Strand displacement and duplex invasion into double-stranded DNA by pyrrolidinyl peptide nucleic acids

Article abstract of DOI:10.1039/c5ob01273b

The so-called acpcPNA system bears a peptide backbone consisting of 4′-substituted proline units with (2′R,4′R) configuration in an alternating combination with (2S)-amino-cyclopentane-(1S)-carboxylic acids. acpcPNA forms exceptionally stable hybrids with complementary DNA. We demonstrate herein (i) strand displacements by single-stranded DNA from acpcPNA-DNA hybrids, and by acpcPNA strands from DNA duplexes, and (ii) strand invasions by acpcPNA into double-stranded DNA. These processes were studied in vitro using synthetic oligonucleotides and by means of our concept of wavelength-shifting fluorescent nucleic acid probes, including fluorescence lifetime measurements that allow quantifying energy transfer efficiencies. The strand displacements of preannealed 14mer acpcPNA-7mer DNA hybrids consecutively by 10mer and 14mer DNA strands occur with rather slow kinetics but yield high fluorescence color ratios (blue:yellow or blue:red), fluorescence intensity enhancements, and energy transfer efficiencies. Furthermore, 14mer acpcPNA strands are able to invade into 30mer double-stranded DNA, remarkably with quantitative efficiency in all studied cases. These processes can also be quantified by means of fluorescence. This remarkable behavior corroborates the extraordinary versatile properties of acpcPNA. In contrast to conventional PNA systems which require 3 or more equivalents PNA, only 1.5 equivalents acpcPNA are sufficient to get efficient double duplex invasion. Invasions also take place even in the presence of 250 mM NaCl which represents an ionic strength nearly twice as high as the physiological ion concentration. These remarkable results corroborate the extraordinary properties of acpcPNA, and thus acpcPNA represents an eligible tool for biological analytics and antigene applications.

Full text of DOI:10.1039/c5ob01273b

Organic &
Biomolecular Chemistry
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PAPER
Strand displacement and duplex invasion into
double-stranded DNA by pyrrolidinyl peptide
nucleic acids†
Cite this: Org. Biomol. Chem., 2015,
3, 9223
1
a
b
a
Peggy R. Bohländer, Tirayut Vilaivan* and Hans-Achim Wagenknecht*
The so-called acpcPNA system bears a peptide backbone consisting of 4’-substituted proline units with
2’R,4’R) configuration in an alternating combination with (2S)-amino-cyclopentane-(1S)-carboxylic acids.
(
acpcPNA forms exceptionally stable hybrids with complementary DNA. We demonstrate herein (i) strand
displacements by single-stranded DNA from acpcPNA–DNA hybrids, and by acpcPNA strands from DNA
duplexes, and (ii) strand invasions by acpcPNA into double-stranded DNA. These processes were studied
in vitro using synthetic oligonucleotides and by means of our concept of wavelength-shifting fluorescent
nucleic acid probes, including fluorescence lifetime measurements that allow quantifying energy transfer
efficiencies. The strand displacements of preannealed 14mer acpcPNA–7mer DNA hybrids consecutively
by 10mer and 14mer DNA strands occur with rather slow kinetics but yield high fluorescence color ratios
(blue : yellow or blue : red), fluorescence intensity enhancements, and energy transfer efficiencies. Fur-
thermore, 14mer acpcPNA strands are able to invade into 30mer double-stranded DNA, remarkably with
quantitative efficiency in all studied cases. These processes can also be quantified by means of fluor-
escence. This remarkable behavior corroborates the extraordinary versatile properties of acpcPNA. In con-
trast to conventional PNA systems which require 3 or more equivalents PNA, only 1.5 equivalents
acpcPNA are sufficient to get efficient double duplex invasion. Invasions also take place even in the pres-
ence of 250 mM NaCl which represents an ionic strength nearly twice as high as the physiological ion
concentration. These remarkable results corroborate the extraordinary properties of acpcPNA, and thus
acpcPNA represents an eligible tool for biological analytics and antigene applications.
Received 22nd June 2015,
Accepted 21st July 2015
DOI: 10.1039/c5ob01273b
www.rsc.org/obc
derivatives in the peptide backbone failed in many cases since
the nucleic acid hybridization properties are deteriorated.
