Chondroitin Sulfate-Coated Heteroduplex-Molecular
Spherical Nucleic Acids
Toni Laine,[a] Prasannakumar Deshpande,[b] Ville Tähtinen,[a] Eleanor T. Coffey,[b] and
Pasi Virta*[a]
Molecular Spherical Nucleic Acids (MSNAs) are atomically uni-
form dendritic nanostructures and potential delivery vehicles
for oligonucleotides. The radial formulation combined with
covalent conjugation may hide the oligonucleotide content and
simultaneously enhance the role of appropriate conjugate
groups on the outer sphere. The conjugate halo may be
modulated to affect the delivery properties of the MSNAs. In
the present study, [60]fullerene-based molecular spherical
nucleic acids, consisting of a 2’-deoxyribonucleotide and a
ribonucleotide sequence, were used as hybridization-mediated
carriers (“DNA and RNA-carriers”) for an antisense oligonucleo-
tide, suppressing Tau protein, (i. e. Tau-ASO) and its conjugates
with chondroitin sulfate tetrasaccharides (CS) with different
sulfation patterns. The impact of the MSNA carriers, CS-moieties
on the conjugates and the CS-decorations on the MSNAs on
cellular uptake and - activity (Tau-suppression) of the Tau-ASO
was studied with hippocampal neurons in vitro. The formation
and stability of these heteroduplex ASO-MSNAs were evaluated
by UV melting profile analysis, polyacrylamide gel electro-
phoresis (PAGE), dynamic light scattering (DLS) and size
exclusion chromatography equipped with a multi angle light
scattering detector (SEC-MALS). The cellular uptake and -
activity were studied by confocal microscopy and Western blot
analysis, respectively.
Introduction
Spherical nucleic acids (SNAs, introduced by Mirkin’s group) are
nanostructural delivery vehicles, consisting of a dense shell of
oligonucleotides attached to a suitable core unit.[1,2] The core
structure can be for example gold,[1,3] silica,[4] liposomes[5] or
proteins.[6] SNAs may undergo class A scavenger receptor–
mediated endocytosis,[7,8] which has been demonstrated by the
successful free uptake of antisense oligonucleotides and
siRNAs[9] in more than 50 different cell lines.[3,10] In addition,
atomically uniform variants of SNAs, i. e. molecular spherical
nucleic acids (MSNAs)[11–18] and other dendritic
oligonucleotides[19] have received growing interest as delivery
vehicles. The lower oligonucleotide density of MSNAs results in
weaker activation of scavenger receptors,[11,20] but MSNAs may
become attractive delivery vehicles when they are conjugated
with cell/tissue-specific ligands. The radial formulation con-
nected to covalent conjugation may hide the oligonucleotide
content and simultaneously enhance the role of appropriate
conjugate groups on the outer surface, which may be tuned to
find enhanced precision delivery for oligonucleotides. Indeed,
our recent in vivo PET-imaging study[15] shows that i. v.-adminis-
trated MSNAs exhibit prolonged circulation times due to
prevented accumulation in the kidney, and liver, in comparison
to linear phosphorothioate oligonucleotides[21,22] and most
nanoparticular delivery vehicles,[23,24] respectively. The biodistri-
bution and targeted delivery could be further adjusted by the
surface decoration (folate[25] and trastuzumab[12] demonstrated)
of MSNAs.
In the present study, [60]fullerene-based MSNAs, consisting
of 2’-deoxy oligoribonucleotide (MSNA1) or oligoribonucleo-
tide-strands (MSNA2), were synthesized and used as hybrid-
ization-mediated carriers (“DNA- and RNA-carriers”) for an
antisense oligonucleotide sequence, suppressing Tau protein,
(i. e. Tau-ASO, cf. ON5–8) (Figure 1). Tau is a protein expressed
in neurons whose hyperphosphorylation and aggregation are
important factors in neurodegeneration caused by several
diseases, including Alzheimer’s disease.[26,27] Regulation of
pathological Tau can prevent symptoms of Alzheimer’s disease
and restore lost function.[28–30] Antisense oligonucleotides
(ASOs) targeting Tau protein have been developed,[29,31,32]
including positive phase I trials in humans with mild Alzheimer’s
disease.[31] Heteroduplex[33,34] Tau-ASOs are assembled on the
MSNAs (MSNA3–10). On the DNA-MSNA carrier (MSNA1), the
release of the active Tau-ASO (consisted of phosphorotioate
backbone) can undergo passive dissociation or DNase-mediated
degradation of the carrier (consisted of native phosphodiester
backbone). On the RNA-MSNA (MSNA2), in turn, RNase H-
mediated degradation of the RNA-based carrier may take place,
releasing the active Tau-ASO-sequence. The impact of the
MSNA carrier and its chemistry (DNA vs RNA, MSNA3 vs
MSNA7) on hippocampal neuron uptake and activity to
[a] T. Laine, Dr. V. Tähtinen, Prof. P. Virta
Department of Chemistry
University of Turku
20500 Turku, Finland
E-mail: pamavi@utu.fi
[b] Dr. P. Deshpande, Prof. E. T. Coffey
Turku Bioscience Centre
University of Turku, Åbo Akademi University
20520 Turku, Finland
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cbic.202400908
© 2024 The Author(s). ChemBioChem published by Wiley-VCH GmbH. This is
an open access article under the terms of the Creative Commons Attribution
License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
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suppress Tau protein were studied in vitro by confocal micro-
scopy and Western blot analysis, respectively. In addition to
MSNA formulation, the role of chondroitin sulfate tetrasacchar-
ides with different sulfation patterns as surface decoration of
MSNAs (MSNA4–6 and MSNA8–10) to the hippocampal neuron
