Transport of Nucleoside Triphosphates into Cells by Artificial Molecular Transporters

Article abstract of DOI:10.1002/anie.201801306

Chemically modified nucleoside triphosphates (NTPs) are widely exploited as unnatural metabolites in chemical biology and medicinal chemistry. Because anionic NTPs do not permeate cell membranes, their corresponding neutral precursors are employed in cell-based assays. These precursors become active metabolites after enzymatic conversion, which often proceeds insufficiently. Here we show that metabolically-active NTPs can be directly transported into eukaryotic cells and bacteria by the action of designed synthetic nucleoside triphosphate transporters (SNTTs). The transporter is composed of a receptor, which forms a non-covalent complex with a triphosphate anion, and a cell-penetrating agent, which translocates the complex across the plasma membrane. NTP is then released from the complex in the intracellular milieu and accumulates in nuclei and nucleoli in high concentration. The transport of NTPs proceeds rapidly (seconds to minutes) and selectively even in the presence of other organic anions. We demonstrate that this operationally simple and efficient means of transport of fluorescently labelled NTPs into cells can be used for metabolic labeling of DNA in live cells.

Full text of DOI:10.1002/anie.201801306

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Accepted Article
Title: Transport of Nucleoside Triphosphates into Cells by Artificial
Molecular Transporters
Authors: Zbigniev Zawada, Ameneh Tatar, Pavle Mocilac, Miloš
Buděšínský, and Tomas Kraus
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201801306
Angew. Chem. 10.1002/ange.201801306
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Transport of Nucleoside Triphosphates into Cells by Artificial
Molecular Transporters
Zbigniew Zawada, Ameneh Tatar, Pavle Mocilac, Miloš Buděšínský and Tomáš Kraus*
Abstract: Chemically modified nucleoside triphosphates (NTPs) are
widely exploited as unnatural metabolites in chemical biology and
medicinal chemistry. Because anionic NTPs do not permeate cell
membranes, their corresponding neutral precursors are employed in
cell-based assays. These precursors become active metabolites after
their enzymatic conversion, which often proceeds insufficiently. Here
we show that metabolically-active NTPs can be directly transported
into eukaryotic cells and bacteria by the action of designed synthetic
nucleoside triphosphate transporters. The transporter is composed of
a receptor, forming a non-covalent complex with a triphosphate anion,
and a cell-penetrating agent, which translocates the complex across
the plasma membrane. NTP is then released from the complex in the
intracellular milieu and accumulates in nuclei and nucleoli in high
concentration. The transport of NTPs proceeds rapidly (seconds to
minutes) and selectively even in the presence of other organic anions.
We demonstrate that this operationally simple and efficient means of
transport of fluorescently labeled NTPs into cells can be used for
metabolic labeling of DNA in live cells.
nucleoside/nucleotide-phosphorylating kinases constitute a very
efficient cellular barrier preventing the interference of unnatural
NTPs with the cell metabolism. Nevertheless, the interest in using
unnatural NTP analogues in chemical biology and medicinal
chemistry fuelled efforts to develop methods that would allow
delivery of NTPs to cells. Most approaches described for
eukaryotic cells to date are based on i) transient disruption of the
plasma membrane by physical and mechanical means,[3] ii)
uptake of nanoparticulate assemblies of NTPs with positively
charged polymers[4] or highly concentrated DNA transfection
reagents,[5] and iii) uptake of prodrugs[6] constituted of NTP
analogues modified with lipophilic diester functions at γ-
phosphates, cleavable by intracellular esterases. In the realm of
prokaryotic cells, bacterial nucleoside triphosphate transporters,
found in some obligate intracellular bacteria,[7] were
heterologously expressed in E. coli and provided the delivery of
unnatural NTPs.[8] Although some of the above-mentioned
approaches undoubtedly proved suitable for particular purposes,
currently there is no versatile, simple and scalable approach for
the direct transport of NTPs to cells allowing enzyme-independent
fast intracellular release of NTPs without affecting cell viabilities.
Control over the transport of ions across the plasma
membrane is a vital function of viable cells. The permeability of a
cell membrane to ions, which are not recognized by membrane-
incorporated transporting proteins, is therefore significantly
limited. Particularly the transport of anionic compounds to cells
represents a great challenge in scientific fields ranging from
fundamental research in chemical biology to medicinal
applications.[1]
Herein, we describe selective, rapid and efficient transport of
NTPs into eukaryotic cells and bacteria by the action of synthetic
nucleoside triphosphate transporters (SNTTs; Figures 1 and 2).
