Cellular delivery of dinucleotides by conjugation with small molecules: targeting translation initiation for anticancer applications

Article abstract of DOI:10.1039/d1sc02143e

Targeting cap-dependent translation initiation is one of the experimental approaches that could lead to the development of novel anti-cancer therapies. Synthetic dinucleoside 5′,5′-triphosphates cap analogs are potent antagonists of eukaryotic translation initiation factor 4E (eIF4E)in vitroand could counteract elevated levels of eIF4E in cancer cells; however, transformation of these compounds into therapeutic agents remains challenging - they do not easily penetrate into cells and are susceptible to enzymatic cleavage. Here, we tested the potential of several small molecule ligands - folic acid, biotin, glucose, and cholesterol - to deliver both hydrolyzable and cleavage-resistant cap analogs into cells. A broad structure-activity relationship (SAR) study using model fluorescent probes and cap-ligand conjugates showed that cholesterol greatly facilitates uptake of cap analogs without disturbing the interactions with eIF4E. The most potent cholesterol conjugate identified showed apoptosis-mediated cytotoxicity towards cancer cells.

Full text of DOI:10.1039/d1sc02143e

Chemical
Science
View Article Online
View Journal | View Issue
EDGE ARTICLE
Cellular delivery of dinucleotides by conjugation
with small molecules: targeting translation
initiation for anticancer applications†
Cite this: Chem. Sci., 2021, 12, 10242
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
Natalia Kleczewska, ‡a Pawel J. Sikorski,‡a Zofia Warminska,ab Lukasz Markiewicz,a
abc
e
Renata Kasprzyk,
Aneta Karpinska,
Natalia Baran,ad Karina Kwapiszewska,
e
c
Jaroslaw Michalski,e Robert Holyst,e Joanna Kowalska
*
a
*
and Jacek Jemielity
Targeting cap-dependent translation initiation is one of the experimental approaches that could lead to the
development of novel anti-cancer therapies. Synthetic dinucleoside 50,50-triphosphates cap analogs are
potent antagonists of eukaryotic translation initiation factor 4E (eIF4E) in vitro and could counteract
elevated levels of eIF4E in cancer cells; however, transformation of these compounds into therapeutic
agents remains challenging – they do not easily penetrate into cells and are susceptible to enzymatic
cleavage. Here, we tested the potential of several small molecule ligands – folic acid, biotin, glucose,
and cholesterol – to deliver both hydrolyzable and cleavage-resistant cap analogs into cells. A broad
structure–activity relationship (SAR) study using model fluorescent probes and cap–ligand conjugates
showed that cholesterol greatly facilitates uptake of cap analogs without disturbing the interactions with
eIF4E. The most potent cholesterol conjugate identified showed apoptosis-mediated cytotoxicity
towards cancer cells.
Received 16th April 2021
Accepted 29th June 2021
DOI: 10.1039/d1sc02143e
rsc.li/chemical-science
masked pyrophosphate bonds are chemically unstable and
undergo rapid hydrolysis under aqueous conditions. Therefore,
Introduction
alternative approaches have been developed for the delivery of
nucleoside oligophosphates into cells, including application of
nanogels,8 nanoparticles,9 and naturally-inspired or articial
molecular transporters.10,11 Meier et al. have recently developed
cell-permeable nucleoside diphosphate and triphosphate pro-
drugs (DiPPro and TriPPro, respectively) that carry highly lipo-
philic masking groups exclusively at the terminal phosphate,
which has yielded a signicant breakthrough in this eld.12–14
However, the issue of delivering dinucleoside oligophosphates
and other structurally related compounds into cells remains
largely unaddressed.15 Here, we explore whether ligand-
mediated cellular delivery of dinucleoside oligophosphates
can resolve this problem.
Nucleoside and dinucleoside oligophosphates, and their
synthetic analogs, are a class of biologically active compounds
which oen act as modulators of metabolic pathways and
protein activity in cells and, therefore, have great therapeutic
potential.1 However, in vivo applications of these compounds
are largely limited due to poor cellular permeability arising
from high polarity and a negative net charge at physiological
pH.2 To date, the cell-permeability issue for nucleotides has
been fully addressed only for nucleoside monophosphates,3,4
which can be disguised as pronucleotides that contain charge-
masking phosphate modications that undergo removal aer
delivery into cells.5–7 However, such an approach is not suitable
for (di)nucleoside di- and triphosphates because fully charge-
As model dinucleotides with highly promising biological
activity in vitro but limited applications in vivo due to perme-
ability issues, we used 7-methylguanosine 50 cap analogs. 7-
Methylguanosine triphosphate is attached to the 50 end of all
eukaryotic messenger RNAs (mRNAs), forming the so called 50
cap.16,17 This cap protects mRNA from premature degradation
and participates in initiation of translation by interacting with
eukaryotic translation initiation factor 4E (eIF4E). eIF4E is one
of the components of translation initiation complex eIF4F,
which guides ribosome recruitment to mRNA during protein
biosynthesis.18 eIF4E has been found to act as a regulator of cap-
dependent translation that strongly affects translation of
aCentre of New Technologies, University of Warsaw Banacha 2c, 02-097 Warsaw,
Poland. E-mail: j.jemielity@cent.uw.edu.pl
bCollege of Inter-Faculty Individual Studies in Mathematics and Natural Sciences,
University of Warsaw, Banacha 2c, 02-097 Warsaw, Poland
cDivision of Biophysics Institute of Experimental Physics, Faculty of Physics University
of Warsaw, Pasteura 5, 02-093 Warsaw, Poland. E-mail: jkowalska@fuw.edu.pl
dFaculty of Biology University of Warsaw, I. Miecznikowa 1, 02-096 Warsaw, Poland
eInstitute of Physical Chemistry Polish Academy of Sciences, Kasprzaka 44/52, 01-224
Warsaw, Poland
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d1sc02143e
‡ These authors contributed equally to this work.
10242 | Chem. Sci., 2021, 12, 10242–10251
© 2021 The Author(s). Published by the Royal Society of Chemistry

View Article Online
Edge Article
Chemical Science
to several prodrugs including tryptamine phosphoramidates of
N7-benzyl GMP (4Ei-1) and 7-Cl-phenylethylene GMP that
release active drugs inside cells.33–35 However, this strategy is
applicable only to monophosphorylated cap analogs, which in
vitro have much lower activity than their di-, and triphosphate
counterparts.36
A particularly promising group of cap-derived eIF4E antag-
onists are oligophosphorylated mono- and dinucleotide cap
analogs that have high affinity for eIF4E and are resistant to
degradation by the cap-specic pyrophosphatase DcpS.37,38
Numerous DcpS-resistant dinucleotide cap analogs have been
designed by replacing one or more oxygen atoms in the
triphosphate bridge with another atom or group of atoms (e.g.
compounds carrying non-bridging g-O-to-S, g-O-to-BH3, b-O-to-
BH3,39,40 bridging b-g-O-to-CH2 or b-g-O-to-NH,41–43 or 50-O-to-S
[50-PSL]44) and have shown superior potency and stability in
rabbit reticulocyte lysates. However, the potential use of these
compounds has never been demonstrated in vivo.