uptake was examined. The glycosaminoglycan chondroitin
sulfate (CS) is a linear polysaccharide important for several
biological processes including cell division, cancer progression
and metastasis, development of the central nervous system and
central nervous system injuries and diseases.[35–37] The specific
sulfation patterns determine the binding of CS to certain
proteins such as neurotrophins, selectins, chemokines and
midkine.[38–41] The ability to interact with certain proteins makes
CS a potential tool for drug delivery. In addition, CS proteogly-
cans have been found to bind to several neuronal
receptors.[42,43] Previously, it has been shown that coating gold
nanoparticles with specific chondroitin sulfates can enhance
their targeting and uptake in neuronal cells.[44] MSNAs are
particularly attractive scaffolds as they can present multiples of
each ligand on their outer surface. This is especially useful with
weakly binding ligands whose binding is multivalent. The
binding of CS to some proteins has been found to be
multivalent as well.[45–47] This has prompted to research on
synthetic analogues, such as polymers[48,49] and
dendrimers,[46,50–52] that present CS-ligands on their outer surface
in an attempt to mimic natural CS polysaccharides.
Results and Discussion
Synthesis of CS-Oligonucleotide Conjugates
The synthesis of the Alexa Fluor (AF488)-labeled CS-oligonu-
cleotide conjugates is outlined in Scheme 1. 5’-(1R,8S,9 s)-
Bicyclo[6.1.0]non-4-yn-9-yl (BCN) modified phosphorothioate
oligonucleotide ON1 was synthesized on a 1 μmol scale on a
commercial amino-modifier solid support using an automatic
DNA/RNA synthesizer and commercially available phosphorami-
dite building blocks. ON1 was conjugated with azidopropyl-
modified chondroitin sulfate (CS) tetrasaccharides[53] 1–3 by
strain-promoted alkyne-azide cycloaddition (SPAAC): ON1 was
incubated with compounds 1–3 (3 equiv; following previously
published procedure[53]) overnight at room temperature. The
resulting CS-oligonucleotide conjugates (ON2–4) were homo-
genized by RP-HPLC and authenticity of the products was
verified by MS (ESI-TOF) spectroscopy (Figures S1–S3 and
Table S1). Isolated yields of ON2–4 varied between 18 and 22%.
Oligonucleotides ON1–4 were labelled with an AF488 fluores-
cent dye: AF488 NHS ester (in DMSO) was added into a solution
of the oligonucleotide (in aqueous borate buffer, pH 8.4) and
the reaction mixture was incubated overnight at room temper-
ature. The resulting AF488-labeled oligonucleotide conjugates
(ON5–8) were purified by RP-HPLC and authenticity of the
products was confirmed by MS (orbitrap) spectroscopy (Figur-
es S4–S7 and Table S1). Isolated yields of ON5–8 varied
between 85% and 91%.
Figure 1. Illustration of the MSNA structures in the study. MSNA1 and MSNA2 are hybridized with ON5–8 to yield MSNA3–10 (cf. Scheme 1). Filled sugar
symbols refer to sulfated N-acetylgalactosamine units.
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Synthesis of MSNAs
[60]fullerene has been shown to be a valuable precursor for
MSNAs. Its reaction with bis(azidoalkyl) malonates via Bingel’s
cyclopropanation[54] offers a radially symmetric 12-valent poly-
azide core (4) that can be conjugated with oligonucleotides
using SPAAC to yield MSNAs.[11] The density of oligonucleotides
on 4 has been shown to be sufficient to activate scavenger A
receptors and facilitate cellular uptake to breast cancer cells
(MCF7), and down regulation of human epidermal growth
factor receptor 2 (HER2) in ovarian cell line (SKOV3).[11] Recently,
we studied biodistribution properties of the [60]fullerene-
derived MSNAs in vivo,[15] and demonstrated their potential
in vitro as antibody-conjugated[12] and glyco-coated drug deliv-
ery vehicles.[14] Bingel’s cyclopropanation with heteroalkyl
malonates offers heterofunctional [60]fullerene-derived core
units, which can be used for the synthesis miktoarm
MSNAs.[13,17,18] These MSNAs have been applied as scaffolds for
abiotic intracellular catalysts[17] and enzyme-resistant
nanoflares,[18] and as more sophisticated gene regulation
agents.[18] Due to the encouraging previous results, 12-valent
polyazide core (4) was used for the synthesis of MSNAs in the
present study (Scheme 2): 5’-BCN-modified 2’-deoxy oligoribo-
nucleotide ON9 and oligoribonucleotide ON10 were synthe-
sized on a 1 μmol scale using an automatic DNA/RNA synthe-
sizer and commercially available phosphoramidite building
blocks. ON9 and ON10 were attached to azide-modified
[60]fullerene core 4 using SPAAC, following the procedure
reported previously by our group.[16] Because of solubility issues,
the C60 core (4) was first conjugated with a sub-stoichiometric
amount (0.3 equivalents) of ON9 or ON10 in a mixture of DMSO
and water. Thereafter, the obtained intermediate product
(Scheme S1, Figures S8 and S9, Table 1) was treated with an
excess of the same oligonucleotide in aqueous solution and in
high salt concentration. This two-step process alleviated the
solubility issues and produced more homogenous MSNAs in
23% (MSNA1) and 14% (MSNA2) overall isolated yields (over
two steps), purified by RP HPLC (Figure S10). Homogeneity,
authenticity and hydrodynamic size of the MSNAs were verified
by polyacrylamide gel electrophoresis (PAGE), size exclusion
chromatography equipped with a multi angle light scattering
detector (SEC-MALS) and dynamic light scattering (DLS),
respectively (cf. Figure 2, Figure 3 and Table 2).