Introduction
5,6
Peptide nucleic acid (PNA) represents a very important nucleic Among several of those PNA architectures reported to date,
acid analog and chemical-biological tool based on its impor- and especially among those with cyclic structures as part of
7
–9
tant features like affinity, sequence selectivity and stability the backbone, the so-called acpcPNA system (Fig. 1) exhibits
1
against degradation by nucleases and proteases. Especially significant features. The peptide backbone consists of 4′-sub-
the uncharged pseudopeptide backbone makes PNA an attrac- stituted proline units with (2′R,4′R) configuration in an alter-
tive scaffold for diagnostic applications. PNA with N-(2-amino- nating combination with (2S)-amino-cyclopentane-(1S)-
ethyl)glycine backbone (aegPNA) is able to form complexes carboxylic acids. As a result of this structural design, acpcPNA
with double-stranded DNA either by binding from outside forms stable hybrids with complementary DNA but signifi-
1
0–13
forming triplexes, or by duplex, double-duplex or triplex cantly less stable hybrids with complementary RNA.
invasion. The attempt to increase affinity and, more impor-
The
1
–4
tantly, sequence selectivity of PNA by incorporation of cyclic
a
Institute of Organic Chemistry, Karlsruhe Institute of Technology (KIT), Fritz-Haber-
Weg 6, 76131 Karlsruhe, Germany. E-mail: Wagenknecht@kit.edu;
Fax: +49-721-608-44825; Tel: +49-721-608-47486
b
Organic Synthesis Research Unit, Department of Chemistry, Faculty of Science,
Chulalongkorn University Phayathai Road, Patumwan, Bangkok 10330, Thailand.
E-mail: vtirayut@chula.ac.th; Fax: +66-2-2187598; Tel: +66-2-2187627
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
Fig. 1 Monomeric unit of DNA (left), aegPNA (middle) and acpcPNA
c5ob01273b
(right) in comparison.
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recognition and binding to DNA shows high sequence selecti- two different DNA strands and yields even better emission
4
4
vity and does not tolerate single base mismatches. The color contrasts as readout.
sequence selectivity can be visualized by fluorescent dyes such In order to apply such fluorescence color changes to directly
thiazole orange and nile red, that were syn- report the dynamics of strand displacements in vitro we syn-
thetically incorporated into acpcPNA. Most importantly, self- thesized the 14mers PNA1 and PNA2 that carry the blue emit-
1
4,15
16
17
as pyrene,
1
1
paired hybrids are not formed by acpcPNA. This feature ting dye 1 in the middle of the sequence (Fig. 2). These
stands in contrast to many other PNA scaffolds that bind to acpcPNA modifications were introduced by post-synthetic
themselves equally or even better than to naturally occurring copper(I)-catalyzed cycloaddition between the precursor of dye
4
nucleic acids.
1 bearing an azide group and the synthesized precursors of
Base and/or PNA backbone modifications are traditionally PNA1 and PNA2 that carry an alkyne functionality. The only
required to destabilize PNA–PNA duplexes in order to allow difference between the two PNA strands is the position of dye
1
8,19
efficient double duplex invasion of DNA.
Alternatively, PNA 1 as modification of the aminopyrrolidinecarboxylic acid
modifications like carcinogenic acridine moieties or nucleo- spacers either on the carboxy or on the amino side of the
base substitutions of cytosine (G-clamp) also promote invasion central thymine unit in the sequence. All synthesized PNA
2
0–23
into mixed sequence B-DNA.
To prevent extensive or bio- strands bear five lysine residues at the C-terminus to enhance
logically harmful modifications, a chiral derivative of PNA solubility. Five DNA counterparts of different lengths were syn-
called γPNA that exhibits even higher DNA affinity than thesized. The shortest one, 7mer DNA1, was not labelled. The
normal PNA with extended strand length was employed. 10mers DNA2 and DNA4 were labelled by the yellow emitting
However, 20 equivalents of γPNA and incubating times of dye 2 attached to the 2′-position of uridine next to the central
8
hours are necessary to accomplish quantitative invasion and adenosine unit (either on the 5′or 3′ side). The longest
2
4
complex formation with double-stranded DNA. Moreover,
limitations for applications in vivo still remain due to the high
affinity of γPNA to both DNA and RNA. In order to demonstrate
the advantages of acpcPNA in this regard we report herein (i)
strand displacements by single-stranded DNA from acpcPNA–
DNA hybrids, and by acpcPNA strands from DNA duplexes,
and (ii) strand invasions by acpcPNA strands into double-
stranded DNA with high efficiency. These processes have high
biological significance; however, we studied them in vitro with
model PNA strands by means of fluorescence color changes as
well as fluorescent intensity enhancements using our concept
of wavelength-shifting nucleic acid probes (“DNA traffic
2
5
lights”).
