019
Email:
Accepted: 01 October 2019
Published: 22 October 2019
Olalla Vázquez* - olalla.vazquez@staff.uni-marburg.de
*
Corresponding author
This article is part of the thematic issue "Molecular switches".
Guest Editor: W. Szymanski
Keywords:
azobenzene; hemithioindigo; peptide nucleic acid (PNA); photoswitch;
visible-light irradiation
© 2019 Zhang et al.; licensee Beilstein-Institut.
License and terms: see end of document.
Abstract
Photoswitchable oligonucleotides can determine specific biological outcomes by light-induced conformational changes. In particu-
lar, artificial probes activated by visible-light irradiation are highly desired in biological applications. Here, we report two novel
types of visible-light photoswitchable peptide nucleic acids (PNAs) based on the molecular transducers: hemithioindigo and tetra-
ortho-fluoroazobenzene. Our study reveals that the tetra-ortho-fluoroazobenzene–PNA conjugates have promising properties (fast
reversible isomerization, exceptional thermal stability, high isomer conversions and sensitivity to visible-light irradiation) as revers-
ible modulators to control oligonucleotide hybridization in biological contexts. Furthermore, we verified that this switchable modi-
fication delivers a slightly different hybridization behavior in the PNA. Thus, both melting experiments and strand-displacement
assays showed that in all the cases the trans-isomer is the one with superior binding affinities. Alternative versions, inspired by our
first compounds here reported, may find applications in different fields such as chemical biology, nanotechnology and materials
science.
Introduction
Light-driven control of oligonucleotide hybridization has photopharmacology [14,15] is an emerging field that highlights
demonstrated an enormous potential to regulate on-demand bio- the importance of reversible photocontrollable drugs in
logical responses such as gene expression [1]. There are indeed tomorrow’s medicine, but photoswitchable antisense research in
a number of successful examples based on photocaged strate- the context of photopharmacology is entirely unexplored.
gies [2-5], in which modified nucleic acids interfere irre- Furthermore, reversible approaches with photoswitches will
versibly with gene expression in vitro [6,7] and in vivo [8-12]. contribute to a better understanding of biological pathways as
Manipulation of gene expression demonstrated therapeutic ap- they would allow precise reversible spatio-temporal activation/
plication – antisense chemistry [13]. Along these lines, deactivation of the desired targets without causing a permanent
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knockout. During the last years, the pioneering structural
studies of reversible photoregulation of DNA/RNA duplex
stability of Asanuma and Komiyama [16,17] have become func-
tional ones, affecting DNA/RNA cleavage [18-20], transcrip-
tion [21-23], and translation [24,25]. Except a handful of cur-
rent examples [22,24,26], most of these photoresponsive oligo-
nucleotides are canonical ones where the classical azobenzene
is the prominently used photoswitch; although spiropyrans [27],
stilbenes [28], diarylethanes [29] and overcrowded alkenes [30]
have also been employed. In vivo application demands the de-
velopment of a new generation of artificial agents to target
DNA/RNA-associated processes. These compounds must be
able to maintain their specificity and effectivity while still being
nuclease resistant, nontoxic and susceptible to light of tissue-
penetrating wavelengths. Peptide nucleic acids (PNAs) [31] are
synthetic nucleic acid analogues, in which nucleobases are
linked to a repeating N-(2-aminoethyl)glycine polyamide back-
bone. The lack of phosphate groups provides them with both
higher binding affinities to complementary DNA or RNA se-
quences and improved mismatch discrimination under physio-
logical conditions than natural ones. Furthermore, PNAs have a
straightforward chemical synthesis by Fmoc-based PNA solid-
phase synthesis and remarkable stability against nuclease- and
protease-mediated degradation [32,33]. In regard to all these
beneficial properties of PNA, they may become a promising al-
ternative to overcome the current limitations of the available
photoswitchable DNA- and RNA-based systems with potential
for in vivo applications too.
