Towards Zwitterionic Oligonucleotides with Improved Properties: the NAA/LNA-Gapmer Approach

Article abstract of DOI:10.1002/cbic.202000450

Oligonucleotides (ON) are promising therapeutic candidates, for instance by blocking endogenous mRNA (antisense mechanism). However, ON usually require structural modifications of the native nucleic acid backbone to ensure satisfying pharmacokinetic properties. One such strategy to design novel antisense oligonucleotides is to replace native phosphate diester units by positively charged artificial linkages, thus leading to (partially) zwitterionic backbone structures. Herein, we report a “gapmer” architecture comprised of one zwitterionic central segment (“gap”) containing nucleosyl amino acid (NAA) modifications and two outer segments of locked nucleic acid (LNA). This NAA/LNA-gapmer approach furnished a partially zwitterionic ON with optimised properties: i) the formation of stable ON-RNA duplexes with base-pairing fidelity and superior target selectivity at 37 °C; and ii) excellent stability in complex biological media. Overall, the NAA/LNA-gapmer approach is thus established as a strategy to design partially zwitterionic ON for the future development of novel antisense agents.

Full text of DOI:10.1002/cbic.202000450

Combining Chemistry and Biology
Accepted Article
Title: Towards Zwitterionic Oligonucleotides with Improved Properties:
the NAA/LNA-Gapmer Approach
Authors: Melissa Wojtyniak, Boris Schmidtgall, Philine Kirsch, and
Christian Ducho
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemBioChem 10.1002/cbic.202000450
Link to VoR: https://doi.org/10.1002/cbic.202000450
01/2020

10.1002/cbic.202000450
ChemBioChem
FULL PAPER
Towards Zwitterionic Oligonucleotides with Improved Properties:
the NAA/LNA-Gapmer Approach
Melissa Wojtyniak,[a] Boris Schmidtgall,[b] Philine Kirsch,[a] and Christian Ducho*[a,b]
In memory of Professor Thomas C. Bruice (1925-2019).
[a]
[b]
M. Wojtyniak, P. Kirsch, Prof. Dr. C. Ducho (0000-0002-0629-9993)
Department of Pharmacy, Pharmaceutical and Medicinal Chemistry, Saarland University
Campus C2 3, 66123 Saarbrücken (Germany)
E-mail: christian.ducho@uni-saarland.de
Dr. B. Schmidtgall, Prof. Dr. C. Ducho
Department of Chemistry, University of Paderborn
Warburger Str. 100, 33098 Paderborn (Germany)
Supporting information for this article is given via a link at the end of the document.
Abstract: Oligonucleotides (ON) are promising therapeutic
candidates, for instance by blocking endogenous mRNA (antisense
mechanism). However, ON usually require structural modifications of
the native nucleic acid backbone to ensure satisfying
pharmacokinetic properties. One such strategy to design novel
antisense oligonucleotides is the replacement of native phosphate
diester units by positively charged artificial linkages, thus leading to
(partially) zwitterionic backbone structures. Herein, we report a
gapmer architecture comprised of one zwitterionic central segment
('gap') containing nucleosyl amino acid (NAA)-modifications and two
outer segments of locked nucleic acid (LNA). This NAA/LNA-gapmer
In order to overcome these hurdles, a large 'toolbox' of
various backbone modifications has been established over the
past decades. According artificial backbone structures can be:
(i) the sole substitution of single atoms within the phosphate
diester unit, e.g. methylphosphonates[5] or the broadly used
phosphorothioates;[6] (ii) replacements of the phosphate diester
with electroneutral groups, for instance with amides[7] or
sulfones;[8] (iii) a complete replacement of the native sugar-
phosphate backbone, e.g. as in peptide nucleic acids (PNAs).[9]
Furthermore, modifications of the (2'-deoxy)ribose units can
also furnish improved properties of ON analogues. For instance,
Wengel and co-workers have developed non-natural locked
nucleic acids (LNAs) to alter the native ON characteristics (cf.
Figure 1A).[10] The LNA modification is characterised by an
additional bridging bond, linking the 2'-oxygen of the ribose with
the 4'-position, thus locking it in the 3'-endo conformation.
Insertion of this rigid sugar results in a significant increase of
binding affinity towards RNA, due to a reduced entropic penalty
upon hybridisation. In addition, the LNA modification was shown
to significantly enhance stability against nuclease-mediated
degradation, thereby improving the overall stability in biological
media. As a result, LNA-modified ON have found pronounced
attention as potential biomedical agents.[10c] Yet, the improved
affinity for duplex formation was also shown to be accompanied
by limited sensitivity towards base mismatches and thus, by
decreased binding selectivity. Furthermore, the high melting
temperatures of LNA-ON in complex with their RNA targets have
been shown to be correlated to increased cytotoxicity resulting
from elevated off-target effects, as well as increased hepatotoxic
risks.[11a] To elucidate this toxic potential, Dieckmann et al. have
developed an in vitro approach to evaluate the hybridisation-
approach furnished
a partially zwitterionic ON with optimised
properties: (i) the formation of stable ON-RNA duplexes with base-
pairing fidelity and superior target selectivity at 37 °C; and (ii)
excellent stability in complex biological media. Overall, the
NAA/LNA-gapmer approach is thus established as a strategy to
design partially zwitterionic ON for the future development of novel
antisense agents.