Hybridization with Complementary MSNA
The hybridization of the oligonucleotide conjugates ON5–8
with MSNA1 and MSNA2 was verified by UV thermal melting
temperature analysis and PAGE. The melting temperature
analysis was performed in 10 mmolL 1 sodium cacodylate
buffer (pH 7.0) with 0.1 molL 1 NaCl using 0.083 μmolL 1 the
MSNA and 12 equivalents (1.0 μmolL 1) of the oligonucleotide.
UV melting profiles of the oligonucleotide/MSNA1 complexes
Scheme 1. A) Structures of the azidopropyl-modified CS tetrasaccharides.[53]
B) Synthesis of AF488-labeled oligonucleotide conjugates ON5–8. The
oligonucleotides are consisted of phosphorothioate backbone. Underlined
letters represent 2’-O-(2-methoxyethyl) ribonucleotides, the rest are 2’-
deoxyribonucleotides. Underlined C is 5-methylated. Reagents and con-
ditions: (i) 3 equivalents of azidopropyl-modified CS tetrasaccharides 1–3,
H2O, overnight at room temperature. (ii) 20 equivalents of AF488 NHS ester,
0.1 M sodium borate (pH 8.4), H2O:DMSO (9 :1, v/v), overnight at room
temperature
Table 1. UV-Thermal melting temperatures of the MSNAs.
Tm/°C
MSNA3 63.7�0.3
MSNA4 63.7�0.3 ( 0.2)
MSNA5 63.6�0.7 ( 0.1)
MSNA6 63.3�0.4 ( 0.4)
MSNA7 73.5�0.2
MSNA8 73.6�0.6 (+0.1)
MSNA9 74.2�0.4 (+0.7)
MSNA10 73.0�0.5 ( 0.5)
ΔTm values in parentheses are compared to either MSNA3 (MSNA4–6) or
to MSNA7 (MSNA8–10). For conditions, see experimental section.
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(MSNA3–6) showed inflection points at 63–64 °C and of the
oligonucleotide/MSNA2 complexes (MSNA7–10) at 73–74 °C.
The heteroduplexes on the RNA carrier (MSNA2) were ca ΔTm=
10 °C more stable than the heteroduplexes on the DNA-carrier
(MSNA1). In each case the melting temperatures were high
enough to convince the stability of the complexes at physio-
logical temperature (Table 1). The impact of the CS moiety on
the thermal stability of the oligonucleotide/MSNA complexes
was marginal (jΔTm j �0.7 °C), as seen by comparing the
melting temperatures of MSNA4–6 (Tm=63.3–63.7 °C) and
MSNA8–10 (Tm=73.0–73.6 °C) to those of MSNA3 (Tm=63.7 °C)
and MSNA7 (Tm=73.5 °C), respectively.
PAGE also confirmed the hybridization (Figure 2). To allow
the hybridization, the samples were prepared in phosphate-
Scheme 2. Synthesis and hybridization-mediated assembly of MSNAs. Letters in bold represent ribonucleotides Conditions: i) ON9 or ON10 (0.3 equivalents/4),
DMSO:H2O (9 :1, v/v), overnight at room temperature; ii) ON9 or ON10 (1.2 or 1.5 equivalents / azide arm of 4), 1.5 M or 0.75 M NaCl (aq), 3 days at room
temperature.
Figure 2. PAGE analysis of MSNAs. The ladder is only a reference and not
suitable for determining the size of MSNAs. For conditions, see experimental
section. (Note: With the given conditions, the excess oligonucleotide is
eluted outside of the gel electropherogram)
Figure 3. SEC-MALS analysis of MSNAs. For conditions, see experimental
section
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buffered saline (PBS), pH 7.4, before loading on the gel. MSNA1
and MSNA2 alone showed a distinct and relatively sharp band
on gel. In both cases, a faster eluting byproduct, most probably
an 11-armed MSNA,[16] was also detected. Nevertheless, MSNA1
and MSNA2 proved relatively pure after single RP-HPLC
purification (Figure S9). Hybridization of the MSNAs with ON5–8
gave rise to a slower eluting and broad band on the gel,
referring to formation of hybridization complexes MSNA3–10.