Results and discussion
Strand displacements
DNA strand displacements are biologically relevant and enzy-
2
6–29
matically driven processes inside living cells.
Furthermore,
3
0
these processes play a crucial role in DNA nanotechnology.
Consecutive annealing and strand displacement steps rep-
resent the methodological basis to dynamically control the for-
mation of highly ordered and self-assembled DNA
3
1–34
nanoarchitectures, such as DNA switches, DNA tweezers,
3
4–38
39–42
DNA-based logic gates,
DNA walkers
and others. In
order to readout the strand displacement dynamics with
acpcPNA not only by conventional fluorescence intensity
changes at a single emission wavelength, we followed our
2
5
concept of wavelength-shifting nucleic acid probes. Five
years ago, we introduced this concept that is based on energy
transfer between thiazole orange and thiazole red as fluo-
rescent DNA base substitutions and applied it for molecular
2
5,43
beacons.
Moreover, we showed that this concept works
Fig. 2 Sequences of PNA1, PNA2, DNA1–DNA6 and structures of
also for pairs of fluorescent dyes attached to the 2′-positions in acpcPNA and of dyes 1–3 as PNA or DNA labels, respectively.
9224 | Org. Biomol. Chem., 2015, 13, 9223–9230
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counterparts, 14mers DNA3 and DNA5, were modified at place the energy acceptor dye 2 into proximity to donor dye 1.
corresponding positions by the red emitting dye 3 (for the If the excitation wavelength is kept constant at 389 nm, the
syntheses of dyes 1–3 and PNA/DNA labelling procedures see efficient (vide infra) energy transfer between these two dyes
4
4,45
ESI†).
causes significant fluorescence color changes from blue to
The dye 1-labeled PNA1 and PNA2 were separately annealed yellow which were observed in all four cases (PNA1-DNA1 and
with the unmodified 7mer oligonucleotide counterstrand PNA2-DNA1, each with DNA2 or DNA4). The second titrations
DNA1 to obtain the starting duplexes for strand displacement with DNA3 or DNA5, respectively, remove dye 2 from the
experiments. Both samples were consecutively titrated with 1.0 hybrids and place the energy acceptor dye 3 into close proxi-
equivalent of DNA2 or DNA4, followed by DNA3 or DNA5, mity to dye 1. As a result the fluorescence color changes from
respectively (Fig. 3). The first titrations with DNA2 or DNA4 yellow to red; again at the same excitation wavelength
(
389 nm).
From these titrations the following two observations have
been made and further investigated. (i) The first strand dis-
placements of 7mer DNA1 by 10mer DNA2 or DNA4 take place
relatively fast (within 5 minutes). The second strand displace-
ments of 10mer DNA2 or DNA4 as part of the intermediate
acpcPNA hybrids by 14mer DNA3 or DNA5 occur significantly
slower than the first displacement processes (approximately
1
h). This can be explained by the increasing affinity of
acpcPNA to longer DNA pieces during the strand displace-
ments and the exceptionally high stability of acpcPNA–DNA
hybrids in general. Melting temperatures of the starting, inter-
mediate and final acpcPNA–DNA hybrids were determined
(
Table 1). The representative titration of hybrid PNA2–DNA1
with DNA2 and DNA3 is shown in Fig. 3 and exhibits a T
increase from 71.5 °C over 77.2 °C to >90 °C. As expected all
PNA–DNA hybrids possess far higher T than comparable
m
m
DNA–DNA duplexes (see ESI†). Both PNA strands, PNA1 and
PNA2, form especially stable hybrids with unmodified DNA
counterstrands as shown by T
m