Introduction
Oligonucleotides (ON) represent attractive candidates for novel
therapeutic agents with respect to their unique binding mode, i.e.,
hybridisation with complementary endogenous nucleic acids.
Hence, they enable interference with protein biosynthesis via
several modes of action: (i) the antisense mechanism;[1] (ii) the
antigene mechanism;[2] and (iii) the RNA interference
mechanism.[3] These unique interaction pathways have already
been proven to be clinically useful. For instance, fomivirsen, an
antiviral antisense oligonucleotide used against cytomegalovirus
retinitis, was approved by the FDA as the very first antisense
drug on the market in 1998.[4a] In the following years, few other
ON were approved for clinical use, such as mipomersen for
treatment of familial hypercholesterolemia,[4b] nusinersen against
spinal muscular atrophy[4c] and eteplirsen to treat Duchenne
muscular dystrophy.[4d] However, the development of ON into
pharmaceuticals is significantly hampered due to several
characteristics of their phosphate diester backbone. First, its
dense negative charge results in restrained cell permeability.
Second, the native phosphate diester linkage is labile towards
nuclease-mediated hydrolysis.
dependent toxicity of high-affinity ON, which confirmed
a
correlation of high melting temperatures (Tm values) and
undesired toxic effects.[11b]
An alternative strategy in the development of backbone-
modified ON involves the replacement of the anionic phosphate
diester unit with artificial, positively charged linkers, thus creating
an either partially zwitterionic[12] or fully cationic[13] ON. This
approach significantly differs from the introduction of positive
charges by modification of the 2'-hydroxy groups (in RNA) or
nucleobases, while leaving the phosphate diester backbone
unchanged. Such strategies also afford zwitterionic structures,
but result in densely charged oligonucleotides.[14] In contrast,
1
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10.1002/cbic.202000450
ChemBioChem
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cationic replacements of the anionic phosphate diester enable
the synthesis of artificial ON with a reduced net charge due to
the compensation for adjacent phosphates. This substitution has
been shown to have a positive effect on the ability to penetrate
biological barriers such as cellular membranes.[15] So far, four
according types of non-natural cationic linkers have been
reported: (i) Letsinger's phosphoramidate linkages[16] that carry a
positively charged head group connected to the phosphate by
an alkyl chain; (ii) the guanidine[17] and (iii) S-methylthiourea[18]
modifications, both first described by Bruice et al.; and the (iv)
nucleosyl amino acid (NAA) modification (Figure 1A), an amide-
derived cationic backbone motif previously reported by our
group.[12,13b,13c,19]
DNA-ON confirmed high stability against cleavage by 3'5'- and
5'3'-exonucleases as well as in more complex biological media
(human plasma, whole cell lysate).[19]
For the future development of NAA-containing partially
zwitterionic ON towards potential biomedical agents, one major
issue was the undesired decrease in thermal stability for
duplexes with complementary RNA strands (as these would
represent the drug targets in antisense applications). Hence, we
have envisioned to overcome this hurdle by using a 'gapmer'
approach,[21] i.e. a hybrid structure with different internucleoside
linkages in the centre of the ON than at its ends. The overall
goal was to obtain a chimeric, partially zwitterionic NAA-
containing ON that exerts high affinity towards its target RNA,
while still showing excellent base-pairing fidelity. LNA units
facilitate the formation of thermally highly stable DNA/RNA
hetero-duplexes (vide supra). Hence, it was planned to exploit
this feature for the design of according NAA-containing gapmers.
We have therefore designed a partially zwitterionic gapmer-ON
consisting of LNA segments at the 3'- and the 5'-ends and a
block of (6'R)-configured NAA-modified DNA filling the 'gap' in
the central section (Figure 1A).
In this work, we report the synthesis of the novel NAA/LNA-
gapmer 1 (with a partially zwitterionic backbone) and its
properties in comparison with DNA/LNA-gapmer
2 (as a
reference ON with a fully anionic backbone, Figure 1B). The
identical base sequence of 1 and 2 has been artificially designed
in order to study the fundamental principles of a zwitterionic
gapmer construct such as 1, i.e. its sequence is not (yet)
designed to target a specific biologically relevant RNA sequence.
The choice of this sequence was based on synthetic
considerations and the goal to obtain an ON containing all four
canonical bases, while having a reasonable G-C content. The
reported results strongly indicate that the NAA/LNA-gapmer
approach is a useful strategy to design partially zwitterionic ON
with improved properties.
Figure 1. A. Schematic representation of the NAA/LNA-gapmer approach. B =
nucleobase, L = LNA-modification, x = NAA-modification. B. Sequences of
novel NAA/LNA-gapmer 1 and DNA/LNA-gapmer 2 (as reference ON). All
non-labelled linkages are native phosphate diesters.