Only very few is known about the reversible hybridization of
PNAs upon irradiation [34-36]. Besides these precedents use
azobenzene-containing PNA to mainly regulate PNA/DNA
triplex helix formation by illumination at low wavelengths
Figure 1: A) Structure of the pioneering azobenzene-modified DNA
[
16] compared with the photoswitchable PNA structure based on a
monomer surrogate. B) Isomerization of the molecular transducers in-
corporated and studied in this project: 1.: tetra-ortho-fluoroazobenze
(oF4Azo) and 2.: hemithioindigo (HTI).
(
360 nm/425 nm) [35,36]. This effect was successfully
exploited for the photocontrol of transcription by T7 RNA poly-
merase in vitro [36]. In fact, such a result opens new avenues frequently overexpressed in tumors and particularly, in prostate
for the investigation of other photoswitchable PNAs and cancer [39].
pursuing visible-light modulation. Herein, we report the design
of a versatile synthetic platform to derivatize PNAs with differ- Results and Discussion
ent photoswitches (Figure 1), which has never been studied in Initially, short 12-mer PNA probes (Table 1) complementary to
the context of PNA. After incorporation, their switching capaci- the HDAC-1 mRNA sequence were synthesized since, in
ties and duplex formation were analyzed.
general, PNAs are active with shorter sequences than the canon-
ical analogues due to the superior binding abilities [40]. Despite
Our group has recently demonstrated that photoresponsive that target specificity may be compromised, 12-mer long se-
peptides can affect the transcription of genes via inhibition of quences are a suitable starting point for our preliminary tests.
histone-modifying enzymes [37]. Repression of enzymes is An overview of all the sequences of this study can be found in
achievable at nucleic acid level too. Therefore, in this project Table 1 and Table 2.
we envision a minimal model based on the previously reported
accessible mRNA region of the class I histone deacetylase Contrary to the previous synthetic approach of azobenzene-con-
HDAC-1: 5’-GUGAGCCAAGAAACACUGCCU-3’ to investi- taining PNAs [35,36,41], in which the preformed monomer
gate our photoswitchable PNAs [38]. Importantly, HDAC-1 is building block was used, we gained versatility using the diver-
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gent linear approach introduced by Seitz (Scheme 1) [42]. This patible with the standard TFA/m-cresol/H2O (90:5:5) acido-
strategy enabled the straightforward access to functionalized lysis yielding the photoswitchable PNA conjugates. These and
PNA via on-resin coupling of the corresponding photoswitch in all further studied PNAs in this work (Table 2) were purified by
good yields. Of note, this post-synthetic modification is com- reversed-phase (RP) HPLC and fully characterized. Analogous
patible with base sensitive compounds, which undergo degrada- compounds lacking the photoswitch were used as controls.
tion under standard Fmoc deprotection conditions. As it is
common for PNAs, our oligomers have an acetylated First, the photochromic behavior of the newly synthesized
N-terminus and a C-terminal carboxamide group. After comple- modified PNAs (Table 1, compound 3 and 4) was investigated
tion of the PNA sequences, the orthogonally protected back- and compared with the corresponding photoswitchable PNA
bone module [2-(N-Alloc)aminoethyl]glycine residue monomers (Table 1, compound 1 and 2).
–
Aeg(Alloc)– was selectively deprotected in the presence of
Pd(OAc)2, Ph3P, NMM, PhSiH3 in CH2Cl2 for 2 h. Subse- Apart from the expected increase of the band at 260 nm due to
quently, carboxy photoswitches were introduced using Oxyma the aromatic base moieties within the PNAs, UV–vis spectros-
and N,N’-diisopropylcarbodiimide (DIC) as coupling agent.