Here, we sought to develop a ligand-based approach for the
delivery of dinucleotide cap analogs into cells that would also be
applicable also to other biologically relevant dinucleoside oli-
gophosphates. As potential transporters, we evaluated several
small molecule ligands previously identied as transporting
vehicles for various types of (macro)biomolecules (Fig. 1). The
tested ligands included folic acid, which uses a receptor-
mediated endocytosis pathway;45 biotin, which is dominantly
taken up by high-affinity biotin transporters;46 glucose, which
enters cells through facilitated diffusion;47 and cholesterol,
which facilitates passive diffusion of small molecules into
cells.48 To select the most active ligands and ideal cell culture
models, we rst studied simple uorescent probes using ow
cytometry, confocal microcopy, and uorescence correlation
spectroscopy (FCS). Based on those studies we synthesized
several cap analogs decorated with select ligands and a uo-
rescent dye to verify that the ligands are capable of transporting
negatively charged dinucleoside oligophosphates into cells.
Aer conrming the proof of concept, we synthesized a series of
cap analogs differing in susceptibility to DcpS and conjugated
those to the most potent ligands to examine their biological
activity both in vitro and towards breast cancer cells. As a result,
we identied several compounds with good cellular perme-
ability, high activity, and stability in vitro, and ability to induce
apoptosis in cancer cells.
Fig. 1 (A) mRNA 50 cap structure, (B) ligand selection process meth-
odology using fluorescent probes, (C) mode of action for translation
inhibitors (cap-ligands) and their cellular delivery and interaction with
eIF4E. The folic acid – receptor-mediated endocytosis pathway and
passive diffusion for cholesterol are displayed. Created with https://
www.BioRender.com.
selected intracellular mRNAs. These transcripts (“weak”
mRNAs) usually have long structured 50-untranslated regions
(UTRs) and, interestingly, many encode proteins responsible for
angiogenesis (VEGF-A), cell proliferation (c-myc), cell survival
(Bcl-2), and other aspects of oncogenesis.19 Increased eIF4E
expression is associated with poor prognosis in cancer
patients20 and induces cancer transformation of primary
epithelial cells and broblasts.21,22 In contrast, downregulation
of eIF4E levels inhibits melanoma proliferation and invasion.23
Therefore, various therapeutic strategies for targeting eIF4E
have been proposed to combat carcinogenesis.24,25 These
include antisense oligonucleotides and siRNAs to modulate
eIF4E mRNA levels,26,27 aptamers which target eIF4E,28,29 and
small molecules to disrupt the stability of the eIF4F complex
either by preventing eIF4E binding to the mRNA 50 cap or by
hindering association of eIF4E with the eIF4G scaffold
protein.30,31
Results and discussion
Synthesis of uorescent probes and their permeability
assessment
We rst veried the ability of select ligands to transport polar
molecules into cells and choose optimal cell culture models for
further experiments. Probes 1–4 were thus designed consisting
of the non-permeable uorescent dye uorescein (FAM) conju-
gated to folic acid, biotin, glucose, or cholesterol via a polyether
linker (Fig. 2A).
The probes were synthesized by taking advantage of N-
hydroxysuccinimide chemistry (NHS) and previously reported
procedures (Fig. 2B and C; ESI†).49–51 The cellular permeability
Synthetic cap analogs have been studied as potential eIF4E
antagonists for nearly three decades,32 but their activity was
limited by poor cell permeability. Of these, 7-alkylaryl 50-
monophosphates were identied as potent eIF4E antagonists.
To enable cellular uptake, the compounds have been converted
© 2021 The Author(s). Published by the Royal Society of Chemistry
Chem. Sci., 2021, 12, 10242–10251 | 10243

View Article Online
Chemical Science
Edge Article
Fig. 3 Initial selection of cell-permeable ligands. (A–D) Specific cell
lines were incubated with FAM-labeled probes and their cellular
uptake were assessed by flow cytometry. Histograms represent
overlaid flow cytometry data as a percentage of unstained and FAM-
ligand (50 nM) – stained cells. (A) Probe 1 uptake in MDA-MB-231 cells
compared to A549 cells. Probe 2–4 uptake in SK-BR-3 and MDA-MB-
231 cells compared to A549 cells, respectively (B–D). Normalized FCS
autocorrelation curves obtained in MDA-MB-231 cells incubated with
(E) FAM-PEG, probe 1 (F) or 4 (G). Confocal imaging/FLIM of a cell
incubated with 50 nM probe 1 (H) or 4 (I). FLIM image colored
according to the scheme: blue – fluorescence lifetime < 2.4 ns
(autofluorescence), red – fluorescence lifetime > 2.6 ns (probe).
Fig. 2 Chemical synthesis of probes 1–4 (A) structures of probes 1-4,
(B) synthesis of compounds 1, 2 and 4 via NHS chemistry: linker:
4,7,10-trioxa-1,13-diaminotridecane (6), ligand-NHS: folate-NHS,
biotin-NHS, cholesteryl hemisuccinate-NHS, (C) synthesis of
compound 3.
of the probes was evaluated in three human cancer cell lines:
MDA-MB-231, which overexpress folate receptor (FR) a for
endosomal uptake of folic acid;52 SK-RB-3, which expresses the
SMVT1 receptor for biotin46 and the GLUT-1 transporter for
glucose;53 and A549, which expresses none of these receptors.54
Potential uptake of probes 1–4 was initially assessed by ow
cytometry. Cells were incubated with the probes for 2 h followed
by ow cytometry analysis of the cells to quantify FAM emission.
Folic acid-conjugated probe 1 efficiently increased FAM emis-
sion of MDA-MB-231 cells, but not A549 cells (Fig. 3A), sug-
gesting that delivery via receptor-mediated endocytosis might
occur indeed in the former case. Neither the biotin-conjugated
probe 2 nor b-glucose-conjugated probe 3 affected the FAM
emission in any of the studied cell lines (Fig. 3B and C). In
contrast, the cholesterol-conjugated probe 4 increased FAM
emission for all studied cells lines (Fig. 3D), suggesting either
internalization via a diffusion-based mechanism or unspecic
binding to cell membrane.
method, which veries probe localization and estimates the size
of uorescent entities. FCS enables the measurement of the
diffusion coefficients for various uorescently-labeled probes in
the cell.55 By knowing the hydrodynamic radius of the studied
probe and employing a cytoplasm viscosity model for various
length scales, one can predict the theoretical coefficient for the
probe freely diffusing in the cytoplasm and compare it to
experimental values.56–58 If the value measured in the cells is
lower than the predicted value, it indicates binding of the probe
to the cellular components. To that end, the hydrodynamic radii
of probes 1 and 4 (Tables S1 and S2†) and cytoplasmic viscosity
of MDA-MB-231 cells were rst established by FCS (Table S3 and
Fig. S1†).