We designed SNTT 1 and its fluorescently labelled analogue 2 as
bifunctional molecules, each consisting of i) a receptor site
forming a highly-stable non-covalent complex with a triphosphate
(tripolyphosphate) moiety and ii) a cell-penetrating agent (CPA)
capable of carrying the whole complex across the plasma
Nucleoside triphosphates (NTPs) are important metabolites
involved in numerous cellular processes. Their chemically
modified analogues have long been exploited as antimetabolites
in chemical biology and medicinal chemistry. Due to their anionic
nature, NTPs do not permeate cell membranes and, therefore,
their corresponding neutral and more readily transportable
precursors (nucleosides or monophosphate prodrug derivatives)
are most commonly employed in cell-based assays as well as in
clinical use. However, nucleosides become active metabolites
after sequential phosphorylation to NTPs by intracellular kinases,
which often proceeds with low efficiency for unnatural
analogues.[2] Apparently, the impermeability of the plasma
membrane to anionic NTPs together with the specificity of
[*]
Dr. Zbigniew Zawada, Dr. Ameneh Tatar, Dr. Pavle Mocilac, Dr.
Miloš Buděšínský, Dr. Tomáš Kraus
Institute of Organic Chemistry and Biochemistry of the Czech
Academy of Sciences
Flemingovo nám. 2, 166 10 Praha 6, Czech Republic
E-mail: kraus@uochb.cas.cz
Figure 1. Hypothesized mechanism of the transport of a modified NTP by
the synthetic nucleoside triphosphate transporter (SNTT).
Supporting information for this article is given via a link at the end
of the document.
1
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membrane. Non-covalent binding of the triphosphate moiety of an
NTP to the transporter offers several advantages over a covalent
linkage. First, the synthesis of NTP-CPA conjugates with
intracellularly-cleavable covalent linkers tailored to individual NTP
structures is avoided. Second, the triphosphate (or phosphonato-
diphosphate in some antiviral drugs) anion is a versatile anchoring
point, since it is the only conserved indispensable moiety of all
metabolically active yet structurally very divergent NTP analogues.
We hypothesized that upon entering the intracellular milieu the
complexed triphosphate moiety would be liberated from the
receptor site by competitive displacement with abundant natural
NTPs such as ATP (Figure 1).
In search of a potent receptor of organic triphosphate anions
operative in isotonic aqueous solutions we found that per-6-
amino-β-cyclodextrin formed a highly stable 1:1 stoichiometric
complex with ATP (Ka= 2ꞏ107 M-1 in aqueous isotonic tricine
buffer; Table S3 in SI). Under these conditions, per-6-amino-β-
cyclodextrin outperformed its N-methylated derivative reported
earlier.[9] The CPA moiety was constructed using an arginine-rich
molecular transporter consisting of eight aminocaproic acid 
arginine pairs; this peptidomimetic was reported to have high
efficiency of transport of a covalently attached fluorescent probe
across cell membranes.[10] SNTT 1 (Figure 2) was then prepared
by photo-initiated addition of thiol-terminated peptidomimetic 5
(Schemes S1 and S2 in SI) to 2-O-allyl-per-6-amino-β-
cyclodextrin (3). SNTT 2 labelled with sulforhodamine B (SRB)
was prepared by an analogous way (Scheme S3 in SI).
Figure 3. Live-cell imaging of the transport of fluorescent triphosphate anions
to eukaryotic cells and bacteria. A) U2-OS cells treated with 10 µM complex of
SNTT 1 and Alexa-488-dUTP for 10 min; B) U2-OS cells treated with 10 µM
Alexa-488-dUTP only (control experiment in the absence of SNTT 1, 10 min);
C) U2-OS cells treated with 10 µM complex of SNTT 1 and SRB-triphosphate
4. D) Bacteria E. coli treated with 40 µM equimolar complex of SNTT 1 and
Cy-3-dUTP for 30 min. Inset in D: 3-D reconstruction of stained E. coli (super-
resolution 3D-SIM). Scale bars: 20 µm (A-C); 2 µm (D); 1 µm (inset in D).