copy of 20 μM solutions in phosphate buffer (10 mM
NaH2PO4, 150 mM NaCl, pH 7.4) confirmed that PNA incor-
We explored two different types of photoswitches (Figure 1B): poration did not significantly affect the photochromism (Figures
) second generation azobenzenes based on the tetra-ortho- S24, S20, S30 and S33, Supporting Information File 1). This
fluoroazobenze (oF4Azo) developed by Hecht [43] and verifies the integrity of the chromophores after the cleavage
) hemithioindigos (HTI) rediscovered by Rück-Braun [44] and from the solid support. Among the initial studied photoswitch-
1
2
Dube [45], which have not been studied in the context of DNA/ able PNAs, the PNA12(oF4Azo) (3) displayed the most promis-
RNA as molecular transducer yet. Both compounds were com- ing properties as reversible modulator of oligonucleotide
Scheme 1: Solid-phase synthesis of photoswitchable PNAs; Aeg = N-(2-aminoethyl)glycine, Bhoc = benzhydryloxycarbonyl.
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Table 1: Isomerization conversions at the photostationary state (PSS).
Compound
Isomer ratioa [%]
Ac-Aeg(oF4Azo)CONH2 (1)
trans
cis
91
90
Ac-Aeg(HTI)CONH2 (2)
trans
cis
50
99
PNA12(oF4Azo) (3):
Ac-ggcagAeg(oF4Azo)gtttct-CONH2
trans
cis
95
82
PNA12(HTI) (4):
Ac-ggcagAeg(HTI)gtttct-CONH2
trans
cis
47
93
aIsomer ratios of a 20 µM solution of the corresponding compound in
phosphate buffer (10 mM NaH2PO4, 150 mM NaCl, pH 7.4) were de-
termined at the isosbestic point (275 nm, for oF4Azo and HTI) by
RP-HPLC; irradiation to obtain the cis-isomer at PSS: 520 nm for
1
0 min and the trans-isomer at PSS: 405 nm for 2 min. Mean values
derived from two independent experiments; Aeg = N-(2-
aminoethyl)glycine.
hybridization. Thus, it displayed the fastest reversible isomeri-
zation (≈2 s for cis → trans and ≈120 s for trans → cis at these
conditions, Figure S24C, Supporting Information File 1) with-
out any signs of photodegradation and photochemical fatigue up
to 20 cycles under visible-light irradiation. Regarding the
photoconversion ratios between isomers (Table 1),
PNA12(oF4Azo) (3) had the best ratios. However, the large sep-
aration between the n → π* bands of the trans and the cis-forms
Figure 2: Time-dependent conversion to the thermodynamically stable
isomer of PNA12(oF4Azo) (3; green triangles) and PNA12(HTI) (4; red
circles). 20 µM solutions of the corresponding compound in phosphate
buffer (10 mM NaH2PO4, 150 mM NaCl, pH 7.4) were irradiated to
obtain maximal cis (oF4Azo, 520 nm, 10 min) or trans (HTI, 405 nm,
2
min) and stored in the dark. PNA12(oF4Azo) (3) was also measured
with continuous heating at 90 °C (gray squares). UV–vis spectra were
collected at different time points during 10 h. Curves derived were
calculate from two independent experiments where the value of the ab-
sorbance at 304 nm (for 3) or at 442 nm (for 4) was measured.
(
Δλ = 69 nm; Figure S24, Supporting Information File 1) did
not lead to the quasi-quantitative conversion for the cis-isomer,
as for the photoswitchable PNA monomer 1 and the molecular
transducer [43,46]. This slightly lower cis ratio was also re-
ported in photoswitchable peptides and DNA binders equipped zation to a complementary DNA. For this purpose, we decided
with oF4Azo [47,48].