The cellular uptake of these probes, along with linker-
functionalized uorescein (FAM-PEG), were then studied by
FCS. MDA-MB-231 cells were incubated for 1 h with 1, 4, or FAM-
PEG and subjected to FCS measurements based on which the
autocorrelation curves were calculated (Fig. 3E–G). For
To further qualitatively and quantitatively evaluate the
uptake of probes 1 and 4 into MDA-MB-231 cells we used FCS
10244 | Chem. Sci., 2021, 12, 10242–10251
© 2021 The Author(s). Published by the Royal Society of Chemistry

View Article Online
Edge Article
Chemical Science
Table 1 Results of FCS measurements in the cytoplasm of MDA-MB-231 cells incubated with fluorescent probesa
Probe
FAM-PEG-FA (1)
FAM-PEG-CHOL (4) m7GpppA-FAM-FA (15) m7GpppA-FAM-CHOL (17)
Dcyto [mm2 sꢀ1], predicted for free probe in cytosol 187
205
203 ꢁ 29
96.6
87.0 ꢁ 43.6
58.5
58.2 ꢁ 12.4
D1 [mm2 sꢀ1], faster component in FCS
D2 [mm2 sꢀ1], slower component in FCS
184 ꢁ 56
1–9 high variability 3–14 high variability 0.1–9.8 high variability 0.1–5 high variability
a
Dcyto – diffusion coefficient in the cytoplasm.
compounds 1 and 4, FCS autocorrelation curves were tted with the FCS measurements did not provide any further details
a two-component free diffusion model (Table 1, Table S1†) differentiating these two potential mechanisms.
while for the FAM-PEG probe, no autocorrelation of uores-
cence signal was detected. For both probes, the diffusion coef-
cients for the rst, faster component (D1) were in line with
Synthesis of uorescent cap–ligand conjugates (m7GpppA-
FAM-ligand) and their cellular permeability
values expected for free diffusion of the probes in cytosol (187
mm2 sꢀ1 and 205 mm2 sꢀ1, respectively, Table 1), whereas the
We next tested whether folic acid and cholesterol may facilitate
delivery of dinucleotide cap analogs into cells. To that end, we
synthesized a series of doubly functionalized cap analogs that
slower components (D2) showed high variability from 1 mm2 sꢀ1
to 14 mm2 sꢀ1. Such D2 values are characteristic for large
proteins and small membrane structures.
were labeled with uorescein and conjugated to either folic
acid, biotin, or cholesterol (15, 16, and 17, respectively;
compound 16 being used as a negative control). The label and
ligand were attached at the position N6 of adenine by means of
NHS and CuAAC chemistry, respectively, and a multivalent
polyamine linker (Scheme 1 and ESI†).
Flow cytometry analysis showed that the cap analog conju-
gated with folic acid (15) potentially penetrated into MDA-MB-
231 cells (FR+), but not A549 cells (FRꢀ) (Fig. 4A and B). This
is consistent with the results obtained for probe 1, and reveals
that the presence of negative charge in the small molecule does
not prevent transport by folic acid receptors.
Compound 17, bearing the cholesterol moiety, penetrated
into both the MDA-MB-231 and A549 cell lines (Fig. 4A and B),
which also agrees with the results obtained for probe 4. As ex-
pected, based on the results for probe 2, neither cell line took up
biotin-conjugated compound 16 (Fig. 4A and B).
No components with D < 0.1 mm2 sꢀ1, which is typically
interpreted as active transport of endosomes, was observed for
any of the probes. To verify localization of probes 1 and 4,
uorescent lifetime imaging (FLIM) was performed (Fig. 3H and
I). FLIM, in contrast to typical confocal microscopy, enables
differentiation of emission coming from the probe (character-
ized by long uorescence lifetimes, t > 2.6 ns) from auto-
uorescence of the cell (t < 2.4 ns). These observations
conrmed the presence of compounds 1 and 4 in MDA-MB-231
cells and revealed mostly cytoplasmatic localization for both.
Overall, these data indicate that both folic acid and cholesterol
are good candidates for studying cap analog delivery into cancer
cells. The initial evaluation in different cultured cells suggested
that cholesterol-conjugated probe 4 is taken up by a receptor-
independent mechanism, whereas uptake of probe 1 requires
the presence of folic acid receptors on the cell surface. However,
Scheme 1 Chemical synthesis of fluorescently labeled cap conjugates. Conditions and reagents: (a) H2O, NaOH to adjust to pH ¼ 8, 72 h, 65 ꢂC,
(b) ZnCl2, DMF, 24 h, rt, (c) 6-carboxyfluorescein-N-hydroxysuccinimide (FAM-NHS), borate buffer (50 mM), pH ¼ 8.4, DMSO, (d) for 15 (with
folate azide) and 16 (with biotin azide): CuSO4 x5H2O, NaAsc, DMSO : H2O, (e) for 17 (with cholesteryl-TEG-azide): Cu(II)-TBTA, NaAsc, trie-
thylammonium acetate pH ¼ 7, DMSO.
© 2021 The Author(s). Published by the Royal Society of Chemistry
Chem. Sci., 2021, 12, 10242–10251 | 10245

View Article Online
Chemical Science
Edge Article
Fig. 4 Cellular uptake of FAM labeled cap analog probes by (A) MDA-MB-231 and (B) A549 cells assessed by flow cytometry. Histograms
represent overlaid flow cytometry data of unstained and m7GpppA-FAM-ligand (50 nM)-stained cells. (C) Probe 15 uptake by MDA-MB-231 cells
(50 nM) in the presence of increasing concentrations of folic acid. Histograms represent overlaid flow cytometry data as a percentage of
unstained and m7GpppA-FAM-FA-stained cells. (D) Uptake of FAM labeled cap analog conjugates by A549 and MDA-MB-231 cells assessed by
confocal microscopy. (E and F) Normalized FCS autocorrelation curves for MDA-MB-231 cells incubated with probes 15 and 17 and their diffusion
coefficients in cytoplasm derived from FCS data. Confocal imaging/FLIM of a cell incubated with 50 nM probe 15 (H) and 17 (G). FLIM image
colored according to the scheme: blue – fluorescence lifetime < 2.4 ns (autofluorescence), red – fluorescence lifetime > 2.6 ns (probe).