Some even slower moving bands, likely caused by aggregation,
were also observed. The bands of the hybridization complexes
remained unchanged even if higher excess of oligonucleotides
were used for the hybridization. A slight contribution of the CS
decoration to the elution of the complexes could be seen.
Further characterization of MSNAs was performed by SEC-
MALS and DLS. Representative SEC-MALS profiles (MSNA1,
MSNA2 and MSNA4 and MSNA8) are shown in Figure 3. Each
chromatogram shows a faster eluting peak, which refers to
partial aggregation of the MSNAs that was also observed on
PAGE (slower eluting bands). Molecular weights (MW) of the
MSNAs, extracted from MALS of the major fraction matched
well with the calculated ones, including also masses for the
hybridization complexes (an average refractive index increment,
dn/dc of 0.1703 mLg 1 used). In fact, the MALS-based molecular
weight was more accurate for the hybridization complexes than
for the carrier MSNAs: MSNA1: MWfound 79.4�0.5 kDa (MWcalcd
84.4 kDa), MSNA2: MWfound 81.9�0.5 kDa (MWcalcd 83.0 kDa),
MSNA4: MWfound 187.5�2.3 kDa (MWcalcd 194.4 kDa), MSNA8:
MWfound 193.6�1.6 kDa (MWcalcd 193.1 kDa). As seen, the differ-
ence between the observed and calculated molecular weights
was less than the mass of a single oligonucleotide, verifying
complete decoration and hybridization of the MSNAs. For the
DLS measurements, samples were prepared in PBS (pH 7.4) and
hydrodynamic size of the MSNAs was determined. For the
carriers: MSNA1 and MSNA2, hydrodynamic size of 7.8 and
7.0 nm were observed. As expected, the hybridization with
ON5–8 increased the size. For MSNA3–6: 12.4–13.2 nm and for
MSNA7–10: 12.2–12.9 nm were observed.
Cellular Uptake and Intracellular Activity of the MSNAs
Hippocampal neurons were prepared from rats and incubated
for 10 days in culture with 120 nM solutions of anti-Tau-
oligonucleotides (ON5–ON8) and 10 nM solutions of the
corresponding MSNA-heteroduplexes (MSNA3–10). Thus, the
effective Tau-ASO concentration in the experiments was the
same (i. e. 120 nM). The obtained uptake data (Figure 4C) is
comparable between the oligonucleotide conjugates (ON5–
ON8) and between the hybridization complexes, i. e. MSNA3–6
and MSNA7–10, but comparison between the conjugates and
MSNAs can be confounded by different dissociation/degrada-
tion rates and fluorescence quenching on the MSNAs. The
signal intensity refers to intact molecules, in which fluorescence
quenching (Figure S11) on MSNAs has been considered. By
examining the oligonucleotides (ON5–ON8) themselves, it
could be seen that the CS moiety and its sulfation pattern had
impact on the uptake. The CS-tetrasaccharide with R1=H and
R2=SO3
 (ON6, cf. Scheme 1) increased the uptake, whereas the
other two CS variants (ON7 and ON8, cf. Scheme 1) decreased
the uptake, compared to ON5. The deep understanding how
different CS structures affect the uptake needs further studies.
However, the sulfate groups more on the outer sphere (ON7
and ON8 vs ON6) seemed to have a negative effect on the
uptake, which may suggest to shift the interest more towards
hyaluronic-like structures (i. e. non-sulfated glycans). The DNA-
MSNA-carrier increased the uptake (MSNA3), whereas the
uptake by RNA-MSNA carrier (MSNA7) remained at a similar
level compared to ON5. However, the aforementioned differ-
ence between the concentrations (10 nM vs. 120 nM) of the
complexes and of the oligonucleotides, as well as the potential
interference in fluorescence intensity, should be considered in
this evaluation. While the CS-conjugate group could show some
beneficial effect on the uptake of oligonucleotides (ON6 vs
ON5), similar effect could not be found on CS-coated MSNAs
(MSNA4–6 and MSNA8–10). However, the uptakes of CS-
decorated MSNA4–6 followed the same trend as observed with
Tau-ASO-conjugates ON6-ON8. The effect of CS-decoration on
the uptake remained modest, considering previous findings
with neurons and CS-coated gold nanoparticles,[44] but may still
encourage evaluation of other CS structures as decorates of
MSNAs. The CS decoration studied in the present study
probably interferes the scavenger receptor A-mediated cellular
uptake of the MSNAs, which cannot be compensated by
beneficial CS-ligand-driven effect on the uptake, if any with
hippocampal neurons (cf. ON6 vs. ON5). After the uptake
experiments, the treated neurons were lyzed and introduced to
Western blot assay. Total Tau level was compared to actine and
normalized to untreated cells (Figures 4D and E). A marked Tau
suppression could be observed in cells treated with ON5,
MSNA3 (i. e. ON5 on the DNA-MSNA carrier) and MSNA7 (i. e.