to measure thermal melting curves. Beforehand we verified the
photostability of cis-PNA12(oF4Azo) (3) in the temperature
Regarding stability, the cis-PNA12(oF4Azo) (3) was stable at ramp from 20 °C to 90 °C by UV–vis spectroscopy. We ob-
least for 24 h at 37 °C, while under the same conditions the served that the cis-isomer was always stable during the whole
thermodynamically unstable isomer of PNA12(HTI) (4) reverted temperature range (Figure S27B, Supporting Information
after 6 h at room temperature in the dark according to UV–vis File 1). Remarkably, its time-dependent conversion to the trans-
measurements (Figure 2 and Figure S32, Supporting Informa- PNA12(oF4Azo) (3) at 90 °C was surprisingly slow (Figure 2
tion File 1). The thermal relaxation of the cis-PNA12(oF4Azo) and Figure S28, Supporting Information File 1) in contrast to
(
3) was slow even at high temperatures (Figures S27 and S28, the reported cis-azobenzene tethered oligonucleotides [49].
Supporting Information File 1). Furthermore, RP-HPLC chro- Therefore, such unique characteristic allowed the irradiation of
matograms of its cis-form did not show any decomposition the photoswitchable PNA before hybridization (see Supporting
under these conditions (Figure S27C, Supporting Information Information File 1 for the detailed procedure). Thermal melting
File 1), unlike when the oF4Azo was grafted onto a pyrrole profiles showed single sigmoidal transitions (Figure 3 and
scaffold [48].
Figures S37–S45, Supporting Information File 1), which
enables the calculation of the melting temperatures (TM) by the
Next, we explored if the inclusion of oF4Azo as monomer analysis of the first derivative [50]. TM values are summarized
surrogate within the PNA sequence could affect its hybridi- in Table 2.
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Figure 3: A) Melting curves of a 1 µM duplex solution in phosphate buffer (10 mM NaH2PO4, 150 mM NaCl, pH 7.4) of the PNA (18 = yellow solid
line; 19 = grey solid line; 9 = brown, cis dashed line, trans solid line; 10 = purple, cis dashed line, trans solid line and the complementary ssDNA
(
5’-GTG AGC CAA GAA ACA CTG CCT-3’)). The melting curves were duplicates from two independent experiments combining 3 cycles of measure-
ments from 20 °C to 95 °C at 260 nm without pre-hybridization. B) Melting temperatures (TM) obtained by the first derivative of the data in A.
Table 2: Melting temperatures (TM) of the duplex between the PNAs and the complementary ssDNA (5’-GTG AGC CAA GAA ACA CTG CCT-3’).
Sequence
TM [°C]
cis
trans
3
5
Ac-ggcagAeg(oF4Azo)gtttct-CONH2
Ac-ggcagAeg(Ac)gtttct-CONH2
50.5 ± 0.04
50.5 ± 0.02
44.3 ± 0.4
6
7
8
9
1
1
Ac-Lys-Aeg(oF4Azo)gcagtgtttcttgg-Lys-CONH2
Ac-Lys-ggAeg(oF4Azo)agtgtttcttgg-Lys-CONH2
Ac-Lys-ggcagtAeg(oF4Azo)tttcttgg-Lys-CONH2
Ac-Lys-ggcagtgtttcttgAeg(oF4Azo)-Lys-CONH2
Ac-Lys-ggcAeg(oF4Azo)gtgtttcAeg(oF4Azo)tgg-Lys-CONH2
Ac-Lys-ggAeg(oF4Azo)agtgAeg(oF4Azo)ttctAeg(oF4Azo)gg-Lys-CONH2
70.8 ± 0.3
61.4 ± 0.5
56.8 ± 0.5
70.4 ± 0.8
70.6 ± 0.2
62.4 ± 0.4
57.1 ± 0.4
72.1 ± 0.4
44.3 ± 0.8
n.c.
0
1
≈40.6a ± 1.0
n.c.