Results
Synthesis of gapmer ON 1 and 2
The NAA internucleoside structure has been formally derived
from the 'high carbon' nucleoside core unit of naturally occurring
muraymycin antibiotics and their synthetic analogues.[12,20] In
terms of conformational flexibility, the NAA-linkage is somewhat
intermediate to the rather flexible phosphoramidates and the
rigid guanidines and S-methylthioureas. Its peptide-like structure
features a primary amino group carrying a positive charge at
physiological pH. This unit can be attached with a specific
spatial orientation, i.e. with stereoselectivity at the 6'-position of
the adjacent 'high-carbon' nucleoside.[12,13b,13c] Partially
zwitterionic ON including up to four NAA-modifications in an
otherwise anionic (i.e. phosphate-based) backbone had
previously been proven to form stable helical duplexes with
complementary DNA. They were also shown to preserve
excellent base-pairing fidelity, as demonstrated by decreasing
melting temperatures due to base mismatches in the DNA
counterstrand. For hybrid duplexes of NAA-containing DNA-ON
with RNA, however, a fairly significant loss in thermal stability
was observed (Tm up to ~4.0 °C/modification). In this context,
the (6'S)-configured NAA-linkage seemed to exert a slightly
greater destabilising effect than the (6'R)-configured
congener.[12a] The biological in vitro evaluation of NAA-modified
It was envisioned to prepare both target ON 1 and 2 (cf. Figure
1) by modified automated solid phase-supported ON synthesis
using phosphoramidite methodology. For the synthesis of the
NAA/LNA-gapmer 1, a 'dimeric' CxT-NAA-phosphoramidite (with
(6'R)-configuration in the NAA-linkage) had to be prepared.[12]
Thus, the aforementioned goal to obtain a gapmer ON with
reasonable G-C content could be reached, as the only
previously reported 'dimeric' NAA-phosphoramidites had been
TxT[12a] and AxT,[12b] respectively (with "x" representing the NAA-
linkage, cf. Figure 1B). The synthesis of this novel CxT-NAA-
phosphoramidite required the preparation of a suitably protected
3'-amino-2',3'-dideoxycytidine building block (Scheme 1).
Different synthetic routes towards such a 3'-aminodeoxycytidine
building block had been described before.[22] With respect to its
elegance and high stereoselectivity, our synthetic strategy was
mainly based on the procedure reported by Richert and co-
workers.[22d]
Thus, 2'-deoxycytidine
3
was N-benzoylated at the
nucleobase using a standard transient protection protocol,[23]
furnishing N-benzoyl-2'-deoxycytidine 4 in 96% yield (Scheme 1).
2
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10.1002/cbic.202000450
ChemBioChem
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Scheme 1. Synthesis of the protected 3'-amino-2',3'-dideoxycytidine building block 8.
In contrast to the recrystallisation procedure described by Ti et
al., compound 4 was purified by column chromatography to
Based on our stereoselective route for the synthesis of
uridine-derived nucleosyl amino acids,[25] we had previously
remove excess benzoic acid. This was followed by a sequence
of two Mitsunobu reactions, with the first one leading to 5'-(p-
bromobenzoyl) protection and the second one enabling ring
closure of the cytosin-2-O and C-3', to give cyclised product 5 in
39% yield. Nucleophilic substitution (SN2) at C-3' with sodium
azide then furnished 3'-azidonucleoside 6 in 66% yield. After
saponification of the p-bromobenzoyl ester, silylation gave 5'-O-
TBDMS-protected derivative 7 in 85% yield over two steps from
6. Finally, reduction of the azido group by transfer
hydrogenation[24] afforded the desired protected 3'-amino-2',3'-
dideoxycytidine derivative 8 in 96% yield (Scheme 1).
developed
the
preparation
of
according
thymidine
derivatives.[12,13b] Hence, this reported protocol was used to
prepare protected nucleosyl amino acid 9 (Scheme 2, reactions
not shown).[12] Thymidine derivative 9 underwent amide coupling
with 3'-aminonucleoside 8 to furnish the bis-silylated NAA-linked
C-T dimer 10 in 78% yield. Fluoride-mediated desilylation gave
diol 11 in a moderate yield of 53%. Attempts to improve this
deprotection protocol for the TBDMS ethers were not successful,
as changes in the reaction conditions always led to more side
reactions or even complete decomposition of starting material 10.
Regioselective 5'-O-dimethoxytritylation afforded 5'-O-DMTr-
protected NAA-linked dimer 12 (64% yield), which was then
Scheme 2. Synthesis of the 'dimeric' NAA-linked C-T phosphoramidite 14 for automated ON synthesis.
3
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10.1002/cbic.202000450
ChemBioChem
FULL PAPER
phosphitylated (using diamidite reagent 13) to give the dimeric
NAA-linked C-T phosphoramidite 14 in 78% yield (Scheme 2).
With the NAA-linked C-T phosphoramidite 14 in hand, both
gapmers 1 and 2 (cf. Figure 1) were assembled on the DNA
synthesiser. Standard protocols were slightly adjusted (see
Supporting Information for details). In general, coupling times for
LNA- and NAA-phosphoramidites were prolonged relative to
their commercially available, unmodified DNA congeners, and
the number of couplings was increased to enhance the step-to-
step yield. With respect to the activator for phosphoramidite
coupling, it was found that benzylmercaptotetrazole (BMT) was
superior to 4,5-dicyanoimidazole (DCI) as it gave higher yields,
in particular when the NAA-linked building block 14 was coupled.