ON5 on the RNA-MSNA carrier). While the most effective Tau-
suppression could be observed with MSNA7, no clear influence
caused by the chemistry of MSNAs (i. e. “DNA vs RNA carrier”)
was observed. These results are positive and may encourage for
further studies evaluating the concept of heteroduplex
ASOs[33,34] on MSNAs. The CS conjugate group on ON6–8 (and
Table 2. Hydrodynamic size of the MSNAs.
hydrodynamic size /nm
MSNA1 7.8�0.5
MSNA3 13.2�1.9
MSNA4 12.7�0.7
MSNA5 12.4�1.2
MSNA6 12.4�1.5
MSNA2 7.0�1.2
MSNA7 12.9�1.8
MSNA8 12.2�0.4
MSNA9 12.7�0.8
MSNA10 12.5�0.8
Conditions: 0.2 μmolL 1 MSNA+12 equivalents (2.4 μmolL 1) of the
oligonucleotides (ON5–8) dissolved in 100 μL of PBS.
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CS-decoration on MSNA4–6 and MSNA8–10) interfered with
Tau suppression. This was somewhat expected within these
heavily-modified demo Tau-ASO-sequences, the primary goal of
which was to evaluate the impact of CS-decoration on the
cellular uptake of MSNAs. Modifications at both terminus of the
ASO are likely to interfere RNase-H-mediated cleavage of the
mRNA target.
Enzymatic Stability of MSNAs
To verify the enzyme-mediated cleavage of MSNAs, they were
incubated with DNase I and RNase H, and the potential
degradation of the MSNAs and simultaneous release of the Tau-
ASOs were monitored by PAGE (Figure S12). MSNAs3–6 (on the
DNA carrier, MSNA1) underwent rapid and complete DNase 1-
catalyzed degradation (1 U DNase I/nmol of the effective
oligonucleotide content, 6 h at 37 °C), whereas MSNAs7–10 (on
the RNA carrier, MSNA2) remained mainly intact even by a
prolonged incubation in the same conditions (10 U DNase I/
nmol of the effective oligonucleotide content, 3 d at 37 °C).
MSNAs7–10, fulfilling better the concept of heteroduplex ASOs,
in turn, proved substrates for RNase H (10 U RNase H/nmol of
the effective oligonucleotide content, 3 d at 37 °C). However,
the degradation of the RNA-carrier (MSNA2) proved relatively
slow (c.a. 60% degradation of MSNA7 after 3 d) and it seemed
to be interfered on CS-decorated MSNAs8–10. The data
obtained by these enzyme experiments were partly in line with
the Western blot assay above. RNase H-activity of the Tau
mRNA-ASO-complex is likely to be the limiting step for the
down regulation of Tau protein, which can be interfered by CS-
moieties on ON6-ON8. In each case, the enzyme-catalyzed
(DNase I or RNase H) degradation of the carrier (MSNA1 or
MSNA2) released intact Tau-ASOs (ON5-ON8) as monitored by
PAGE (Figure S12).
Conclusions
The applicability of [60]fullerene-based molecular spherical
nucleic acids (MSNAs) as hybridization-mediated carriers (DNA-
and RNA-based carriers, MSNA1 and MSNA2) for an antisense
oligonucleotide, suppressing Tau protein, (i. e. Tau-ASO) and its
conjugates with chondroitin sulfate tetrasaccharides (CS) with
different sulfation patterns (three different variants) was dem-
onstrated. For the characterization of the obtained heterodu-
Figure 4. Uptake of ON5–8 and MSNA3–10 in neurons. A–B) Maximum intensity projections of five Z-sections of 0.4 μm each through nuclei of hippocampal
neurons treated at 7 days in culture with 120 nM ON5–8 and 10 nM MSNA3–10 for 10 days are shown (the effective ASO concentration of 120 nM in each
case). Signal for the conjugates is shown in cyan while nuclei are highlighted in magenta. Scale=10 μm. C) Mean somatic intensity of AF488 signal relative to
ON5 from 3–6 cells + / S.E.M for each conjugate is shown. The signal intensity refers to intact molecules (fluorescence quenching on MSNAs considered). D)
Total Tau levels relative to actin and normalized to untreated cells analyzed by western blotting of Hippocampal neurons treated with indicated ASO
conjugates and MSNAs as in A–B. Mean data + / S.E.M from 3 repeats is shown. Significance was determined using Student’s t-test. *p<0.05; **p<0.01;