To additionally verify that cellular uptake of compound 15 is cell is a compound successfully delivered to the cytoplasm. The
dependent on FR expression on the cell membrane, we per- slower components (D2) showed high variability from 0.1 mm2
formed a ow-cytometry based competition assay (Fig. 4C). The sꢀ1 to 9.8 mm2 sꢀ1, which is characteristic for large proteins and
uptake of compound 15 was inhibited in a dose-dependent small membrane structures. Components with D < 0.1 mm2 sꢀ1
,
manner by free folic acid in the cell culture medium, indi- typically assigned to endosomes, have not been found in the
cating an FR-specic mechanism. The uptake of compounds 15, FCS signals for any of the studied probes.
16, and 17 was independently studied by confocal microscopy
(Fig. 4D) and veried by checking the orthogonal axis views
(Fig. S2†). The results were generally consistent with the ow
cytometry data, with compound 15 permeating only into MDA-
Aer identifying the ligands capable of transporting the cap
MB-231 (FR+) cells, compound 17 permeating into both cell
lines, and compound 16 being non-permeable.
FCS measurements performed in MDA-MB-231 cells (Fig. 4E
particular, we focused on tailoring the ligand attachment site
and F) revealed primarily cytoplasmic localization for both
compounds 15 and 17. The diffusion coefficients (D1) of the
major, faster components were in line with values expected for
free diffusion of the probes in the cytosol (Table 1, Table S1†),
suggesting that the most abundant uorescent species in the
Cap analog conjugates with folic acid and cholesterol inhibit
cap-dependent translation in vitro
analogs into cells, we focused on optimizing the structure of the
cap residue to maximize its translation inhibitory properties. In
for optimal interaction with eIF4E and decreasing susceptibility
to cleavage by DcpS – the pyrophosphatase that rapidly
degrades native cap residues in the cell. Therefore, we synthe-
sized a series of cap analogs carrying amino-alkyl linkers at
either the N6-position of adenosine or the ribose moiety of
10246 | Chem. Sci., 2021, 12, 10242–10251
© 2021 The Author(s). Published by the Royal Society of Chemistry

View Article Online
Edge Article
Chemical Science
Scheme 2 Chemical syntheses and applied conditions and reagents: (a) ZnCl2, DMF, (b) NHS-activated ligand (a ¼ FA – folate, b ¼ Chol –
cholesteryl hemisuccinate), borate buffer pH ¼ 8.4, DMSO. Phosphorothioates (18, 22, 24, 26, 29, 31) were obtained as a mixture of diaste-
reoisomers (D1/D2).
guanosine and optionally introduced a g-phosphorothioate (g-
We next assessed the affinity of these conjugates to eIF4E. To
PS) modication that prevents the cleavage by DcpS40 this end, a recently developed uorescence intensity binding
(compounds 21, 22, 28, and 29, Scheme 2). Phosphorothioate assay based on a pyrene-labeled probe (PyFLINT-B) was
cap analogs (22, 29) exist as two P-diastereomers (D1/D2) that applied.59 The PyFLINT-B assay is based on measuring changes
were difficult to separate by RP HPLC. Since the initial results of pyrene uorescence in a pyrene-labeled probe upon its
showed that the affinities of the diastereomers for eIF4E were competitive displacement from eIF4E by the studied
very similar (Fig. S3†), we decided to use diastereomeric compounds and allows for fast and reliable measurement of
mixtures of 22 and 29 in further studies.
apparent dissociation constants, based on which equilibrium
Folic acid or cholesterol were then attached to these cap dissociation constants (KD) can be calculated. The KD values of
analogs using NHS-chemistry47,48 to obtain eight different cap– all studied ligands, along with the reference compounds
ligand conjugates (23a–b, 24a–b, 30a–b, and 31a–b). The m7GpppA and m7GpppG, are shown in Table 2, while the
conjugates were puried by RP HPLC and their m/z values were binding curves are shown in the ESI (Fig. S2†). The affinity of
conrmed by high-resolution mass spectrometry (HR-MS, ESI†). m7GpppG for eIF4E as determined by PyFLINT-B was 2-fold
Table 2 Biological properties of synthetized cap analogs
IC50 [mM] (translation inhibition)b
IC50 [mM] (cytotoxicity)c
a
No.
Compound
KD [mM]
21
m7GpppA
0.429 ꢁ 0.079
0.884 ꢁ 0.148
0.433 ꢁ 0.102
0.39 ꢁ 0.09
0.051
78. ꢁ 26
[30
nd.
nd.
[ 30
m7GpppA-HDA
23.9 ꢁ 5.5
10.8 ꢁ 1.7
28.1 ꢁ 4.2
12.6 ꢁ 2.1
29.9 ꢁ 3.3
nd.e
22
m7GpSppA-HDA-D1/D2
m7GpppA-HDA-FA
23a
23b
24a
24b
m7GpppA-HDA-CHOL
m7GpSppA-HDA-FA D1/D2
m7GpSppA-HDA-CHOL D1/D2
m7GpppG
8.28 ꢁ 1.87
0.30 ꢁ 0.06
[30
nd.e
5.20 ꢁ 0.78
0.216 ꢁ 0.035
0.485 ꢁ 0.092
0.242 ꢁ 0.045
0.12 ꢁ 0.02
0.043
39.6 ꢁ 13.7
10.9 ꢁ 1.4
6.0 ꢁ 0.8
16.1 ꢁ 3.5
26.1 ꢁ 5.0
4.2 ꢁ 0.6
20.5 ꢁ 5.7
[500
[30
28
29
30a
30b
31a
31b
m7GpppG-L6N-20-O/30-O
m7GpSppG-L6N-20-O/30-O D1/D2
m7GpppG-L6N -20-O/30-O-FA
m7GpppG-L6N -20-O/30-O-CHOL
m7GpSppG-L6N -20-O/30-O-FA D1/D2
m7GpSppG- L6N 20-O/30-O-CHOL D1/D2
Folic acid
nd.
nd.
[30
10.90 ꢁ 2.81
0.10 ꢁ 0.02
[30
0.026
6.52 ꢁ 1.73 mM [6.04 ꢁ 1.58]d
>10
0.94
[30
[30
Cholesterol
99. ꢁ 20.
a
Dissociation constants (KD) for eIF4E–cap analog complexes determined using the PyFLINT-B method at 20 ꢂC. Data shown are means ꢁ SD of
three independent experiments; for compounds 23b, 30b, and 31b, only one repetition was performed. b From cap-dependent translation inhibition
in rabbit reticulocyte lysate (RRL). c From resazurin cell viability assay in MDA-MB-231 cells. d Value in brackets is for K562 cells. e The values were
not determined due to limited available material.