Apart from this, standard solvents and reagents for solid phase-
supported DNA synthesis and the usual basic workup procedure
were used. Purification of gapmers 1 and 2 was achieved by
polyacrylamide gel electrophoresis (PAGE) with urea as
chaotropic component. The identities of gapmers ON 1 and 2
were confirmed by high resolution mass spectrometry (see
Supporting Information).
coefficient , Tm values were calculated and used to describe
the melting temperature of all studied duplexes. Furthermore,
T37°C values were calculated to investigate the hybridisation at
a physiologically relevant temperature of 37 °C (for more details
see Supporting Information). The obtained results are given in
Table 1, with selected melting curves shown in Figure 2.
Satisfactorily, the duplex stability of the NAA/LNA-gapmer 1
(cf. Figure 1) with fully complementary DNA was equal to the
according native DNA/DNA duplex (Tm = 48.5 °C, entries 9 vs.
1, Table 1), whereas control gapmer 2 furnished an even higher
value (ΔTm = +4.5 °C, entry 5). This was also the case for the
2-RNA duplex, for which an even stronger increase in melting
temperature was observed (ΔTm = +17.0 °C, entry 6). This was
anticipated as the LNA segments in gapmer 2 were supposed to
stabilise duplexes with native RNA.[10] However, for the
hybridisation of the complementary RNA strand with NAA-
containing gapmer 1, this overall stabilising effect was not found.
Instead, a slight decrease in the melting temperature was
observed (ΔTm = -6.0 °C, entry 10). This decrease in duplex
stability is equivalent to ca. -5.8 °C per NAA-modification (entry
10 vs. entry 6), which is similar to the previously described
destabilising effect of the NAA-modification on DNA-RNA hybrid
duplexes (~3.5 °C per NAA-modification for a comparable type
of sequence).[12a] The presence of the stabilising LNA units then
limits the overall destabilisation of the 1-RNA duplex to a formal
value of -1.5 °C per NAA-modification (entry 10 vs. entry 2).
Therefore, the results obtained with ON 1 demonstrated that the
gapmer architecture with two flanking LNA segments indeed
furnished an improved duplex stability for hybridisation with RNA.
For a more detailed analysis of the hybridisation properties
of gapmer 1, we considered the overall ratio of hybrid duplex to
free single strands at physiological human body temperature
(37 °C), as this property is decisive for any potential in vivo
NAA/LNA-gapmer 1 shows superior hybridisation properties
at physiologically relevant temperature
One main goal of the reported gapmer approach was to
enhance the stability of NAA-containing DNA-RNA hybrid
duplexes, while retaining base-pairing fidelity. Therefore, melting
temperature experiments with fully complementary strands as
well as with mismatched RNA-ON X (base mismatch opposite of
one of the LNA segments: 5'-AAUCUAGAGAGAGACCU-3') and
mismatched RNA-ON Y (base mismatch opposite of the central
NAA gap: 5'-AAUCUAGAGGGAGAUCU-3') were performed. To
eliminate the temperature dependency of the extinction
Table 1. Tm values (in °C) of native DNA (entries 1-4), DNA/LNA-gapmer 2 (entries 5-8) and NAA/LNA-gapmer 1 (entries 9-12) in complex
with either complementary DNA, complementary RNA, mismatched RNA X, and mismatched RNA Y, respectively. L = LNA-modification,
x = NAA-modification (cf. Figure 1). All non-labelled linkages are native phosphate diesters. Mismatches in RNA-ON X and Y are highlighted in
bold and underlined.
ΔTm [°C][a]
T37°C
T37°C [%]
1-T37°C [%]
#
duplex
Tm [°C]
1
2
3
4
DNA+DNA
DNA+RNA
DNA+X
48.5
55.5
50.0
52.0
-----
-----
-----
-----
0.97
1.00
0.97
0.98
97
100
97
3
0
3
2
DNA+Y
98
5
6
7
8
gapmer 2+DNA
gapmer 2+RNA
gapmer 2+X
53.0
72.5
62.5
68.5
4.5
0.98
1.00
1.00
1.01
98
2
0
0
0
17.0
12.5
16.5
100
100
100
gapmer 2+Y
9
gapmer 1+DNA
gapmer 1+RNA
gapmer 1+X
48.5
49.5
37.0
43.0
0.0
-6.0
-13.0
-9.0
0.96
0.98
0.51
0.86
96
98
51
86
4
2
10
11
12
49
14
gapmer 1+Y
[a] ΔTm values were calculated based on the difference to native duplexes, i.e. Tm (gapmer+counterstrand) - Tm (native DNA+counterstrand).
4
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10.1002/cbic.202000450
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see Figure S7, Supporting Information). Furthermore, in all of
these experiments, the mismatch-mediated destabilisation of
duplex structures was most pronounced when the mismatched
base was placed opposite the LNA segment, i.e. with RNA-ON X
as counterstrand (entries 3, 7, and 11). Interestingly, NAA/LNA-
gapmer
1 proved to be highly sensitive towards both
mismatched RNA-ON X and Y (entries 11 and 12 vs. entry 10).
In this case, mismatch recognition was found to be even better
than for the native DNA strand (entries 3 and 4 vs. entry 2), as
expressed by a stronger mismatch-induced destabilisation of the
duplex structures. Again, the duplex-to-single strand ratio at
37 °C was determined from the melting curve data (vide supra).
Both reference ON (gapmer 2 and native DNA) showed a large
percentage of duplex structures at 37 °C (T37°C = 97-100%),
both with X and Y as counterstrands (entries 3, 4, 7, and 8).