of >90 °C (see ESI†), whereas
singly and doubly modified acpcPNA–DNA hybrids exhibit
lower Tm values. The singly modified duplex DNA6–DNA3 exhi-
bits a T
m
which is around 50 °C lower in comparison to the
corresponding acpcPNA–14mer DNA hybrid (see ESI†) and
hence, affirms strong binding between acpcPNA and DNA even
Table 1 Color contrasts, fluorescence intensity increase factors, energy
transfer efficiencies and melting temperatures T
m
of PNA/DNA hybrids
Fluorescence
intensity
increase factor
Energy
transfer
efficiency
c
Color
contrast
T
m
a
b
Duplex
[°C]
PNA1–DNA1
PNA1–DNA2
PNA1–DNA3
PNA1–DNA4
PNA1–DNA5
PNA2–DNA1
PNA2–DNA2
PNA2–DNA3
PNA2–DNA4
PNA2–DNA5
DNA6–DNA3
—
1 : 34
1 : 141
1 : 38
1 : 173
—
1 : 20
1 : 47
—
—
71.0
78.2
> 90
> 90
> 90
71.5
77.2
> 90
82.7
> 90
32.7
d
16.6
21.1
15.0
8.9
0.82 ± 0.13
0.99 ± 0.13
0.79 ± 0.12
0.98 ± 0.16
—
0.78 ± 0.11
0.98 ± 0.05
0.75 ± 0.10
0.98 ± 0.07
—
e
f
g
—
d
10.8
20.1
9.8
19.8
—
e
f
1 : 49
1 : 223
—
Fig. 3 Optical spectra of the representative strand displacement experi-
ment with PNA2–DNA1 (2.5 µM in 10 mM Na–P buffer, pH 7) by con-
secutive addition of DNA2 (1.0 equiv.) and DNA3 (1.0 equiv.): UV/Vis
absorption (top), fluorescence (middle, λexc = 389 nm) and time-depen-
g
i
a
b
See definition in text. Calculated by fluorescence lifetimes (see ESI).
c
–1
dent fluorescence intensity changes (dye 1: λem = 478 nm, dye 2: λem
=
λ = 260 nm, 5–95 °C, interval: 0.5 °C min , 2.5 μM duplex in 10 mM
d
e
f
g
Na–P
i
, pH 7.
I
478 nm/I560 nm
.
I
478 nm/I606 nm. I478 nm/I579 nm
.
I
478 nm
/
560 nm, dye 3: λem = 606 nm) after addition of DNA2 (0–41 min, and
600 nm
I .
DNA3 (41–138 min).
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in the presence of an attached dye. In case of the majority of
acpcPNA–10mer DNA hybrids, e.g. PNA1–DNA2, the Tm values
show that the fluorescent dye labels are destabilizing and
require carefully chosen placements in complementary
1
7
hybrids. Especially the dye attachment in the hybrid PNA1–
DNA4 causes less destabilizing effects and gives a T of
90 °C. (ii) Large fluorescence color ratios blue : yellow/blue :
m
>
red and fluorescence intensity enhancements were accom-
plished by an efficient energy transfer (vide infra) from donor
dye 1 to both acceptor dyes 2 and 3, respectively (Table 1). The
highest blue : red contrast (I478 nm/I600 nm) of 1 : 223 is obtained
during displacement of 10mer DNA4 by 14mer DNA5 yielding
hybrid PNA2–DNA5. The enhancements of the yellow and red
Fig. 4 Fluorescence intensity change during the strand displacement
of DNA6–DNA3 (2.5 µM in 10 mM Na–P buffer, pH 7) by PNA1 (1.0
i
fluorescence signals were quantified by the fluorescence inten- equiv.), λexc = 389 nm, for UV/Vis absorption spectra and time-depen-
dent fluorescence intensity changes see ESI.†
sity increase factors (Table 1) that were determined for both
acceptor dyes 2 or 3, respectively, at their corresponding emis-
sion maximum. The factors represent the ratios of the fluo-
rescence intensities of the acceptor dyes after excitation at the
absorption maximum of donor dye 1 to the fluorescence inten-
triplex formation, triplex invasion, duplex invasion and double
sities of the acceptor dyes upon excitation at the same wave-
4
,46
duplex invasion.
In order to confirm that acpcPNA is able
length but in the absence of the donor dye 1. The latter
fluorescence intensities were obtained with single-stranded
DNA1 to DNA5. The largest fluorescence enhancement factor
of 21.1 was obtained during the strand displacement of PNA1–
DNA2 by DNA3.
