Facile functionalization of peptide nucleic acids (PNAs) for antisense and single nucleotide polymorphism detection

Article abstract of DOI:10.1039/c7ob01592e

In this report, we show how a convenient on-resin copper-click functionalization of azido-functionalized peptide nucleic acids (PNAs) allows various PNA-based detection strategies. Firstly, a thiazole orange (TO) clicked PNA probe facilitates a binary readout when combined with F/Q labeled DNA, giving increased sensitivity for antisense detection. Secondly, our TO-PNA conjugate also allows single nucleotide polymorphism detection. Since antisense detection is also possible in the absence of the TO label, our sensing platform based on azido-d-ornithine containing PNA even allows for additional and more advanced functionalization and sensing strategies.

Full text of DOI:10.1039/c7ob01592e

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Facile functionalization of peptide nucleic acids (PNAs) for
antisense and single nucleotide polymorphism detection†
Digvijay Gahtory,a Merita Murtola,b Maarten M. J. Smulders,a Tom Wennekes,a,c Han Zuilhof,a,d
*
Received 00th January 20xx,
Accepted 00th January 20xx
Roger Strömbergb* and Bauke Albadaa*
DOI: 10.1039/x0xx00000x
www.rsc.org/
In this report, we show how a convenient on-resin copper-click
functionalization of azido-functionalized peptide nucleic acids
(PNAs) allows various PNA-based detection strategies. Firstly, a
thiazole orange (TO) clicked PNA probes facilitates a binary readout
when combined with F/Q-labeled DNA, giving increased sensitivity
for antisense detection. Secondly, our TO-PNA conjugate also
allows for the detection of single-nucleotide polymorphism
detection. Since antisense detection is also possible in the absence
of the TO-label, our sensing platform based on azido-D-ornithine
containing PNA, even allows for additional and more advanced
functionalization and sensing strategies.
Fig. 1 Structural comparison of a thiazole orange (red structure) fluorophore linked to
PNA by an amide on Aeg (a) and DOrn (b), respectively, versus (c) triazole linkage.
Since the discovery of peptide nucleic acids (PNAs),1 they have
been employed for numerous applications in molecular
biology,2 antisense technology,3 and nucleic acid detection.4
Generally, the PNA structure is composed of a polyamide
backbone consisting of N-(2-aminoethyl)glycine (Aeg)5 or other
amino acid units,6 with the nucleobases linked via an amide
linkage. This bio-mimetic structure makes them compatible
with biology, yet renders them resilient to enzyme-catalyzed
degradation.7 Furthermore, DNA-PNA duplexes have higher
melting points than similar DNA-RNA or DNA-DNA duplexes.8
The biomimetic properties of PNA have led to a growing interest
in their utilization as carriers for the delivery of antisense
DNA/RNA molecules for inhibition of gene expression, for
example using cell penetrating peptide (CPP)-PNA conjugates.9
PNA has even been used as a basis for artificial RNases.10
Seitz et al. reported that replacing one of the nucleobases with
an intercalating fluorophore provides so-called forced
intercalation probes that possess an excellent single-nucleotide
polymorphism (SNP) detection ability.11 Specifically, it was
shown that replacement of a PNA backbone unit to which
thiazole orange (TO) is anchored from Aeg to D
-ornithine (DOrn)
increased the SNP detection ability.12,13 In this approach, TO is
still linked to the PNA backbone via an amide-group (Figure 1a
and b). Although click-chemistry has been widely applied in the
past decade,13 e.g. on incorporated azido-functionalized
nucleobases14 or via a flexible azido/alkyne tether15, this
chemistry was not yet applied to attach forced intercalation
probes to PNA backbone. In this study, we assess the extent to
which the triazole linkage is also a good isostere for amide
bonds in this setting,16 specifically related to two sensing
applications for TO-functionalized PNA.
a.Laboratory of Organic Chemistry, Wageningen University and Research,
Stippeneng 4, 6708 WE Wageningen, The Netherlands. E–mail:
han.zuilhof@wur.nl, bauke.albada@wur.nl
Here, we report a click strategy for resin-bound PNA using
-
azido -ornithine, that can be incorporated in the PNA chain
D
b.Department of Biosciences and Nutrition, Novum, Hälsovägen 7, 141 83
Huddinge, Sweden. E-mail: roger.stromberg@ki.se
during standard Fmoc-based solid-phase PNA synthesis. Once
incorporated, the azido group allows direct attachment of