Only the NAA/LNA-gapmer 1 was partially dissociated from the
mismatched RNA-ON under these conditions (entries 11 and 12).
In the presence of Y ,14% of gapmer 1 was not bound to the
RNA-ON, and with X, the unbound fraction of the gapmer was
even ~50%. This selectivity in RNA binding led to the conclusion
that the hybridisation properties of NAA/LNA-gapmer 1 are
superior relative to both the reference gapmer 2 and to native
DNA (vide infra), even though the presence of the NAA-
modification furnished a moderate decrease of thermal duplex
stability.
Figure 2. A. Melting curves of native DNA, DNA/LNA-gapmer 2, and
NAA/LNA-gapmer 1 in complex with complementary DNA. B. Melting curves
of native DNA, DNA/LNA-gapmer 2, and NAA/LNA-gapmer 1 in complex with
complementary RNA. All depicted curves are the average of technical
triplicates.
Circular dichroism spectra of both gapmers in complex with
DNA or RNA demonstrate structural resemblance to a DNA-
RNA heteroduplex
In order to elucidate the structural properties of the partially
zwitterionic NAA/LNA-gapmer 1, circular dichroism (CD) spectra
of duplexes of 1 either with complementary DNA (Figure 3A) or
RNA (Figure 3B) were recorded and compared to the respective
application of antisense ON. Thus, the y-values of the -curve
for T = 37 °C were determined for every thermal denaturation
experiment (T37°C values, Table 1, also see Supporting
Information for the exact procedure). These values correspond
to the amount of duplex present at this given temperature and
can therefore also be expressed as a percentage of hybridised
(i.e. bound) ON (T37°C values in %). Correspondingly, the term
1-T37°C provides the unbound, single-stranded ON fraction
(Table 1). It was found that hybridisation of native DNA and RNA
of the given sequence occurred with 100% at 37 °C (entry 2,
Table 1). This was also the case for the mixture of reference
gapmer 2 and complementary RNA under these conditions
(entry 6). For the NAA/LNA-gapmer 1 and complementary RNA,
almost quantitative (98%) hybridisation at 37 °C was determined,
with only ~2% single-stranded fraction (entry 10). This
demonstrated the possibility to achieve excellent target
engagement by such an NAA-containing gapmer under
physiologically relevant conditions.
We then studied the base-pairing fidelity of NAA/LNA-
gapmer 1, i.e. its hybridisation properties with RNA-ON
containing a single base mismatch. Therefore, gapmer 1 as well
as both reference ON (2 and native DNA, respectively) were
investigated for duplex formation with two different mismatched
RNA-ON: (i) the aforementioned RNA strand X having a non-
matching base opposite one of the LNA segments of 1; (ii) the
aforementioned RNA strand Y having the mismatch opposite the
zwitterionic NAA-modified gap of 1. In all resultant cases (i.e.
with gapmers 1 and 2 as well as with native DNA), a decrease in
thermal duplex stability due to the introduction of base
mismatches was observed (entries 3 and 4 vs. entry 2; entries 7
and 8 vs. entry 6; entries 11 and 12 vs. entry 10, Table 1; also
spectra of DNA/LNA-gapmer
counterstrands.
2
with complementary
For the duplexes of either gapmers 1 or 2 with DNA, CD
signals indicated more resemblance between both gapmer-
containing duplexes than to the unmodified DNA-DNA duplex of
the same sequence (Figure 3A). In particular regarding the
distinct negative signal at 210 nm and the following positive
amplitude at 225 nm - which were almost non-existent for the
DNA-DNA reference duplex - duplexes containing gapmers 1
and 2 showed pronounced similarities. This hints towards
substantial structural differences in the conformation of both
gapmer-DNA duplexes as compared to the native DNA-DNA
helix. However, when according duplexes with a complementary
RNA counterstrand were studied, striking similarities to the
unmodified DNA-RNA congener were observed (Figure 3B). In
this case, the CD signals of the 1-RNA duplex almost perfectly
superposed with the native DNA-RNA duplex. This was also the
case for reference gapmer 2, with the slight difference that the
negative signal at 210 nm was stronger than for the other two
duplexes. Furthermore, the CD spectra of the 1-RNA and 2-RNA
duplexes show some resemblance to the spectra of the 1-DNA
and 2-DNA congeners, respectively (Figure 3B vs. Figure 3A).
Overall, these results therefore demonstrate that both gapmers
1 and 2, either in complex with DNA or RNA, furnish duplexes
with topologies similar to a DNA-RNA heteroduplex. Remarkably,
this finding was independent of the charge pattern in the ON
backbone, i.e. the partially zwitterionic nature of gapmer 1.
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Figure 4. A. Effect of human plasma (HP) on native DNA as well as on
gapmers 1 and 2, respectively, over a time course of eight hours (analysis by
urea-PAGE). B. Effect of human whole cell lysate (WCL) on native DNA as
well as on gapmers 1 and 2, respectively, over a time course of eight hours
(analysis by urea-PAGE).
Figure 3. A. CD spectra of native DNA, DNA/LNA-gapmer 2, and NAA/LNA-
gapmer 1 in complex with complementary DNA. B. CD spectra of native DNA,
DNA/LNA-gapmer 2, and NAA/LNA-gapmer 1 in complex with complementary
RNA. All depicted curves are the average of technical triplicates.