to undergo double duplex invasion and to follow this process
by fluorescence spectroscopy, dsDNA7 to dsDNA10 were syn-
thesized (Fig. 5). They were labeled by the blue emitting dye 1
or the red emitting dye 3, respectively, at the central A
(
dsDNA7, dsDNA9) or at the U on the 3′-side (dsDNA8,
In order to calculate energy transfer efficiencies, fluore-
scence lifetimes of dye 1 (λexc = 389 nm, λem = 478 nm) were
determined for all modified PNA–DNA hybrids (see ESI†). In
the absence of energy acceptor, dye 1 in hybrids of PNA1 and
PNA2 with full length DNA counterstrands (see ESI†) exhibits
lifetimes of approximately 1.6 ns and 1.5 ns, respectively. Due
to the presence of acceptor dyes 2, or 3, respectively, these life-
times are significantly shortened to the sub-ns range in all
doubly labeled acpcPNA–DNA hybrids yielding excellent
energy transfer efficiencies between 75% and 99%.
dsDNA10). PNA3 to PNA5 contain 14mer sequences that are
complementary to the central part of the sequences of dsDNA7
to dsDNA10, and again 5 lysine residues to get enough solubi-
4
7,19
lity. Based on studies in other PNA systems
it can be
assumed that the lysine residues facilitate the strand invasion
process by stabilizing the PNA–DNA duplex and destabilizing
the PNA–PNA self duplex, although whether their presence is
essential for the invasion is difficult to confirm due to the poor
solubility of dye-labeled PNA without lysine. The stability of
these double strands is only slightly diminished, if at all, by
The following strand displacement experiment representa-
tively shows the ability of acpcPNA to displace one DNA strand
out of the 14mer duplex DNA6–DNA3. The recorded fluo-
rescence of dye 3 shows a significant intensity increase due to
the fact that the unmodified DNA6 is replaced by PNA1
bearing the energy donor which is dye 1 (Fig. 4). This clearly
demonstrates that strand displacement of DNA6 by PNA1
occurs efficiently within several minutes due to the large
increase in stability from 32.7 °C (DNA6–DNA3) to >90 °C
the attached dyes: the T
and +0.1 °C, if compared to a 30mer, completely unmodified
DNA double strand (DNA16–DNA15, T = 70.4 °C, see Table S5
m
difference is only between −1.7 °C
m
in ESI†). PNA3 was modified with the yellow emitting dye 2
that serves as energy acceptor for dye 1 in dsDNA7 and
dsDNA8, whereas PNA4 is modified with the green emitting
dye 4 that represents the energy donor for dye 3 in dsDNA9
and dsDNA10 (for synthesis of dye 4, see ESI†). With respect to
the biological context, PNA3 and PNA4 are complementary to
the TIAM1 sequence that is regulated by miRNA-21 and
(
PNA1–DNA3). This kind of strand displacement is an impor-
tant prerequisite for the in vitro strand invasion experiments
described in the second part below.
4
6
miRNA-31 in colon carcinoma cells. The unmodified PNA5
serves as the fourth strand that was originally thought to be
essential for the double duplex invasions. PNA6 bears a non-
sense sequence for control experiments. Invasions were
Strand invasions
Antigene targeting of double stranded (ds) DNA by PNA is an detected by blue : yellow or green : red fluorescence color con-
important goal for biological applications inside living cells. It trasts (e.g. I475 nm/I585 nm) and by fluorescence intensity
1
0,11
was shown
that the conformationally restricted acpcPNA is increase factors (Table 2) that were determined similarly as
not able to form triplex structures like conventional PNA that described above, for both acceptor dyes 2 or 3, respectively, at
binds to dsDNA in several different binding modes, including their corresponding emission maximum. The factors represent
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Table 2 Color contrasts, fluorescence intensity in-crease factors and
energy transfer efficiencies of acpcPNA strand invasion into dsDNA
Fluo-rescence
intensity
increase factor
Energy
transfer
efficiency
Invasion
complex
Color
contrast
a
b
c
NaCl
d
dsDNA7–
PNA3–PNA5
dsDNA8–
PNA3–PNA5
dsDNA9–
PNA4–PNA5
dsDNA9–
PNA4
+
−
+
−
+
−
+
1 : 2.1
18.1
19.6
14.6
20.8
16.6
23.2
14.4
17.4
1.8
0.67 ± 0.09
0.74 ± 0.12
0.66 ± 0.15
0.76 ± 0.12
0.97 ± 0.04
0.99 ± 0.01
0.94 ± 0.03
0.94 ± 0.13
—
e
1 : 7.5
f
1 : 1.4g
1 : 4.8
h
1 : 43.0i
1 : 87.0
h
1 : 23.7i
−
+
1 : 23.0
j
dsDNA9–
1 : 2.2
PNA6
dsDNA10–
PNA4–PNA5
k
+
−
1 : 28.6l
10.0
13.3
0.93 ± 0.06
0.97 ± 0.02
1 : 61.1
a
b
c
2
50 mM.