© 2021 The Author(s). Published by the Royal Society of Chemistry
Chem. Sci., 2021, 12, 10242–10251 | 10247

View Article Online
Chemical Science
Edge Article
higher than that of m7GpppA, which is in agreement with consequently affinity and susceptibility of cap analogs to DcpS
previous ndings by other methods.60 The introduction of an is usually decreased upon functionalization of ribose or
aminohexyl linker at the N6 position of adenosine (compound nucleobase moieties.40,63,64
21) resulted in about 2-fold decrease in affinity for eIF4E (KD
Therefore, it is plausible that the incorporation of a linker at
0.88 ꢁ 0.15 mM for m7GpppA-HDA (21) vs. KD 0.43 ꢁ 0.08 mM for the N6-position of adenine or the 20-O/30-O-position of ribose in
m7GpppA). A similar thermodynamic effect was observed upon the cap decreased its susceptibility to DcpS, thereby stabilizing
introduction of the linker at the ribose moiety of guanosine it in the lysate. The presence of g-PS conferred high inhibitory
(compound 28) (KD 0.485 ꢁ 0.092 mM for m7GpppG-L6N (28) vs. potency for all tested compounds. Compounds 22 and 29
KD 0.216 ꢁ 0.035 mM for m7GpppG).
bearing this modication had lower IC50 values than their
The presence of the g-PS moiety increased affinity to eIF4E unmodied counterparts (IC50 23.9 ꢁ 5.5 mM for m7GpppA-HDA
by about 2-fold (KD 0.88 ꢁ 0.15 mM for m7GpppA-HDA (21) vs. KD (21) vs. IC50 10.8 ꢁ 1.7 mM for m7GpSppA-HDA D1/D2 (22) and
0.43 ꢁ 0.10 mM for m7GpSppA-HDA D1/D2 (22) and KD 0.485 ꢁ IC50 10.9 ꢁ 1.4 mM for m7GpppG-L6N (28) vs. IC50 6.0 ꢁ 0.8 mM
0.092 mM for m7GpppG-L6N (28) vs. KD 0.242 ꢁ 0.045 mM for for m7GpSppG-L6N-20-O/30-O D1/D2 (29)). This observation is in
m7GpSppG-L6N-20-O/30-O D1/D2 (29)). Interestingly, the intro- agreement with our previous studies showing that a non-
duction of appropriate ligands (FA or Chol; compounds 23a to bridging g-O-to-S modication in the triphosphate bridge
31b) alleviated the destabilizing effect introduced by the ami- decreases the susceptibility of cap analogs to DcpS (in addition
noalkyl linker, suggesting it was at least partly caused by elec- to stabilizing the cap-eIF4E complex).40 Interestingly, despite
trostatic repulsion of the positively charged amino group with a signicant increase in affinity for eIF4E, the presence of
positively charged surfaces in eIF4E. KD values for compounds ligands did not enhance inhibitory properties of cap analogs in
bearing the folate modication were from 1.5-fold to 4-fold RRL compared to corresponding compounds with aminohexyl
higher than corresponding unconjugated caps (e.g. KD 0.433 ꢁ linkers. Compounds 23a, 23b, 30a, and 31a had IC50 values
0.102 mM for m7GpSppA-HDA-D1/D2 (22) vs. KD 0.30 ꢁ 0.06 mM almost unchanged compared to values for the cap analogs with
for m7GpSppA-HDA-FA D1/D2 (24a)). Unexpectedly, the incor- aminoalkyl linkers, whereas compounds 24a, 30b, and 31b were
poration of the cholesterol moiety resulted in even greater up to 3-fold weaker inhibitors. Overall, all cap–ligand conju-
stabilization of the cap-eIF4E complex ranging from 9-fold to gates studied exhibited sufficient potency and stability in RLL to
17-fold improvement (e.g. KD 0.242 ꢁ 0.045 mM for m7GpSppG- be considered as suitable candidates for evaluation in cell
L
6N-20-O/30-O D1/D2 (29) vs. KD 0.026 mM for m7GpSppG-L6N-20-O/ culture experiments.
30-O-CHOL D1/D2 (31b)). This might be due to additional
hydrophobic interactions with eIF4E provided by the cholesteryl
moiety.
Cholesterol cap analogs inuence cell viability and their
potency depends on a 50,50-triphosphate bridge modication
In order to evaluate the ability of cap analogs to target eIF4E
in a more complex biological system, we studied their potency Knowing the in vitro properties of the synthesized conjugates,
to inhibit cap-dependent translation in rabbit reticulocyte lysate we next analyzed their cytotoxicity towards a breast cancer cell
(RRL) programmed with capped luciferase-encoding mRNA.61 line (MDA-MB-231). We rst veried whether the non-
We have previously shown that the ability of cap analogs to uorescent cap analogs entered the cells. To that end, an
inhibit translation in RRL correlates qualitatively with their immunouorescent assay using a cap-specic antibody was
affinity for eIF4E, but may also be affected by DcpS-like activity, employed.10 Visualization of immunostained cells clearly
which is also present in the lysate.62 Therefore, the compounds showed that cap analog conjugates were taken up by MDA-MB-
were tested in an experimental set up in which both factors 231 (FR+) cells regardless of the attached ligand (Fig. 5A).
could be taken into account. Namely, the compounds were
Next, cytotoxicity of the compounds (23a, 23b, 24a, 24b, 30a,
incubated in RRL for 1 h followed by the addition of mRNA and 30b, 31a, 31b, m7GpppA, m7GpppG, free folic acid, and free
luciferase activity measurement aer another hour. It was ex- cholesterol moiety) towards MDA-MB-231 cells was determined
pected that 1 h pre-incubation in RRL will relatively decrease by resazurin assay. Only the cholesterol conjugates signicantly
the inhibitory potency of cap analogs susceptible to DcpS, inuenced cell viability (Fig. 5B and S5†). The compounds
compared to analogs that are resistant to DcpS.62
carrying the g-PS moiety in the 50,50-triphosphate bridge (24b
The IC50 values reasonably correlated with KD values from and 31b) had cytotoxic effects about 2-fold stronger than
PyFLINT-B assay (Table 2), albeit with some important unmodied counterparts (IC50 8.3 ꢁ 1.9 mM for 23b vs. IC50 5.2
exceptions. For instance, the introduction of hexamine linkers ꢁ 0.8 mM for 24b and IC50 10.9 ꢁ 2.8 mM for 30b vs. IC50 6.0 ꢁ
resulted in stronger inhibition compared to unmodied 1.6 mM for 31b), clearly showing that stabilizing the cap against
reference compounds [IC50 23.9 ꢁ 5.5 mM for m7GpppA-HDA DcpS is benecial for the biological activity of the cap analogs in
(21) vs. IC50 78.1 ꢁ 25.6 mM for m7GpppA and IC50 10.9 ꢁ living cells (Fig. 5C). Next, we checked whether cholesterol cap
1.4 mM for m7GpppG-L6N (28) vs. IC50 39.6 ꢁ 13.7 mM for analogs exhibit cytotoxic activity towards other cancer cell lines.
m7GpppG; Table 2, Fig. S4],† despite their lower affinity for To that end, a leukemia cancer cell line (K562) was treated with
eIF4E. This observation conrms that factors other than cap analog 31b. Resazurin assay conrmed the cytotoxicity of
affinity for eIF4E, and DcpS-susceptibility in particular, inu- this compound towards the K562 cell line (Fig. 5D; IC50 6.5 ꢁ 1.7
ence the inhibitory properties under the conditions of our mM) at a level comparable to MDA-MB-231 cells (IC50 6.0 ꢁ 1.6
experiment. DcpS exhibits
a tight cap-binding mode, mM; Table 2).