Both gapmer ON show excellent stability in biological media
Figure 5A shows the results of the incubation of the labelled
RNA with all three aforementioned strands, respectively, and
RNase H over a time course of 60 min. Furthermore, the bottom
panel depicts the influence of RNase H on the 32P-labelled RNA
strand without the presence of any counterstrand as an
additional control experiment. Analyses of the assay mixtures
were carried out by urea-PAGE and autoradiography. As
anticipated,[28] DNA/LNA-gapmer 2 induced the activation of
RNase H and led to complete degradation of the parent RNA
strand within the first 5 min of incubation (Figure 5A, degradation
product label b). The timepoint at 0 min shows the intact RNA
(Figure 5A, label a) as well as a band of the 2-RNA duplex (label
x) which was still present despite the addition of urea as
chaotropic agent. Similar observations were made for
unmodified DNA that likewise triggered rapid degradation after
5 min, but led to the formation of an additional degradation
Stability in biological media is crucial for any potential in vivo
application of antisense ON. Therefore, we have tested the
stability of both gapmers 1 and 2 in pooled human plasma (HP,
Figure 4A) as well as in whole cell lysate (WCL) of the human
U937 cell line (Figure 4B). Unmodified DNA served as a positive
(i.e., degradable) control in both assays.
We had previously reported that NAA-modified ON have
potentially excellent stabilities in such media, dependent on the
position of the cationic NAA-linkage.[19] LNA-modified ON are
also known to show an improved stability against nuclease-
mediated degradation[26a] and also in human serum, relative to
unmodified controls.[26b] In this work, we have aimed to verify the
thus anticipated stability of gapmers
1 and 2 in the
aforementioned biological media. Analysis by urea-PAGE
demonstrated that both gapmers 1 and 2 show excellent stability
against cleavage in HP (Figure 4A) as well as in WCL (Figure
4B) over a time course of eight hours, while an unmodified DNA-
ON of the same sequence was completely cleaved.
product. In contrast, single-stranded RNA without
a
complementary counterstrand remained stable against RNase H
over the observed timeframe. Interestingly, NAA/LNA-gapmer 1
also induced RNase H-mediated cleavage of the target RNA,
although no uniform phosphate diester backbone was present in
its structure. However, the rate of degradation appeared to be
significantly slower than for reference gapmer 2.
Duplex
formation
of
NAA/LNA-gapmer
1
with
complementary RNA results in moderate activation of
RNase H
We therefore performed a second set of experiments to
observe RNase H-mediated degradation over an extended time
period (Figure 5B). After 24 h, degradation of the labelled RNA
was clearly detectable in the assay mixture containing
NAA/LNA-gapmer 1, yet nearly the exact same outcome was
observed without the presence of 1. To rule out general
instability of the chosen RNA sequence over such an extended
time period, another control experiment was included: 32P-
In order to determine whether NAA/LNA-gapmer 1 is capable of
triggering RNase H-mediated cleavage of complementary RNA,
an assay employing a 5'-32P-labelled RNA strand was used.[27]
Duplexes of native DNA as well as of DNA/LNA-gapmer 2,
respectively, with this RNA strand served as controls, with the
DNA-RNA heteroduplex representing a positive control, i.e. a
system furnishing RNase H-catalysed RNA degradation.
6
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10.1002/cbic.202000450
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Figure 5. A. RNase H-mediated degradation of 32P-labelled RNA upon hybridisation with 1, 2, native DNA, and without any counterstrand over 60 min (urea-
PAGE). B. RNase H-mediated degradation of 32P-labelled RNA with 1, without any counterstrand, and without both counterstrand and RNase H over 24 h (urea-
PAGE). x: heteroduplex of 2 and RNA, a: intact RNA, b: RNA degradation products.
labelled RNA was incubated over the full time period (i.e. up to
24 h) in the absence of both a counterstrand and RNase H.
However, no RNA cleavage was detected under these
conditions (Figure 5B). It has to be noted that the degradation
signals observed for the duplex of 1 and 32P-labelled RNA after
30 and 60 min (Figure 5A) could not be visualised in the
prolonged incubation experiment (Figure 5B) due to differences
in signal intensity.
Furthermore, gapmer 1 was shown to be sensitive towards
single base mismatches in the counterstrand at 37 °C.