I
sample/IdsDNA (definition see text). Calculated by
d
h
e
fluorescence lifetimes (see ESI).
I
475 nm/I585nm iI477 nm/I580 nm
.
.
.
f
j
g
I
I
472 nm/I583 nm
477 nm/I613 nm. I529 nm/I611 nm. I528 nm/I608 nm.
.
I
475 nm/I574 nm
.
I
528 nm/I604 nm. I528 nm/I600 nm
k
l
90 minutes without NaCl (see ESI†). Although NaCl-free con-
ditions show comparable kinetics, they yield stronger increase
of emission intensities and considerably higher color contrasts
blue : yellow. Obviously the dye interactions with DNA in the
hybrids with DNA are salt dependent and alter the fluo-
rescence intensities. Further titrations revealed that the fluo-
rescence color change is maximized upon addition of 3.0
equiv. of acpcPNA (see ESI†). Hence, full double-duplex inva-
sions into dsDNA7 and dsDNA8, respectively, were carried out
by addition of 3.0 equiv. of PNA3 together with PNA5. In
absence of acceptor dyes, the blue emitting dye 1 exhibit life-
times of approximately 2.4–2.6 ns in dsDNA7 and dsDNA8.
The lifetimes are shortened down to 0.6–0.9 ns in the invasion
Fig. 5 Schematic drawing of double duplex invasion, sequences of
PNA3–PNA6, structure of dye 4 and sequences of dsDNA7–dsDNA10 complexes with PNA3 and PNA5, which yields energy transfer
(
from top to bottom).
efficiencies in the range from 66% to 76% (see ESI†). More-
over, strand invasion efficiencies could be determined based
on these energy transfer efficiencies. As fluorescence reference
for complete invasion, reference hybrids of PNA3 with full-
the ratios of the fluorescence intensities of the acceptor dyes length complementary DNA strands bearing dye 1 at the
after excitation at the absorption maxima of donor dyes 1 or 4, central A were annealed. The energy transfer efficiencies of
respectively, to the fluorescence intensities of the acceptor these hybrids were measured and represent 100% reference
dyes upon excitation at the same wavelength but in the values. Remarkably, invasion efficiencies of approximately
absence of the donor dyes 1 or 4. The latter fluorescence inten- 99% could be accomplished in all cases (see ESI†). Best results
sities were obtained with the non-invaded dsDNA7 to with this dye combination were accomplished e.g. with
dsDNA10.
dsDNA7 in absence of NaCl (Fig. 6). The invasion by PNA3
Energy transfer efficiencies (Table 2) of all invasion com- together with PNA5 exhibits an intensity increase of the yellow
plexes were calculated from the corresponding fluorescence emission by a factor of 19.6, a color contrast blue : yellow
lifetimes of donor dyes 1 (λexc = 370 nm, λem = 475 nm) and 4 (I477 nm/I580 nm) of 1 : 7.5 and an energy transfer efficiency of
(
λexc = 435 nm, λem = 528 nm). All strand invasion experiments 74%.
were performed in presence and absence of 250 mM NaCl
Similarly, double-duplex invasion kinetics of dsDNA9 and
since we assumed that duplex invasions by acpcPNA might be dsDNA10 after adding 1.0 equivalent PNA4 together with PNA5
salt-dependent processes. At first, we studied kinetics of were recorded. It is important to note that titrations revealed
strand invasion into dsDNA7 and dsDNA8 by 1.0 equiv. of that 1.5 equiv. PNA4 and PNA5 were sufficient for this type of
PNA3 together with 1.0 equiv. of PNA5. Constant fluorescence strand invasions to obtain maximal fluorescence change (see
intensities are reached within 60 minutes with NaCl and ESI†). Hence, quantification of energy transfer efficiencies,
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double duplex invasions. Hence, the latter result demonstrates
that single stranded acpcPNA may be sufficient to open double
stranded DNA without requiring the other acpcPNA strand.