***p<0.001. (E) Representative images for treatment in D are shown.
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plex Tau-ASO-MSNAs, PAGE, SEC-MALS, UV-melting profile
analysis and DLS were applied. SEC-MALS proved a valuable
tool, which offered molecular mass analysis for the hybrid-
ization-mediated complexes of ca. 190 kDa. The hippocampal
neuron uptake and activity suppressing Tau protein of the CS-
Tau-ASO-conjugates (ON6–ON7) and of the corresponding
heteroduplex MSNAs (MSNA4–6 and MSNA8–10) were studied
in vitro by confocal microscopy and Western blot analysis,
respectively. The impact of the MSNA carriers, CS-moieties on
the conjugates and the CS-decorations on the MSNAs were
evaluated. One of the CS-Tau-ASO-conjugates enhanced the
uptake to neuron cells (ON6), as did the DNA-based hetero-
duplex MSNA carrier (MSNA3), but the other two CS-ASO-
conjugates and MSNA-formulations of the conjugates had
negative or marginal effect on the uptake. These results, while
modest, may still prompt to evaluate other CS halos on the
MSNAs. Previous findings with CS-coated nanoparticles have
been encouraging, and imply that the neuronal uptake is
sensitive to the CS structure.[44] A significant suppression of Tau
protein could be observed by Western blot assay in neuron cells
treated with Tau-ASO without CS-conjugation (ON5) and its
heteroduplexes on DNA- and RNA-based MSNAs (MSNA3 and
MSNA7). The suppression of Tau protein was interfered by CS
conjugation on oligonucleotides (ON6–ON8) and on the
corresponding MSNAs (MSNAs4–6 and MSNAs8–10). This
finding was supported by enzymatic stability tests (DNase I and
RNase H) of MSNAs. Accordingly, RNase H-activity of the Tau
mRNA-ASO-complex may be the limiting step of the down
regulation, which was interfered by CS-moieties on ON6–ON8.
Despite the interfering effect of the CS conjugation with these
model compounds, the results are encouraging and may
prompt for further studies with more potent ligand decoration.
By applying the concept of heteroduplex ASO on MSNAs,
making them substrates for RNase H, may open new possibil-
ities for MSNA-derived drug delivery vehicle design.
Experimental Section
General Remarks
RP-HPLC analysis and purification of the oligonucleotide conjugates
was performed by an analytical RP C18 (250×4.6 mm, 5 μm) column
unless stated otherwise. A flow rate of 1.0 ml/min, a detection
wavelength of 260 nm and gradient A, B or C were employed.
Gradient A: 0–70% MeCN in 0.1 M aqueous triethylammonium
acetate (pH 7) over 25 min. Gradient B: 0–95% MeCN in 0.1 M
aquous triethylammonium acetate (pH 7) over 25 min. Gradient C:
40–100% MeCN in 50 mM aquous triethylammonium acetate
(pH 7) over 20 min The mass spectra were recorded using either MS
(orbitrap) or MS (ESI-TOF) spectrometer. The isolated yields of the
oligonucleotide conjugates were determined according to their UV
absorbance at 260 nm.
Synthesis of Oligonucleotide Conjugates
5’-BCN-modified oligonucleotides ON1, ON9 and ON10 were
synthesized on a 1.0 μmol scale using an automatic DNA/RNA
synthesizer. Commercially available solid supports (including the 3’-
aminomodifier) and phosphoramidite building blocks of 2’-deoxy,
2’-O-(2-methoxyethyl) and 2’-O-TBDMS nucleosides, a 5’-amino
modifier and 2-(bicyclo[6.1.0]non-4-yn-9yl)ethan-1-ol were used for
the assembly. ON1, ON9 and ON10 were released from the
supports by the usual ammonolysis protocol (followed by TBDMS
removal in case of ON10) and homogenized by RP-HPLC using
gradient A.
CS-oligonucleotides conjugates ON2–4 were synthesized as follows
(Scheme 1): ON1 (88 nmol in 27 μL H2O) was combined with azide-
modified CS tetrasaccharide 1 (263 nmol in 128 μL of H2O). The
reaction was incubated at room temperature overnight and the
resulting conjugate ON2 was purified by RP-HPLC using gradient A
(Figure S1). The HPLC fractions were collected and lyophilized to
dryness to yield 19 nmol (22%) of ON2. ON3 and ON4 were
synthesized following the same procedure in 18% and 22% yields,
respectively. The authenticity of the products was verified by MS
(ESI-TOF, Table S1, Figures S1–S3).
AF488-labelling of the oligonucleotides was performed as follows:
ON2 (19 nmol) was lyophilized to dryness and dissolved in 20 μL of
sodium borate buffer (0.1 molL 1, pH 8.4). AF488 NHS ester
(380 nmol in 2 μL DMSO) was added, the reaction was incubated at
room temperature overnight, and the resulting oligonucleotide
ON6 was purified by RP-HPLC using gradient B. The HPLC fractions
were collected and lyophilized to dryness to yield 17 nmol (88%) of
ON6. ON5, ON7 and ON8 were prepared by the same protocol.
Isolated yields varied between 85% and 91% (Table S1). The
authenticity of the products was verified by MS (ESI-TOF, Table S1,
Figures S4-S7).