1
1
1
1
1
1
2
3
4
5
6
7
Ac-Lys-Aeg(Azo)-gcagtgtttcttgg-Lys-CONH2
Ac-Lys-ggAeg(Azo)agtgtttcttgg-Lys-CONH2
Ac-Lys-ggcagtAeg(Azo)tttcttgg-Lys-CONH2
Ac-Lys-ggcagtgtttcttgAeg(Azo)-Lys-CONH2
Ac-Lys-ggcAeg(Azo)gtgtttcAeg(Azo)tgg-Lys-CONH2
Ac-Lys-ggAeg(Azo)agtgAeg(Azo)ttctAeg(Azo)gg-Lys-CONH2
71.5b ± 0.7
62.7b ± 0.3
57.3b ± 0.3
70.0b ± 0.8
42.1b ± 0.6
n.c.
71.5 ± 0.4
63.0 ± 0.3
58.0 ± 0.3
73.2 ± 0.3
44.5 ± 0.6
n.c.
1
1
8
9
Ac-Lys-ggcagtgtttcttgg-Lys-CONH2
Ac-Lys-tgagtgcgtctgttg-Lys-CONH2
74.8 ± 0.3
n.c.
2
0
Ac-ggcagtgtttct-CONH2
64.5c
aThis value might be approximate according to its 1st derivative (Figure 3B); bPNA and ssDNA (5’-GTG AGC CAA GAA ACA CTG CCT-3’) were
isomerized to the cis-form after hybridization, following reported procedures [36]; ctheoretical value calculated by PNA Bio Tools
(
http://www.pnabio.com); n.c. = not calculated. Mean values derived from two independent experiments; Aeg = N-(2-aminoethyl)glycine.
The incorporation of the visible-light responsive azobenzene The potential of the functionality of our compounds could be
PNA 3) stabilized the duplex in comparison with the Aeg(Ac)- enhanced by both changing the localization and the number of
(
modified analogue 5. However, the TM of 3 is 14 °C lower than incorporated photoswitches. To try to maintain the cooperative
the one calculated for the unmodified PNA analogue (20). base pairing, we synthesized longer PNA conjugates (6–11, 18
Unfortunately, no difference between isomers was observed. and 19) with flanking lysines, which improved the solubility of
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the probes. In addition, we compared our probes with the ones between isomers were also qualitatively consistent with a slight
that contained the classical unmodified azobenzene moiety improvement for the case of the dual-labelled PNA(oF4Azo) 10.
(
Azo) (PNA 12–17). The lower stability of the unmodified cis-
Azo forced us to perform the isomerization after duplex hybrid- To corroborate our results, we developed a strand-displacement
ization, which resulted in only a constant 36% cis-isomer rate. assay using fluorescence as readout. We designed a system
The obtained TM values suggested that the localization of the based on three molecules (Figure 4): a black hole quencher
oF4Azo affects both the duplex stability and isomer differences. (BHQ)-labelled single-strand (ss) DNA template, a complemen-
Thus, the incorporation of oF4Azo near the center of the PNA tary fluorescein (FAM)-labelled ssDNA and the PNA of
(
7 and 8) sequence dramatically destabilized the PNA/DNA interest. In this framework, the PNA could bind the BHQ-
duplex (ΔTM = 18 °C), which agrees with excellent capacity of ssDNA; thus, its addition to the quenched BHQ/FAM-DNA
PNAs for effectively discriminate between single base duplex would trigger an exchange reaction and the release of
mismatches. However, when the exchanged base is at either the the FAM-ssDNA. The FAM probe and the PNAs shared the
N- or C-terminus of the PNA (6 and 9) the effect is not that same 15 base-pair sequence (Table S2, Supporting Information
dramatic. As reported in the precedent of azobenzene-PNA File 1). Fluorescence spectroscopy determined the hybridi-
[
51], the C-terminus modification displayed the highest isomer zation degree and, in turn, the effect of the photoswitch.
difference but modest, in our case. Furthermore, always when a
clear difference between isomers was detected, the duplexes
containing the trans-oF4Azo were more stable than those with
the cis-form. In addition, we tested PNAs with the photoswitch
at two (10) and three positions (11). The TM uniformly de-
creased with the number of oF4Azo to the point that the probe
Figure 4: Outline of the displacement assay principle, in which a
photoswitchable PNA probe (blue) hybridizes to a complementary
quencher-labelled single-stranded (ss) DNA (black) and replaces a
fluorescent-labelled ssDNA (grey); F = fluorescein; Q = black hole
quencher; blue rectangle = tetra-ortho-fluoroazobenzene moiety.