thiazole orange (TO), illustrating its convenience as handle for
PNA diversification. We used an on-resin copper-catalyzed
azide-alkyne click (CuAAC) reaction to attach the dye to the
resin-bound PNA chain. The resulting TO-PNA construct was
then tested as a sensing module in (i) FRET-assisted antisense
c. Department of Chemical Biology and Drug Discovery, Utrecht Institute for
Pharmaceutical Sciences and Bijvoet Center for Biomolecular Research, Utrecht
University, Utrecht, The Netherlands
d.Department of Chemical and Materials Engineering, King Abdulaziz University,
Jeddah, Saudi Arabia.
†
Electronic Supplementary Information (ESI) available: Synthesis of alkyne-
derivatized thiazole orange
1
, additional 1H NMR spectra, ESI-MS, HPLC traces and
fluorescence data. See DOI: 10.1039/x0xx00000x
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Fig. 2 Normalized fluorescence spectra showing the binding of antisense DNA D1 to TO-
labeled PNA P3 (λex = 510 nm).
Scheme 1. General scheme for synthesis of DOrn-containing PNA (P1) and on-resin click
with thiazole orange (TO) to give clicked PNA (P2), which after acidic cleavage and
deprotection yields soluble TO-PNA P3; a similar procedure leads to SNP-sensor P4.
5’- and 3’-ends of the oligonucleotide chain, respectively. The
oligonucleotide sequence was chosen so that it would form a
circular DNA-PNA heterodimer in which the labeled termini of
the DNA bind to the middle section of the PNA sequence, close
to the TO-moiety (see Scheme 2a); an internal stretch of ssDNA
would provide the sensing sequence located at the opposite
side of the circle with respect to the TO-label. Specifically, the
overall design provides significant binding to two 7 bp-long
termini of P1 while providing a ssDNA docking site of 7 bp for an
detection, and (ii) single-nucleotide mismatch detection. For
comparison, the azido-PNA precursor was tested in regular
antisense detection.
First, we prepared an DNA-binding motif based on PNA. As
shown by Seitz et al., a D-ornithine linker successfully mimics the
six-atom long building block that normally forms the PNA
backbone, facilitating optimal PNA nucleobase alignment for
hybridization with DNA or RNA.11 Accordingly, resin-bound PNA
incoming oligonucleotide that would be complementary to D1
.
In this configuration, the fluorophore and black hole quencher
are in close proximity to each other and to the TO-dye. As a
result, the FRET from TO to Cy5 is quenched by the Iowa Black
quencher. In the presence of a DNA trigger, however, the F/Q-
labelled DNA strand should be released from the DNA-PNA
heterodimer via a competitive hybridization stimulus, resulting
in the formation of dsDNA and a concomitant separation of Cy5
from the TO-label and from the Iowa Black quencher, leading to
an increase in the fluorescent signals emanating from both D1
and P3. As an alternative to the direct antisense detection, the
presence of a TO-moiety in the middle of a PNA-strand would
also facilitate direct detection of single-nucleotide
polymorphisms (Scheme 2b). Alternative to the application of
the azido-moiety for conjugation to a dye that allows advanced
sensing approaches, the azido group could also function as a
handle for further functionalization, e.g. with a bioactive
moiety.
that contained azido-
standard procedures (Scheme 1 and see ESI,
crude HPLC traces of P1). C- and N-terminal lysine residues were
incorporated in the longer PNA sequences P1 and P3 in order to
increase the solubility of the PNA-sequence; the azido-D-
ornithine residue was sandwiched in between two adenine
bases to achieve a low fluorescence F0 in the single stranded
form after functionalization with TO.11b Following this, resin-
bound PNA underwent a facile CuAAC with alkyne-derivatized
D
-ornithine (P1) was prepared using
†
Figure S1–S3 for
thiazole orange 1 (see ESI for synthesis; ex = 510 nm, em = 530
nm), to yield resin-bound TO-PNA conjugate P2. After acidic
removal of TO-labeled PNA P2 from the resin, TO-PNA
conjugates P3 and P4 were obtained in good yields (40% after
HPLC purification) (Scheme 1 and see ESI,† Figure S4–S6 for
HPLC traces and absorbance spectrum of conjugate P3).
For DNA detection, we pursued partial hybridization of the
PNA strand to a sequence D1 that was labelled with a
fluorophore-quencher pair of Cy5 (
and black hole quencher Iowa Black (Q) (
The sensing ability of the DNA-PNA duplex was tested using
an antisense sequence that binds to the normal luciferase
mRNA, i.e. 5´-TTCTTTATGTTTTTGGCGTCT-3´; this sequence was