10248 | Chem. Sci., 2021, 12, 10242–10251
© 2021 The Author(s). Published by the Royal Society of Chemistry

View Article Online
Edge Article
Chemical Science
(propidium iodide) staining. This analysis detects changes in
plasma membrane integrity during cell apoptosis that result in
the appearance of phosphatidylserine on the outer leaet of the
membrane.
Annexin V has a strong, calcium-dependent affinity for
phosphatidylserine and, therefore, can be used as a marker of
apoptosis. PI is a DNA binder and can be used to detect genomic
DNA released from necrotic cells. Double staining with PI and
Annexin V enables differentiation and quantication of living,
apoptotic, and necrotic cells. Fig. 5E shows representative dot
plots from Annexin V/PI staining, whereas Fig. 5F shows the
percentages of apoptotic cells. These data reveal that compound
31b indeed induces apoptosis in a dose-dependent manner
(30.5 ꢁ 6.5% and 60.8 ꢁ 3.2% apoptosis in the presence 5 mM
and 20 mM cap analog, respectively).
Conclusions
We synthesized a series of uorescent probes and doubly
functionalized cap analogs, which allowed us to examine the
potential of four pre-selected ligands (folic acid, biotin, glucose,
and cholesterol) to transport polar, negatively charged mole-
cules into cells. Only folic acid and cholesterol facilitated
cellular uptake of uorescent probes and cap analogs in the
studied models. The lack of observable cellular uptake for
glucose and biotin conjugates may result from insufficient
transport efficiency for these ligands in the studied cell lines or
disturbance of the transport by cargo attachment.
Fig. 5 Cellular response to cap analog conjugates. (A) Verification of
cellular delivery of cap conjugates. MDA-MB-231 cells after 2 h
treatment with 0.5 mM compound were fixed and stained with anti-cap
antibody. (B) Viability of MDA-MB-231 cells treated with different
concentrations of selected compounds. After 72 h incubation, cells
viability was assessed with the resazurin assay. Data points were
normalized to mock treated cells for each time point and represent
mean value ꢁ SD from at least two independent biological replications
with two technical replicates per treatment. (C) IC50 values of
cholesterol cap analog conjugates calculated based on the cell viability
assay presented in (B), bars represent mean value ꢁ SD. Statistical
significance: * p < 0.05, ** p < 0.01 (Welch's t test). (D) Viability of K562
cells treated with different concentrations of selected compounds.
After 72 h incubation, cells viability was assessed with the resazurin
assay. Data points were normalized to mock treated cells for each time
point and represent mean value ꢁ SD two independent biological
replications with two technical replicates per treatment. (E) Apoptosis
assessed by Annexin V/propidium iodide staining. K562 cells were
treated with 5 mM and 20 mM 31b for 20 h and examined by flow
cytometry. Viable cells are in the (I) lower left quadrant, early apoptotic
are in the (II) lower right quadrant, late apoptotic cells are in the (III)
upper right quadrant and non-viable necrotic cells are in the (IV) upper
left quadrant. (F) Apoptotic cell fraction calculated based on the
Annexin V/propidium iodide staining presented in (E). Bars represent
mean value ꢁ SD from at least three independent biological replica-
tions. Statistical significance: * p < 0.05, *** p < 0.001 (Welch's t test).
We next optimized the structure of the cap moiety for
conjugation with folic acid and cholesterol to maximize its
translation inhibitory properties. To that end, we synthesized
a set of cap-derived eIF4E antagonists with folic acid and
cholesterol attached in two different positions of cap analogs (at
the nucleobase and the ribose moiety) and optionally carrying
a g-PS modication that prevents cap degradation by DcpS. We
tested the cytotoxicity of these compounds towards cancer cells.
Both cholesterol and folate conjugates were efficiently taken up
by cancer cells, but cytotoxic effects were only observed for
compounds conjugated with cholesterol. The cytotoxicity of
these conjugates was notably enhanced by introducing the g-PS
modication in the 50,50-triphosphate bridge. The possible
explanations of this phenomenon include either strong specic
binding to folic acid receptors and resulting entrapment of the
cap analogs in residual endosomal structures or competitive
binding of the conjugates to other folate recognizing proteins,
which prevents binding by eF4E. If this is the case, the issue
may be resolved in the future by application of cleavable
conjugates that release free cap structures following cell entry.
Moreover, the designed cap analogs can serve as a platform to
test the potential of other ligands to deliver impermeable
compounds to mammalian cells. Importantly, dual labeling of
cap analogs enables not only intracellular visualization and
tracking but also opens up new possibilities to test combina-
tions of cell penetrating ligands, which could lead to the
development of superior delivery strategies such as a dual
labeling approach that could bypass the “endosome escape”
issue. Further studies, including in vivo evaluation on animal
Cholesterol cap analogs induce apoptosis in a leukemia cell
line
Most anticancer drugs currently used in clinical oncology
exploit apoptotic mechanisms to trigger cancer cell death.65
Therefore, to verify whether cholesterol-conjugated cap analogs
induce apoptosis in cancer cells, K562 cells were incubated with
the most potent compound (31b), followed by a more detailed
cell viability analysis. The ability of 31b to induce apoptosis was
determined by ow cytometry using Annexin V and PI
© 2021 The Author(s). Published by the Royal Society of Chemistry
Chem. Sci., 2021, 12, 10242–10251 | 10249

View Article Online
Chemical Science
Edge Article
models, are required to fully assess the anticancer activity and 13 T. Gollnest, T. D. de Oliveira, D. Schols, J. Balzarini and
selectivity of cap–cholesterol conjugates.
C. Meier, Nat. Commun., 2015, 6, 8716.
14 S. Warnecke and C. Meier, J. Org. Chem., 2009, 74, 3024–
3030.
Data availability
15 S. F. Dowdy, Nat. Biotechnol., 2017, 35, 222–229.
All data needed to evaluate the conclusions are present in the 16 J. D. Lewis and E. Izaurde, Eur. J. Biochem., 1997, 247, 461–
paper and in the associated supplementary material.
469.
17 M. Warminski, P. J. Sikorski, J. Kowalska and J. Jemielity,
Top. Curr. Chem., 2017, 375, 16.
18 A. Batool, S. Aashaq and K. I. Andrabi, J. Cell. Biochem., 2019,
120, 14201–14212.