The duplex of the 14mer PNA with DNA containing a single
mismatch base was still quite stable under the experimental
conditions, therefore the invasion also proceeded with dsDNA
target containing a mismatch at the invasion site (data not
shown). In order to rule out that unspecific PNA aggregation to
dsDNA occurs instead of the expected invasion, a random
sequence and non-sense PNA6 was synthesized and modified
with the blue donor dye 1 (Fig. 5). A rather small fluorescence
enhancement after addition of 1.5 equiv. non-sense PNA6 to
dsDNA9 was observed (Fig. 6, Table 2). As proven in strand dis-
placement experiments above, an efficient energy transfer is
only possible between dye 1 and dye 3 if they get in close proxi-
mity to each other. Thus, the very small intensity increase
factor of 1.8 in case of PNA6 clearly excludes non-specific
PNA–DNA aggregation.
In order to support the duplex invasions by PNA3 and
PNA4, melting temperatures of dsDNAs, acpcPNA–DNA-
hybrids, their DNA–DNA-analogs and of complexes invaded
by 1.0 equiv. acpcPNA were determined, in the absence or
presence of NaCl, respectively (see ESI†). Fig. 7 representatively
displays the normalized absorption changes during T
m
deter-
Fig. 6 Representative fluorescence spectra before and after double
mination of the complex dsDNA8 invaded by only 1.0 equiv.
PNA3 together with PNA5 without NaCl. It should be noted
here that strand invasion with 1.0 equiv. of PNA3 is not com-
plete. Accordingly, discrete melting temperature regions for
each part of the invading complex are explicitly visible: (i)
Comparison 8mer dsDNA13 (overhang on both ends of
dsDNA8) that invasion takes place without dissociation of the
remaining duplex regions on both sides of invasion, and with
duplex invasion: dsDNA7 (2.5 µM in 10 mM Na–P
by 3.0 equiv. PNA3 together with PNA5, in absence and presence of
NaCl (250 mM), λexc = 391 nm (top); dsDNA9 (2.5 µM in 10 mM Na–P
buffer, pH 7) invaded by 1.5 equiv. PNA4 together with PNA5 in absence
and presence of NaCl (250 mM), by 1.5 equiv. PNA4 without PNA5, and
by 1.5 equiv. non-sense PNA6, λexc = 391 nm (bottom).
i
buffer, pH 7) invaded
i
fluorescence intensity increase factors and color contrasts a locally displaced complementary strand. (ii) The melting
green : red were carried out under these conditions, in absence transition of dsDNA8 which corresponds to the non-invaded
and presence of NaCl, respectively (Table 2).
Remarkably, energy transfer from donor dye 4 to acceptor
dye 3 occurs with 97–99% efficiency which yields very large
increase factors and color contrasts. Here again, invasion
efficiencies of 99% were achieved in all cases (see ESI†).
Especially the modification at the central adenosine by dye 3
(dsDNA9) and invasion in the absence of NaCl turned out to
be the optimal condition for double duplex invasion (Fig. 6).
After addition of 1.5 equiv. of PNA3 together with PNA5 an
increase factor of 23.3 and a color contrast green : red (I528 nm
/
I600 nm) of 1 : 87 could be accomplished. The observed vari-
ation of intensity enhancements and color contrasts could be
assigned to the different anchor point of the post-synthetic
DNA labeling by dye 3. Reference measurements with corres-
ponding DNA duplexes bearing the dye 3 modification at an
adenosine (dsDNA9) vs. uridine (dsDNA10) clearly show
approximately 20% stronger fluorescence intensities (see
ESI†). Importantly, the possibility of strand invasion of
dsDNA9 by using only one PNA strand (PNA4) yields a D-loop
and could be evidenced by energy transfer efficiency of 94%
and invasion efficiency of 96% (see ESI†) which is only slightly
Fig. 7 Representative, normalized melting temperature determination
of dsDNA8 invaded by 1.0 equiv. PNA3 and PNA5 (blue line), and
dsDNA9 invaded by PNA4 only (gray line), and those of references
dsDNA8 (green dotted), 8mer dsDNA13 (black dotted) and DNA18–
lower compared to 99% invasion efficiencies obtained for PNA3-hybrid (light blue dotted), in absence of NaCl.
9228 | Org. Biomol. Chem., 2015, 13, 9223–9230
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duplex vanishes with more equivalents of PNA3/PNA5. (iii) The
Tm of the DNA18–PNA3 hybrid is >90 °C represents the Tm and
the exceptionally stability of the invaded duplex (for sequences
of dsDNA13 and DNA18 see ESI†).
Notes and references
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