ex = 650 nm,

em = 668 nm)
max = 667 nm), at the
2 | J. Name., 2012, 00, 1-3
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chosen to be applied in CPP-PNA delivery vectors.9a Initially, we
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Realizing that it would be beneficial to be able to directly
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tested the sensing ability of the azido-functionalized PNA P1 in monitor the association and dissociationDoOf Il:a1b0e.1l0e3d9/DCN7OABt0o15t9h2eE
combination with F/Q-functionalized DNA D1. Mixing D1 and P1 PNA, we attached TO (1) to the resin-bound PNA (P2), resulting
indeed resulted in a 6-fold decrease in fluorescence intensity at in TO-PNA (P3), allowing us to measure FRET between TO and
667 nm when compared to D1 alone (Figure S7). Upon addition Cy5 (Scheme 2a). Indeed, although the fluorescence intensity
of D2, which is fully complementary to D1, a 2-fold increase in for TO in the D1
fluorescence was observed at em = 667 nm when compared to stranded P3, a clear FRET from TO to Cy5 was observed (Figure
D1 alone, along with a shift of λem to a higher wavelength ( 2, red trace). Upon addition of fully complementary D2, the
-P3 duplex was much lower than in the single-


=
695 nm). Interestingly, at 695 nm the difference in intensities fluorescence intensity for both TO and Cy5 increased multiple
was significantly higher, i.e. 25-fold. Comparing the intensity of times (Figure 2, blue trace), signaling displacement of DNA
the D1-P1 duplex at 667 nm with that of the D1-D2 duplex at strand D1 from the TO-labeled PNA strand P3. As the
695 nm displays a 10-fold increase in fluorescence as result of displacement of P3 from D1 by D2 leads to the hydrophobic
the sensing event. For further discussions regarding the sensing ssPNA P3, we tentatively attribute the increase in Cy5
ability of the azido-functionalized PNA P1, see ESI (Figure S7 and fluorescence to aggregation of the hydrophobic PNA P3 to the
accompanying discussion).
Cy5 dye, which is also hydrophobic.
AFM analysis of the D1 P3 duplex revealed dotted structures
Figure S9), indicating formation of the expected
D2. The circular DNA-PNA hybrid structures, and ruling out
P1 shows that the considerable contribution from linear structures to the
former duplex was more tightly associated than the latter observed FRET signal in D1 P3. Since PNA-oligonucleotide
In addition to this antisense detection approach, we observed
-
a difference of 10 °C in TM value (ΔTM) between the two (see ESI,
complexes (see ESI, Figure S8), i.e. D1 P1 and D1
higher TM for D1 D2 when compared to D1
†
†
-
-
a
-
-
-
hybrid duplex. Although PNA-DNA duplexes usually have a hybrids usually offer higher stability towards enzymatic
higher TM than DNA-DNA duplexes that have the same degradation,7 this should allow utilization of our circular PNA-
sequences, the lower TM of the D1
by the presence of 7 additional base-pairs in D1
compared to D1 P1
-
P1 duplex can be explained DNA/RNA complexes for cellular delivery based on our
-
D2 when conjugation strategy.
-
.
Scheme 2. Overview of the sensing approaches that are described in this study. The solid wavy arrows indicate the irradiation and emission event of a fluorophore (F or TO), the
dotted wavy arrow indicates low intensity emission due to quenching by a nearby organic black hole quencher (Q, panel a) or the larger mobility of the TO-dye (in panel b). a)
Schematic depiction of the FRET-assisted antisense detection system based on TO-labelled PNA P3. Hybridization with F and Q labelled DNA sensor strand D1, the dyes Cy5 and TO
are in close proximity of the quencher, leading to reduced fluorescence of TO, which results in poor FRET and concomitant lower emission from Cy5. Fluorescence of both TO and
Cy5 is restored after replacement of the PNA P3 with a fully complementary target strand D2. b) Schematic representation of hybridization between TO-PNA (P3) and
matched/mismatched DNA, showing the single nucleotide polymorphism (SNP) detection approach in which the TO label senses the match/mismatch pair of its neighboring base.
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Lastly, we studied the performance of a TO-labeled PNA, P4
,
In conclusion, we report
a
novel strategy for PNA
in single-nucleotide polymorphism (SNP) detection.10a We functionalization that allows straightforward on-resin click
envisioned that our novel triazole-linked TO-PNA conjugate modification. We demonstrate the applicability of the method
could, in principle, also be used for mismatch detection by clicking TO at an internal position on the PNA. The azido- or
(Scheme 2b). We chose a reported DNA sequence D6 (X = T) for TO-modified PNA is then hybridized to a fluorescent DNA strand
which up to 3-fold match/mismatch discrimination using FIT- to form a circular complex, allowing direct (via azido-PNA P1
)
PNA at 25 °C was reported.17 Upon hybridization of PNA P4 to and TO-assisted (via TO-PNA P3) sensing of the target DNA
complementary D6 (X = T) we observed a 5-fold enhancement strand. In addition, we determined the SNP detection ability of
of fluorescence (Figure 3a). In comparison, changing the a triazole linked dye on a FIT-PNA (P4) to be up to 3.5-fold match
adjacent pyrimidine nucleobase into a mismatched pyrimidine vs mismatch. Since PNA-DNA hybrids generally show a higher
(X = C, in D7) increased the fluorescence only 1.5-fold. Thus, we stability towards exonuclease degradation, we believe that this
observed about 3.5-fold discrimination ability for the A-C work also provides a platform for different cellular delivery
match/mismatch pair, indicating good match/mismatch applications. Evaluation of PNA-conjugates as a vector and
discrimination ability. Importantly, we observed a similar protection agent for the delivery of an antisense sequence is
discriminating ability for other mismatches (X = G and A, D8 and ongoing.
D9, respectively) as well, indicating the general applicability of
We thank NanoNextNL (program 5A), a micro and
our method (Figure 3b).
nanotechnology consortium of the Government of The
Netherlands and 130 partners for funding of this project. We
also thank Dr. Aart van Amerongen, Adrie Westphal, Antoine
Moers, and Rickdeb Sen for fruitful discussions.
a)
1.0
P4-D6 (X = T)
P4-D7 (X = C)
0.8
P4
Notes and references
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