Author contributions
JK, JJ, NK, and PJS designed the study. NK and ZW performed
the syntheses. PJS, NB, and LM performed biological assays. RK 19 S. Uttam, C. Wong, T. J. Price and A. Khoutorsky, Front.
performed the binding assay. KK, AK, JM, RH performed FCS Genet., 2018, 9, 470.
and FLIM studies. NK, PS, JK, and JJ prepared the rst dra of 20 B. Culjkovic and K. L. Borden, J. Oncol., 2009, 2009, 1–12.
the manuscript. All authors reviewed, edited, and approved the 21 A. Lazaris-Karatzas, K. S. Montine and N. Sonenberg, Nature,
nal version of the manuscript.
1990, 345, 544–547.
22 S. Avdulov, S. Li, V. Michalek, D. Burrichter, M. Peterson,
D. M. Perlman, J. C. Manivel, N. Sonenberg, D. Yee,
P. B. Bitterman and V. A. Polunovsky, Cancer Cell, 2004, 5,
553–563.
Conflicts of interest
There are no conicts to declare.
23 C. E. Joyce, A. G. Yanez, A. Mori, A. Yoda, J. S. Carroll and
C. D. Novina, Cancer Res., 2017, 77, 613–622.
24 J. Chu and J. Pelletier, Cold Spring Harbor Perspect. Biol.,
2017, 10, a032995.
Acknowledgements
This work was supported by the National Science Centre Poland
(UMO-2016/20/S/ST5/00364 to NK and 2018/31/B/ST5/03821 to 25 A. Fan and P. P. Sharp, J. Med. Chem., 2021, 64, 2436–2465.
JK), the Foundation for Polish Science (TEAM/2016-2/13 to JJ) 26 J. R. Graff, B. W. Konicek, T. M. Vincent, R. L. Lynch,
and the National Centre for Research and Development Poland
(LIDER/10/0033/L-9/17/NCBR/2018 to KK). Figures were created
with https://www.BioRender.com.
D. Monteith, S. N. Weir, P. Schwier, A. Capen, R. L. Goode,
M. S. Dowless, Y. Chen, H. Zhang, S. Sissons, K. Cox,
A. M. McNulty, S. H. Parsons, T. Wang, L. Sams,
S. Geeganage, L. E. Douglass, B. L. Neubauer, N. M. Dean,
K. Blanchard, J. Shou, L. F. Stancato, J. H. Carter and
E. G. Marcusson, J. Clin. Invest., 2007, 117, 2638–2648.
Notes and references
1 M. K. Yates and K. L. Seley-Radtke, Antiviral Res., 2019, 162, 27 F. Zhou, M. Yan, G. Guo, F. Wang, H. Qiu, F. Zheng,
5–21.
Y. Zhang, Q. Liu, X. Zhu and L. Xia, Med. Oncol., 2011, 28,
1302–1307.
28 W. M. Guo, K. W. Kong, C. J. Brown, S. T. Quah, H. L. Yeo,
S. Hoon and Y. Seow, Mol. Ther.–Nucleic Acids, 2014, 3, e217.
29 S. Miyakawa, RNA, 2006, 12, 1825–1834.
2 J. Lipfert, S. Doniach, R. Das and D. Herschlag, Annu. Rev.
Biochem., 2014, 83, 813–841.
3 C. Meier, M. Lorey, E. De Clercq and J. Balzarini, J. Med.
Chem., 1998, 41, 1417–1427.
4 I. Lefebvre, C. Perigaud, A. Pompon, A.-M. Aubertin, 30 N. J. Moerke, H. Aktas, H. Chen, S. Cantel, M. Y. Reibarkh,
J.-L. Girardet, A. Kirn, G. Gosselin and J.-L. Imbach, J. Med.
Chem., 1995, 38, 3941–3950.
A. Fahmy, J. D. Gross, A. Degterev, J. Yuan, M. Chorev,
J. A. Halperin and G. Wagner, Cell, 2007, 128, 257–267.
5 U. Pradere, E. C. Garnier-Amblard, S. J. Coats, F. Amblard 31 R. Cencic, D. R. Hall, F. Robert, Y. Du, J. Min, L. Li, M. Qui,
and R. F. Schinazi, Chem. Rev., 2014, 114, 9154–9218.
6 F. Pertusat, M. Serpi and C. McGuigan, Antivir. Chem.
Chemother., 2012, 22, 181–203.
I. Lewis, S. Kurtkaya, R. Dingledine, H. Fu, D. Kozakov,
S. Vajda and J. Pelletier, Proc. Natl. Acad. Sci. U. S. A., 2011,
108, 1046–1051.
7 A. Kraszewski, M. Sobkowski and J. Stawinski, Front. Chem., 32 E. Darzynkiewicz, I. Ekiel, S. M. Tahara, L. S. Seliger and
2020, 8, 595738. A. J. Shatkin, Biochemistry, 1985, 24, 1701–1707.
8 S. Vinogradov, A. Zeman, E. Batrakova and A. Kabanov, J. 33 Z. Ahmad, B. A. Jacobson, M. W. McDonald, N. Vattendahl
Controlled Release, 2005, 107, 143–157.
9 H. Hillaireau, T. Le Doan, M. Appel and P. Couvreur, J.
Controlled Release, 2006, 116, 346–352.
Vidal, G. Vattendahl Vidal, S. Chen, M. Dillenburg,
A. M. Okon, M. R. Patel, C. R. Wagner and R. A. Kratzke,
Cancer Chemother. Pharmacol., 2020, 85, 425–432.
10 M. Zochowska, A.-C. Piguet, J. Jemielity, J. Kowalska, 34 C. R. Wagner, V. V. Iyer and E. J. McIntee, Med. Res. Rev.,
E. Szolajska, J.-F. Dufour and J. Chroboczek, Nanomedicine,
2015, 11, 67–76.
2000, 20, 417–451.
35 B. Ghosh, A. O. Benyumov, P. Ghosh, Y. Jia, S. Avdulov,
P. S. Dahlberg, M. Peterson, K. Smith, V. A. Polunovsky,
P. B. Bitterman and C. R. Wagner, ACS Chem. Biol., 2009,
4, 367–377.
ˇˇ´
11 Z. Zawada, A. Tatar, P. Mocilac, M. Budesınsk´y and T. Kraus,
Angew. Chem., Int. Ed., 2018, 57, 9891–9895.
12 C. Meier, Antiviral Chem. Chemother., 2017, 25, 69–82.
10250 | Chem. Sci., 2021, 12, 10242–10251
© 2021 The Author(s). Published by the Royal Society of Chemistry

View Article Online
Edge Article
Chemical Science
36 J. Zuberek, J. Jemielity, A. Jablonowska, J. Stepinski, 51 S. C. Kinsky, J. E. Loader and A. L. Benson, J. Immunol.
M. Dadlez, R. Stolarski and E. Darzynkiewicz, Biochemistry,
2004, 43, 5370–5379.