Experiments with mismatched RNA X resulted in only 50%
formation of the 1-RNA duplex at this temperature. With a
different mismatched RNA Y, 14% single-stranded gapmer 1
was determined (cf. Table 1). Interestingly, this selectivity was
not observed with the DNA/LNA-gapmer 2 (i.e. the polyanionic
reference ON) that showed no sensitivity towards base
mismatches in the RNA counterstrand at 37 °C. The same was
true for unmodified DNA. Binding to RNA off-targets with
sequences similar to the actual target mRNA can lead to
potential side effects in the pharmaceutical application of
antisense ON.[29] It is therefore of great relevance that
backbone-modified ON structures show some sequence
selectivity in their hybridisation properties under physiologically
relevant conditions (i.e. at 37 °C). With respect to this
consideration, the hybridisation properties of NAA/LNA-gapmer
1 are superior relative to both the reference gapmer 2 and to
native DNA, even though the presence of the NAA-modification
furnished a decrease of thermal duplex stability. Furthermore,
these results give rise to the general question if Tm values might
be overrated in the evaluation of hybridisation properties of
backbone-modified ON with respect to their potential application
as antisense agents. We herein present an alternative
parameter for such an evaluation: the target selectivity of the
investigated ON (with a suitable length for an antisense agent)
at 37 °C. This appears to be superior to a 'the more stable the
better' approach for studying the physicochemical hybridisation
properties with RNA counterstrands. This hypothesis is also
supported by recent findings by Dieckmann et al. who could
demonstrate a direct correlation of high Tm values and the
hepatotoxic potential of high-affinity ON due to enhanced off-
target effects.[11b] Of course, such an improved target selectivity
due to decreased duplex stability can in principle also be
Discussion
In previous studies,[12a,19] we had reported the generally
favourable properties of ON with partially zwitterionic NAA-
modified backbone structures, i.e. their formation of stable,
helical duplexes with complementary DNA, their retention of
base-pairing fidelity and their high stability in biological media.
However, duplex formation with RNA had been observed to be
moderately hampered, i.e. decreased thermal stabilities (up
to -4.0 °C/NAA-modification[12a]) had been observed. Thus, we
have now aspired to modify such zwitterionic NAA-ON in a way
that furnishes satisfactory binding affinity towards RNA without
compromising base-pairing fidelity, hence potentially enabling an
efficient and selective target engagement with endogenous RNA.
These considerations have led to the design of NAA/LNA-
gapmer 1, in which strongly RNA-binding LNA segments were
combined with a zwitterionic NAA-modified gap unit.
Following the successful synthesis of 1 (and an according
DNA/LNA-gapmer 2 as a reference with a uniformly anionic
backbone), UV-monitored thermal melting studies revealed an
improved binding affinity to complementary RNA, relative to
previously studied NAA-modified ON (formally -1.5 °C/NAA-
modification due to the stabilising effect of the LNA units). As a
result, almost quantitative formation of the 1-RNA duplex at
physiologically relevant 37 °C could be demonstrated.
achieved by alternative means, e.g. designing
a shorter
7
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10.1002/cbic.202000450
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sequence of the antisense ON or a lower number of LNA units.
However, we herein demonstrate that the NAA-modification is a
useful addition to the 'toolbox' of ON modifications to achieve
this goal.
approach furnished
characteristics: gapmer
a
zwitterionic ON
1 with optimised
1
showed superior hybridisation
properties with RNA at physiologically relevant 37 °C as well as
excellent stability in biological media. In a cellular setting, a
gapmer of type 1 might then mainly exert biological activity via a
'steric block' mechanism, even though 1 apparently activated
RNase H-mediated RNA degradation on a very moderate level.
Overall, the NAA/LNA-gapmer approach is thus established as a
strategy to design partially zwitterionic ON for the future
development of novel antisense agents with a reduced overall
charge in the backbone. This is anticipated to furnish improved
pharmacokinetic properties of according ON-based drug
candidates. Based on the favourable characteristics of 1, the
stage is now set for the development of synthetic methodology
for the efficient preparation of zwitterionic gapmers of type 1 with
biologically relevant base sequences. This will enable future
studies on their antisense efficacy in cellulo, with the goal to
establish zwitterionic backbone architectures of oligonucleotide
analogues as a viable strategy for the development of novel
antisense agents.
CD spectroscopic analyses confirmed helical topologies for
the gapmers
1
and 2, respectively, in complex with
complementary DNA or RNA, with the resultant helical
structures being similar to a DNA-RNA heteroduplex in all cases.
This uniform topological preference was obviously caused by the
presence of the LNA segments in the gapmer sequences.[30]
Notably, the partially zwitterionic backbone structure of 1 had no
distorting influence on the topologies of its duplexes formed with
either DNA or RNA counterstrands. In addition, high stability
against degradation of the gapmers in human plasma and whole
cell lysate was observed.
The activation of RNase H generally represents a desirable
(though not essential feature) of antisense ON, and the
NAA/LNA-gapmer 1 was therefore investigated with respect to
this property. In a first set of experiments, the RNase H-
mediated cleavage of
a
complementary 32P-labelled RNA
counterstrand was studied over a period of 60 min. Within this
time, the two reference ON (i.e. gapmer 2 and unmodified DNA)
both induced rapid degradation of the target RNA by RNase H.
Surprisingly, some degradation of the labelled RNA (albeit rather
slowly) was also detected in presence of NAA/LNA-gapmer 1,
although 1 possesses no uniform phosphate diester backbone
(cf. Figure 5). This suggested a very moderate activation of
RNase H due to the presence of 1 in the assay mixture. In order
to further study this effect, the assay was then performed again
with a significantly extended incubation period (up to 24 h). Even
though this led to stronger signals for the RNA degradation
products with gapmer 1, this result could not solely be linked to
the influence of the partially zwitterionic gapmer: the same
experimental setup with the absence of 1 resulted in a rather
Experimental Section
Synthesis of oligonucleotides: Based on our previous syntheses of
NAA-modified ON,[12] gapmer ON 1 and 2 were assembled by automated
solid phase-supported DNA synthesis. This required the chemical
synthesis of 'dimeric' NAA-linked phosphoramidite 14 (see Supporting
Information for detailed synthetic procedures). Unmodified DNA
phosphoramidites (Glen Research) and LNA phosphoramidites
(QIAGEN) were commercially purchased. After completion of ON
synthesis and basic workup under standard conditions, purification of 1
and 2 was achieved by urea polyacrylamide gel electrophoresis (urea-
PAGE, Figure S1, see Supporting Information for detailed procedures).