Synthesis of MSNA1 and MSNA2
To a solution of 4 (120 nmol in 130 μL DMSO), 5’-BCN-modified
oligonucleotide ON9 (40 nmol in 20 μL H2O) was added. The
reaction mixture was gently shaken overnight at room temperature
and introduced to RP-HPLC using gradient C (Figure S9). The HPLC
fractions were collected and lyophilized to dryness to yield 19 nmol
(47%) of the monofunctionalized [60]fullerene-oligonucleotide
intermediate product [MS (ESI-TOF), Figure S9]. The intermediate
product (9.0 nmol) was mixed with ON9 (130 nmol, 1.2 equivalents
of ON9 / azide arm) in 1.5 M aqueous NaCl solution and the
reaction mixture was gently shaken for 72 h at room temperature.
The resulting MSNA1 was purified by RP-HPLC using an analytical
Phenomenex Aeris WIDEPORE XB C18 200 Å (150×4.6 mm, 3.6 μm)
column with a linear gradient of 5–45% MeCN in 50 mM aqueous
triethylammonium acetate (pH 7) over 30 min (Figure S10). The
HPLC fractions were collected and lyophilized to dryness to yield
4.4 nmol (48%) of MSNA1. A slightly modified procedure was used
for the synthesis of MSNA2 (cf. supporting information), obtained
in 14% overall isolated yield (over the two steps). The homogeneity
and authenticity of MSNA1 and MSNA2 was confirmed by PAGE
and SEC-MALS (cf. Figure 1 and Figure 3). The hydrodynamic sizes
of MSNA1 and MSNA2 were determined by DLS (cf. Table 2).
PAGE Analysis of MSNAs
Native 6% Tris base, boric acid, ethylenediaminetetraacetic acid
(EDTA), and acrylamide (TBE) gel were used for assessing the purity
of the MSNAs and their hybridized complexes with oligonucleotides
ON5–8. A pre-cast gel cover (10 cm ×10 cm in size, Thermo Fisher
Scientific) was fixed into a vertical electrophoresis chamber, and the
running buffer (90 mM Tris, 90 mM borate, and 2 mM EDTA, pH 8.3)
was filled into the chamber. Samples were prepared by mixing 2 μl
of 0.05 μM MSNA in PBS (pH 7.4) with 2 μl of TBE sample buffer. For
the hybridization complexes (MSNA3-MSNA10), 24 equivalents
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(1.2 μM, corresponding to a 2-fold excess) of the complementary
oligonucleotides ON5–8 were used. The samples were loaded onto
the gel and electrophoresed at constant 100 V for 50 min. After
completion, the gel was removed from the chamber and stained
with SYBRTM gold Nucleic Acid Stain (Thermo Fisher Scientific). Each
gel was run with a 100 bp ladder in one of the wells.
Melting Temperature Analysis of MSNAs
The thermal melting curves of the hybridized MSNA/oligonucleo-
tide complexes were measured at 260 nm with a PerkinElmer
Lambda 35 UV-vis spectrometer equipped with a multiple cell
holder and a Peltier temperature controller. The measurements
were performed in 10 mmolL 1 sodium cacodylate buffer (pH 7.0)
in 0.1 M aqueous NaCl. MSNA concentration in each sample was
0.083 μmolL 1 and 12 equivalents (1.0 μmolL 1) of complementary
oligonucleotides were used. The temperature was changed at a
rate of 0.5 °C/min (10–90 °C). Each sample was heated up and
cooled down three times and Tm-values were determined as the
maxima of the first derivatives of the melting curves.
DLS Experiments
The hydrodynamic size of the MSNAs were measured using a
Zetasizer Nano ZS90 (Malvern Instruments Ltd. UK). The measure-
ments were performed at room temperature in PBS (pH 7.4). The
MSNA concentration in each sample was 0.2 μmolL 1 and 12 equiv-
alents (2.4 μmolL 1) of complementary oligonucleotides ON5–8
were used in the hybridized samples. Each sample was measured
six times and the hydrodynamic diameter was determined as the
average of the measurements.
SEC-MALS Experiments
SEC-MALS was performed using a 1260 Infinity II HPLC system
(sampler, pump, and UV-VIS detector; Agilent Technologies, Santa
Barbara, CA, USA) equipped with a miniDAWN light scattering
detector and Optilab refractive index detector (Wyatt Technologies,
Santa Barbara, CA, USA). An Advance Bio SEC 300 Å 2.7 μm
4.6×300 mm column (Agilent, Santa Clara, CA, USA) and 150 mM
sodium phosphate, pH 7.0, as mobile phase eluting at a rate of
0.2 mLmin 1 and run time of 20 min were used for each experi-
ment. For each run, 40 μL of MSNA sample (0.08–0.16 μg/μl) was
loaded onto a pre-equilibrated column. The refractive index was
used for the molecular weight calculations using an average
refractive index increment (dn/dc) of 0.1703 mL/g.