1
1 with three oF4Azo behaved as the scramble PNA control 19;
this means, it did not form a stable duplex. More interestingly,
we could observe an improved TM difference between the
trans- and cis-PNA15(oF4Azo)2 10 (up to ≈3.7 °C), which was
the best for our system.
After optimization, we found the following conditions: 0.75 μM
Azobenzene (Azo)-containing PNAs behaved similarly to quenched double-stranded (ds) DNA, 2 equiv PNA for 8 hours
PNA(oF4Azo) (Table 2), which verified that the fluorine substi- at 37 °C, as the best ones for our assay performance. As ex-
tution did not affected the binding properties. The differences pected, the formed FAM/BHQ-dsDNA quenched effectively the
Table 3: Normalized increase of fluorescence signal derived from the strand-displacement assays.a
Compound
Fluorescence increase at 37 °C [%]
Fluorescence increase at 30 °C [%]
15-mer FAM-ssDNA
11-mer FAM-ssDNA
cis
trans
cis
trans
6
7
8
9
1
1
36.8 ± 1.2
43.1 ± 1.7
44.0 ± 2.0
46.1 ± 1.3
n.d.
36.3 ± 1.3
45.9 ± 1.0
45.0 ± 1.3
49.0 ± 1.5
n.d.
69.9 ± 3.4
78.1 ± 4.3
67.0 ± 0.9
84.7 ± 2.1
37.8 ± 2.2
n.d.
75.8 ± 2.6
82.1 ± 1.2
72.7 ± 1.9
82.7 ± 3.5
46.3 ± 2.2
n.d.
0
1
n.d.
n.d.
1
1
8
9
98.8 ± 1.4
17.0 ± 0.4
94.5 ± 3.1
27.9 ± 3.2
FAM-ssDNA
backgroundb
100
0
100
0
BHQ/FAM-dsDNA
24.5 ± 0.3
36.6 ± 0.3
aPercentages calculated according to the measured endpoint (8 h) fluorescence intensity values and considering the FAM-ssDNA intensity as 100%;
bit represents the measurement of the PBS buffer: 140 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, 1.8 mM KH2PO4, pH 8.0. Mean values derived from
two independent experiments; n.d. = no displacement observed.
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fluorescence of FAM-ssDNA (Figure S46, Supporting Informa- (Figure 5B) without compromising the specificity of the system.
tion File 1), which was quantitatively restored in the presence of More importantly, this new set up led to slightly higher differ-
the unmodified PNA 18. The effect of the scrambled PNA 19 is ences between isomers and pointed out the dual-labelled probe
the opposite, i.e., signal decrease. We determined a slightly 10 as the best one, which is consistent with the melting experi-
lower fluorescence value than in the case of BHQ-ssDNA; this ments (Table 2 and Figure 3). Among the single mutated PNAs,
can be probably attributed to the quenching ability of both PNA those with lower TM, displayed the best performance in our
probes [52]. Along these lines, the quenching ability of oF4Azo- kinetics studies (Figure 5B).
containing PNA was evaluated (Figure S46, Supporting Infor-
mation File 1; Table 3) and neglected because of the low Finally, in an attempt of gaining insight into the general low
impact.