The identities of both gapmers 1 and 2 were confirmed by high resolution
mass spectrometry (Table S1, Figures S2 and S3, Supporting
Information). All other oligonucleotides were commercially purchased
(Sigma-Aldrich).
similar outcome. To exclude
a general instability of the
radiolabelled RNA strand, an additional control containing only
the target RNA without RNase H was also incubated for 24 h,
but no degradation was recorded. This demonstrated that the
employed preparation of RNase H displayed some sort of
unspecific nuclease activity. Overall, it was concluded that the
aforementioned very moderate activation of RNase H by gapmer
1 became indistinguishable from background reactions over
longer incubation periods. This suggests that an NAA/LNA-
gapmer of type 1 might exert a potential antisense effect by
'steric block' (i.e. binding to endogenous RNA) rather than by
'catalytic' RNA degradation, as the latter would probably require
a more efficient activation of RNase H. It should also be noted
that this part of the reported work generally proves the high
relevance of a rigorous set of control experiments in RNase H
assays. Otherwise, it is unclear if the described alleged
activation of RNase H was genuine or actually resulted (at least
partially) from unspecific nuclease activity. This actually might be
the case for several reports on 'RNAse H activation' by modified
ON structures in the literature.
Melting temperature experiments: To determine the binding affinity of
the NAA/LNA-gapmer
1
towards complementary DNA (5'-
AATCTAGAGAGAGATCT-3') and RNA (5'-AAUCUAGAGAGAGAUCU-
3'), a 1 M solution of the gapmer/counterstrand duplex in phosphate
buffer (10 mM NaH2PO4, pH 7.4 with 100 mM NaCl) was prepared. Prior
to measurements, the solution was heated to 90 °C and subsequently
cooled down to rt to enable duplex formation. Then, three heating and
cooling cycles ranging from 25 °C to 90 °C with a heating rate of
0.7 °C/min were performed and changes in the absorption at  = 260 nm
were recorded using
a Cary 100 UV-Vis spectrometer (Agilent
Technologies). Hyperchromicity was plotted against temperature to
obtain melting curves, and Tm values were calculated as the maximum of
the first derivative. To eliminate the temperature dependency of the
extinction coefficient , Tm values were calculated and used to describe
the melting temperature of all studied duplexes. Furthermore, T37°C
values were calculated to investigate the hybridisation at
a
physiologically relevant temperature of 37 °C. Details on the method for
the compilation of the -curves are provided in the Supporting
Information (Figures S4 to S6). A similar protocol was used for studies on
mismatch sensitivity. Therefore, base mismatches were introduced in the
RNA counterstrand opposite of either one of the LNA segment (5'-
AAUCUAGAGAGAGACCU-3', mismatched RNA-ON X) or the NAA gap
(5'-AAUCUAGAGGGAGAUCU-3', mismatched RNA-ON Y, mismatches
are highlighted in bold and are underlined in the sequences, also see
Figure S7, Supporting Information). To obtain reference Tm values,
according melting temperature experiments were conducted with
Conclusion
In summary, we report the synthesis and properties of a new
type of gapmer oligonucleotide architecture featuring a partially
zwitterionic backbone structure. This NAA/LNA-gapmer
8
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DNA/LNA-gapmer 2 and a native DNA-ON with the same sequence (5'-
AGATCTCTCTCTAGATT-3').
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CD-spectroscopic analysis of duplex structures: Circular dichroism
(CD) spectra of duplexes containing the NAA/LNA-gapmer 1 were
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at 25 °C in a cuvette with a length of 1 cm and a wavelength range of
200-320 nm. Every sample was scanned 10 times with a scanning speed
of 200 nm/min, a bandwidth of 5 nm, response time of 2 s and a data
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 against the recording wavelength . Reference spectra were recorded
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2 or a native DNA-ON with the same sequence (5'-
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Stability assays in biological media: The stabilities of both gapmer ON
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H
assays: An RNA-ON with
a
sequence (5'-
AAUCUAGAGAGAGAUCU-3') complementary to the gapmer sequence
was radiolabelled at the 5'-end using -32P-ATP (Hartmann Analytics,
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Acknowledgements
We thank the Deutsche Forschungsgemeinschaft (DFG, grant
DU 1095/2-1), the Fonds der Chemischen Industrie (FCI,
Sachkostenzuschuss) and the Studienstiftung des deutschen
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Keywords: oligonucleotides • antisense • backbone modification
• zwitterions • gapmers
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10
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Entry for the Table of Contents
One approach towards backbone-modified oligonucleotides is the introduction of (partially) zwitterionic structural motifs. In this work,
we report a 'gapmer' architecture comprised of a zwitterionic central 'gap' (with cationic NAA linkages) and two outer segments of
locked nucleic acid (LNA). This NAA/LNA-gapmer approach furnished a partially zwitterionic oligonucleotide with promising properties
for potential biomedical applications.
Institute and/or researcher Twitter usernames: @ducholab
11
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