Transporting properties of SNTT 1 were initially tested using
U2-OS cells and fluorescently labelled NTPs (Alexa-488-dUTP,
ATTO-495-dUTP, ATTO-488-dUTP, Cy3-dUTP, Alexa-647-dUTP
and Alexa-488-UTP, Fl-UTP; Figure S5 in SI) as well as a non-
nucleoside triphosphate 4 (Figure 2). In order to reduce the
competition between triphosphate moieties of NTPs and anions
(in particular phosphates and carbonates abundantly present in
most culture media) for binding to SNTT 1, short time (seconds to
minutes) transport experiments were carried out in tricine-
buffered isotonic saline solution. Long time treatments (1-12
hours) were performed in L-15 medium (1.78 mM total phosphate,
no carbonate, with or without serum). At the onset, U2-OS cells
were treated with a 10 µM complex of SNTT 1 and Alexa-488-
dUTP in tricine buffer at room temperature and the staining
process was monitored by confocal microscopy. We observed
rapid transport of Alexa-488-dUTP to the intracellular milieu -
approximately one half of the cell population was stained after a
20-second treatment and virtually all cells were stained after a 2-
minute treatment (Section 2.2.2 and Video S1 in SI). High
fluorescence intensity was observed in all cells after 10 minutes
of staining (Figure 3A). Intensities of fluorescence varied among
the individual cells and were significantly higher in the nucleoli and
nuclei than in the cytosol. Under these conditions, the observed
intracellular concentration of Alexa-488-dUTP reached values in
the range of 100-500 µM (see section 2.2.8 in SI). After removaltra
of the SNTT 1/Alexa-488-dUTP solution by washing the cells with
a complete medium, the intracellular fluorescence decreased
within several hours (Video S2 in SI), spontaneous efflux of the
NTP being one of the observed mechanisms. Decrease of the
concentration of SNTT 1 to 2 μM resulted in lower yet still
detectable fluorescence after a 10-minute treatment. When
complete L-15 medium containing 10% serum was employed in
place of tricine buffer (10 μM complex, 10 min), intracellular
fluorescence intensity decreased by 50% (Figure S12 in SI).
Interestingly, transport to U2-OS cells was also observed with
only 1 μM complex of SNTT 1 and NTP (Cy3-dUTP) in L-15
Figure 2. Structures of transporters SNTT 1, SRB-labelled analogous SNTT 2
and SRB-labelled triphosphate 4.
2
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medium without serum applied for 16 hours (Figure S13A in SI)
or with 2 μM complex in L-15 with 10% serum applied for 90 min
(Figure S13B in SI). In both cases Cy3-dUTP was found to be
incorporated in nuclear DNA (vide infra). Non-nucleoside SRB-
triphosphate 4 was transported as well (Figure 3C), supporting
the evidence for the key-role of the triphosphate moiety – rather
than the nucleoside residue – in the transport process. When the
treatment of cells was performed at 4 °C, the intracellular
fluorescence intensity dropped approximately by half (Figure S8
in SI) as compared to that observed at room temperature,
suggesting that the direct translocation rather than endocytosis
(inactive at 4°C) is involved in the mechanism of transport. Control
experiments showed no intracellular staining when a 10 μM
solution of Alexa-488-dUTP alone (Figure 3B) or mixed with free
Next, the selectivity of transport of NTPs by SNTT 1 in the
presence of competing fluorescent anions was tested (Section
2.2.4 and Figure S18 in SI). When a mixture of Alexa-488-dUTP,
Cy3-dUTP and SNTT 1 (each component at 10 μM) was applied
to U2-OS cells, both green and red fluorescence of Alexa-488-
dUTP and Cy3-dUTP, respectively, could be detected inside cells.
However, when Alexa-488-dUTP was replaced with calcein
(green fluorescent tetraanionic dye), only the red fluorescence
was observed in the intracellular milieu with
a confocal
microscope under the same settings, indicating that Cy3-dUTP
but not calcein was transported. Likewise, treatment of cells with
an analogous mixture of sulforhodamine B (red fluorescent
dianionic dye), Alexa-488-dUTP and SNTT 1 resulted in the
detection of green fluorescence of Alexa-488-dUTP only. The
inability of sulforhodamine B to pass the cell membrane even in
receptor
2-O-allyl-per-6-amino-β-cyclodextrin
(3)
and/or
peptidomimetic 5 was applied (Figure S15 and section 2.2.2 in SI). the presence of SNTT 1 stands in striking contrast to the smooth
transport of the analogous sulforhodamine B-triphosphate 4 (cf.