37 M. Ziemniak, M. Strenkowska, J. Kowalska and J. Jemielity,
Future Med. Chem., 2013, 5, 1141–1172.
Methods, 1983, 65, 295–306.
52 J. D. Seitz, J. G. Vineberg, E. Herlihy, B. Park, E. Melief and
I. Ojima, Bioorg. Med. Chem., 2015, 23, 2187–2194.
53 R. L. A, F. W. Zhang and D. Gius, Br. J. Cancer, 2002, 87,
805–812.
38 M. Kalek, J. Jemielity, Z. M. Darzynkiewicz, E. Bojarska,
J. Stepinski, R. Stolarski, R. E. Davis and E. Darzynkiewicz, 54 D. Ren, F. Kratz and S.-W. Wang, Biochem. Eng. J., 2014, 89,
Bioorg. Med. Chem., 2006, 14, 3223–3230. 33–41.
39 J. Kowalska, A. Wypijewska del Nogal, Z. M. Darzynkiewicz, 55 R. Machan and T. Wohland, FEBS Lett., 2014, 588, 3571–
J. Buck, C. Nicola, A. N. Kuhn, M. Lukaszewicz, J. Zuberek, 3584.
M. Strenkowska, M. Ziemniak, M. Maciejczyk, E. Bojarska, 56 T. Kalwarczyk, N. Ziȩbacz, A. Bielejewska, E. Zaboklicka,
´ˇ
´
´
R. E. Rhoads, E. Darzynkiewicz, U. Sahin and J. Jemielity,
K. Koynov, J. Szymanski, A. Wilk, A. Patkowski, J. Gapinski,
Nucleic Acids Res., 2014, 42, 10245–10264.
H.-J. Butt and R. Hołyst, Nano Lett., 2011, 11, 2157–2163.
40 J. Kowalska, M. Lewdorowicz, J. Zuberek, E. Grudzien- 57 T. Kalwarczyk, K. Kwapiszewska, K. Szczepanski,
Nogalska, E. Bojarska, J. Stepinski, R. E. Rhoads,
E. Darzynkiewicz, R. E. Davis and J. Jemielity, RNA, 2008,
14, 1119–1131.
K. Sozanski, J. Szymanski, B. Michalska, P. Patalas-
Krawczyk, J. Duszynski and R. Holyst, J. Phys. Chem. B,
2017, 121, 9831–9837.
41 A. M. Rydzik, M. Kulis, M. Lukaszewicz, J. Kowalska, 58 K. Kwapiszewska, T. Kalwarczyk, B. Michalska,
´ ´
K. Szczepanski, J. Szymanski, P. Patalas-Krawczyk,
J. Zuberek, Z. M. Darzynkiewicz, E. Darzynkiewicz and
J. Jemielity, Bioorg. Med. Chem., 2012, 20, 1699–1710.
42 J. Jemielity, J. Kowalska, A. M. Rydzik and E. Darzynkiewicz,
New J. Chem., 2010, 34, 829.
43 A. M. Rydzik, M. Warminski, P. J. Sikorski, M. R. Baranowski,
S. Walczak, J. Kowalska, J. Zuberek, M. Lukaszewicz,
´
T. Andryszewski, M. Iwan, J. Duszynski and R. Hołyst, Sci.
Rep., 2019, 9, 5906.
59 R. Kasprzyk, B. J. Starek, S. Ciechanowicz, D. Kubacka,
J. Kowalska and J. Jemielity, Chem. -Eur. J., 2019, 25, 6728–
6740.
E. Nowak, T. D. W. Claridge, E. Darzynkiewicz, M. Nowotny 60 A. Niedzwiecka, J. Marcotrigiano, J. Stepinski, M. Jankowska-
and J. Jemielity, Nucleic Acids Res., 2017, 45, 8661–8675.
44 B. A. Wojtczak, P. J. Sikorski, K. Fac-Dabrowska, A. Nowicka,
M. Warminski, D. Kubacka, E. Nowak, M. Nowotny,
Anyszka, A. Wyslouch-Cieszynska, M. Dadlez, A.-C. Gingras,
P. Mak, E. Darzynkiewicz, N. Sonenberg, S. K. Burley and
R. Stolarski, J. Mol. Biol., 2002, 319, 615–635.
J. Kowalska and J. Jemielity, J. Am. Chem. Soc., 2018, 140, 61 J. Jemielity, T. Fowler, J. Zuberek, J. Stepinski,
5987–5999.
M.
Lewdorowicz,
A.
Niedzwiecka,
R.
Stolarski,
45 Y. Lu and P. S. Low, Adv. Drug Delivery Rev., 2012, 64, 342–
352.
46 W. X. Ren, J. Han, S. Uhm, Y. J. Jang, C. Kang, J.-H. Kim and
J. S. Kim, Chem. Commun., 2015, 51, 10403–10418.
E. Darzynkiewicz and R. E. Rhoads, RNA, 2003, 9, 1108–1122.
62 J. Kowalska, M. Lukaszewicz, J. Zuberek, M. Ziemniak,
E. Darzynkiewicz and J. Jemielity, Bioorg. Med. Chem. Lett.,
2009, 19, 1921–1925.
47 E. C. Calvaresi and P. J. Hergenrother, Chem. Sci., 2013, 4, 63 M. Gu, C. Fabrega, S.-W. Liu, H. Liu, M. Kiledjian and
2319.
C. D. Lima, Mol. Cell, 2004, 14, 67–80.
48 D. Irby, C. Du and F. Li, Mol. Pharm., 2017, 14, 1325–1338.
49 M. Warminski, P. J. Sikorski, Z. Warminska, M. Lukaszewicz,
A. Kropiwnicka, J. Zuberek, E. Darzynkiewicz, J. Kowalska
and J. Jemielity, Bioconjugate Chem., 2017, 28, 1978–1992.
64 A. Wypijewska del Nogal, M. D. Surleac, J. Kowalska,
M.
Lukaszewicz,
J.
Jemielity,
M.
Bisaillon,
E. Darzynkiewicz, A. L. Milac and E. Bojarska, FEBS J.,
2013, 280, 6508–6527.
ˆ
50 A. F. Trindade, R. F. M. Frade, E. M. S. Maçoas, C. Graça, 65 G. Pistritto, D. Trisciuoglio, C. Ceci, A. Garu and G. D'Orazi,
C. A. B. Rodrigues, J. M. G. Martinho and C. A. M. Afonso,
Aging, 2016, 8, 603–619.
Org. Biomol. Chem., 2014, 12, 3181–3190.
© 2021 The Author(s). Published by the Royal Society of Chemistry
Chem. Sci., 2021, 12, 10242–10251 | 10251