Primary Neuronal Cultures and Confocal Imaging
Hippocampal neurons were prepared from rats as described
previously.[55] At 7 days in vitro, neurons were treated with 120 nM
of the indicated conjugates and 10 nM of the MSNAs for 10 days
(the effective ASO concentration of 120 nM in each case). Cells
were washed with PBS and fixed with 4% cold paraformaldehyde
in phosphate buffered saline and stained with Hoechst-33342
(1 :2000). Z-sections of 0.4 μm through nuclei of 17 day in vitro
hippocampal neurons were obtained using Zeiss 880 LSM using the
63X oil objective (1.4 NA) using 488 nm (2.3% laser power) for
conjugates or 405 nm laser lines (18% laser power) for Hoechst-
33342. Scan used 2-line averaging with minimum pixel dwell of
0.24 μs and pixel size of 0.06 μm×0.06 μm. Maximum intensity
projections of 5 sections through nuclei were generated using the
software Zen from Zeiss and the mean somatic intensities were
measured using Fiji.
Western Blotting
Hippocampal neurons treated with conjugates and MSNAs as above
were lyzed in 1x Laemmli buffer supplemented with 1 mM MgCl2,
50 U/ml Benzonase, 1× Roche cOmplete, Mini, protease inhibitor
cocktail and 1× Phosphatase inhibitor cocktail 1 (P2850, Sigma).
Lysates were resolved on 10% SDS PAGE gels. Membranes were
probed with Tau (#4019, Cell Signalling Technology) and Actin
(#A3853, Sigma-Aldrich). IRDye® 680RD Donkey anti-Mouse IgG
Secondary Antibody (#926-68072, Licor) was used to detect the
signal and images were acquired using the Odyssey western blot
imager. Densitometry was performed using the image analysis
software Fiji.
Enzymatic Stability Tests
10 mM tris-HCl (pH 7.5) with 2.5 mM MgCl2, 0.1 mM CaCl2 and 4 mM
NaCl was used for DNase I experiments, and 20 mM tris-HCl (pH 7.8)
with 40 mM KCl, 8 mM MgCl2, 1 mM dithiotreitol and 4 mM NaCl for
RNase H experiments. MSNAs3–6 were treated with DNase I (1 U/
nmol of the effective oligonucleotide content). MSNAs7–10 were
treated with DNase I (10 U/nmol of the effective oligonucleotide
content) and RNase H (10 U/nmol of the oligonucleotide content).
The solutions (18.8 μL containing 38 pmol of oligonucleotide) were
incubated at 37 °C and 2 μL samples were taken at appropriate
time intervals. The samples were added into TBE sample buffer
(2 μL) on ice and analyzed by gel electrophoresis (6% TBE
acrylamide gel electrophoresed at constant 100 V for 50 min)
(Figure S12). The extent of MSNA7 degradation was quantified by
the image analysis software Fiji.
Supporting Information
Synthesis of MSNA2, RP HPLC profiles and MS spectra of the
oligonucleotide conjugates ON2–ON4, ON5–ON8, RP HPLC
profiles of MSNA1 and MSNA2 and fluorescence quenching on
MSNAs and PAGE analysis of the enzymatic stability experi-
ments are described in the Supporting Information.
Acknowledgements
The research was supported by the Academy of Finland
(decision number 308931) and Business Finland Ecosystem
project (448/31/2018). Doctoral Programme in Exact Sciences
(EXACTUS), University of Turku and The Finnish Cultural
Foundation are acknowledged.
Conflict of Interests
The authors declare no conflict of interest.
Data Availability Statement
The data that support the findings of this study are available
from the corresponding author upon reasonable request.
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Keywords: Molecular Spherical Nucleic Acids · Heteroduplex
ASOs · Chondroitin sulfates · Nanoparticles · Drug Delivery
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Manuscript received: November 1, 2024
Accepted manuscript online: November 15, 2024
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Wiley VCH Donnerstag, 28.11.2024
2499 / 385178 [S. 9/10] 1
ChemBioChem 2024, e202400908 (9 of 9) © 2024 The Author(s). ChemBioChem published by Wiley-VCH GmbH
ChemBioChem
Research Article
doi.org/10.1002/cbic.202400908
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nloaded from
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istry-europe.onlinelibrary.w
iley.com
/doi/10.1002/cbic.202400908 by D
uodecim
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edical Publications Ltd, W
iley O
nline Library on [05/12/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W
iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
RESEARCH ARTICLE
[60]fullerene-based molecular
spherical nucleic acids (MSNAs) were
used as hybridization-mediated
carriers for an antisense oligonucleo-
tide, suppressing Tau protein, and its
conjugates with chondroitin sulfate
(CS) tetrasaccharides. The impact of
the MSNA-carriers and the CS-halo on
the cellular uptake and suppression of
Tau-protein was studied with hippo-
campal neurons in vitro.
T. Laine, Dr. P. Deshpande, Dr. V.
Tähtinen, Prof. E. T. Coffey, Prof. P.
Virta*
1 – 10
Chondroitin Sulfate-Coated Hetero-
duplex-Molecular Spherical Nucleic
Acids
Wiley VCH Donnerstag, 28.11.2024
2499 / 385178 [S. 10/10] 1
 14397633, 0, D
ow
nloaded from
 https://chem
istry-europe.onlinelibrary.w
iley.com
/doi/10.1002/cbic.202400908 by D
uodecim
 M
edical Publications Ltd, W
iley O
nline Library on [05/12/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W
iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License