photoresponsivity, we performed CD (Figure S53, Supporting
Information File 1) and UV–vis (Figures S54 and S55, Support-
Regarding the photoswitchable PNAs, we observed a correla- ing Information File 1) experiments. The joint-evaluation of
tion between the TM and the increase of fluorescence in the dis- these results suggested that the photoswitch is located inside of
placement-strand assays. Thus, the general trend is: the higher the PNA–DNA duplex (Figure S53, Supporting Information
the TM is, the better is their strand-exchange ability (higher File 1) but it may not effectively intercalate since pre-hybridi-
exchange and faster) (Table S3, Supporting Information File 1). zation did not affect the isomerization efficiency (Figure S55,
PNAs 10 and 11 bearing two and three oF4Azo moieties, re- Supporting Information File 1). This would explain the low ob-
spectively, did not cause an increase of fluorescence. While served photoresponsivity.
such result was expected for 11, according to the melting exper-
iments, the unfortunate outcome of 10 could be probably attri- Conclusion
buted to its low TM in comparison with the quenched dsDNA We have successfully synthesized two novel types of visible-
one (≈40 °C versus 61.5 °C; Table 2, Figure S37, Supporting light photoresponsive PNAs by coupling on-resin the corre-
Information File 1). This would also explain the incomplete dis- sponding molecular transducer. In particular, we focused on the
placement (<50%, Table 3, left) for all modified PNAs. In order tetra-ortho-fluoroazobenzene (oF4Azo) and the hemithioindigo
to improve our method, we tested a shorter 11-mer FAM- (HTI) photoswitches; the latter has not been studied in the
ssDNA provided with a toehold under lower temperatures (30 context of photoregulation of oligonucleotides before. The
°
C). This overhang together with the length of the labelled UV–vis measurements of these probes suggested that the
oligonucleotide must accelerate the strand exchange [53]. PNA(oF4Azo) displayed superior photochemical properties to
Indeed, the displacement was facilitated, reaching almost quan- control oligonucleotide hybridization by irradiation. Thus, for
titative exchanged (70–85%; Table 3, right) with faster kinetics the case of the PNA(HTI) just 47% of trans-isomer was
Figure 5: Time-dependent fluorescence signals from two independent experiments at 520 nm of 0.75 μM FAM/BHQ-dsDNA solutions in PBS buffer
(
140 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, 1.8 mM KH2PO4, pH 8.0) with 2 equiv of the corresponding PNAs 6–11, 18 and 19 placed in 96-well
plates and covered with paraffin oil; fluorescence intensities were measured every 5 min during 12 h at 30 °C; PNA code: 6: blue, 7: green, 8:
maroon, 9: brown, 10: purple, 11: coral, 18: yellow and 19: gray; solid line for trans-isomers and dashed line for cis-isomers. Black lines controls:
solid: FAM-ssDNA without PNA and under the same conditions; dashed: BHQ/FAM-dsDNA.
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detected upon irradiation at 405 nm. In addition, the stability of References
this isomer is compromised. On the contrary, PNA(oF4Azo)
had better conversions and high stability of the non-thermody-
namically isomer, even at 90 °C. Both melting experiments and
strand displacement assays demonstrated that mismatches had a
dramatic effect on the binding affinity of the PNA, which was
slightly compensated by the incorporation of oF4Azo. We ob-
served modest, yet clear, differences in the formation of PNA/
DNA duplexes depending on the number and the localization of
the oF4Azo. Further work on increasing the photoresponsivity
by exploring different connectors and approaches beyond the
base-surrogate approaches is necessary. However, we first
demonstrated the great potential oF4Azo in the context of both
PNA and oligonucleotide hybridization. Thus, we believe that
the excellent photochemical properties of the oF4Azo together
with the use of the visible-light irradiation may overcome some
of the current limitations for in vivo photoregulation of gene
expression and related enzymatic reactions in the near future.
Besides, having at our disposal such antisense probes, whose
activation is reversibly controlled, will contribute to the deci-
phering of biological pathways. Furthermore, the exceptional
stability of the cis-isomer may open new venues of this artifi-
cial photoswitchable oligonucleotide in other fields different
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