We compared the transport efficiency of SNTT 1 with that of
Figures S18C, S18L in SI and Figure 3C). Collectively, these
per-6-(2-aminoethylamino)-β-cyclodextrin (AEA-β-CD), which
competition experiments provide the evidence that SNTT 1
was recently reported[11] to provide endocytic uptake of NTPs to
selectively transports molecules containing triphosphate moieties
MCF-7 cells. The experiments were performed in both short (10
without creating pores in the plasma membrane, which would be
min) and long (5-16 hours, reported in ref.[11]) time periods in
accessible to other tested fluorescent anions.
tricine buffer and L-15 medium (with or without serum; see
detailed protocols in section 2.2.5 in SI), respectively. Relative
fluorescence of Cy3-dUTP transported with AEA-β-CD to U2-OS
cells was lower by factor 25  260 (Figure S21 in SI) as compared
to transport provided by SNTT 1 and was mostly located on cell
membranes (Figures S19H, S20A and S20B in SI), while it was
absent in nuclei. The action of AEA-β-CD resembles control
experiments performed with the receptor 3 itself (Figure S15 C),
highlighting the key role of the CPA in the design of efficient
transporters.
The non-toxicity of the intracellular delivery systems is of
crucial importance for their applications, and is often limiting.
Treatment of several cell types with SNTT 1 without an NTP under
the conditions employed in staining experiments followed with
viability assays (a 3-day XTT assay; Table S4 in SI) revealed that
SNTT 1 did not decrease cell viabilities. The cell cultures treated
with a complex of SNTT 1 and a fluorescent NTP exhibited
attributes of normal behaviour such as adherence of cells to glass
surface (U2-OS cells, Video S5 in SI) or frequent mitosis (Figure
4; Videos S6 and S7 in SI).
Transport of NTPs by SNTT 1 into other cell lines required
variable concentration of the complex: HeLa (10 μM), TZM-bl (10-
20 μM) Hep-G2 (40 μM), HUVEC (50 μM), Vero (60 μM).
Preliminary investigations revealed that the transporter SNTT 1
was able to translocate Cy3-dUTP even into prokaryotic
unicellular organisms: the Gram-negative bacteria E. coli, (30 min,
40 μM; Figure 3D; Videos S3 and S4 in SI) and Gram-positive
Mycobacterium smegmatis (3 min; 26 μM SNTT 1, 6 μM Cy3-
dUTP, 20 μM Fluorescein-12-UTP; Figure S14 in SI).
In order to gain insight into the role and fate of the transporter
itself, the solution of SRB-labelled transporter (2) and Alexa-488-
dUTP (10 μM) was applied onto U2-OS cells. Tracking of signals
of both fluorescent labels in the intracellular milieu revealed
significant co-localization of Alexa-488-dUTP and the SNTT 2
(Figures S16A and S16B in SI). The intensity maxima of SNTT 2
is localized in nuclei and nucleoli suggesting that positively
charged arginine oligomers of SNTT 2 non-covalently bind to
negatively charged nucleic acids. We noticed that during mitosis
the fluorescence of SNTT 2 in the metaphase plate (Figure S16E)
decreased relative to that of Alexa-488-dUTP (Figure S16C). This
indicates that non-covalently bonded SNTT 2 was displaced from
chromosomal DNA while the fluorescent nucleotide remained due
to its incorporation to DNA (vide infra). Cells also showed
punctuated fluorescent patterns (Figure S22A in SI) in the nuclei,
typical for fluorescently-labelled DNA located in nuclear foci. The
incorporation of Alexa-647-dUTP into DNA after transport by
SNTT 1 was evidenced by the measurement of excitation and
emission spectra (Figure S22B in SI) of the isolated DNA.
Figure 4. Confocal time-lapse live-cell imaging of mitosis of a TZM-bl cell;
the DNA was labelled with Cy3-dUTP. Cy3 fluorophore is represented with
yellow colour for better visibility. Scale bar: 20 µm.
We hypothesized that a fluorescently labelled dNTP capable
of incorporation into DNA (e.g. Cy3-dUTP, Figure 4) at sufficient
rate could be advantageously employed in cell proliferation
assays in place of currently widely used 5-bromo-2′-deoxyuridine
or 5-ethynyl-2′-deoxyuridine-based post labelling methods.[12] To
prove this concept, U2-OS cells were treated with 10 μM Cy3-
dUTP / SNTT 1 solution in tricine buffer for a short time period (3
min) and allowed to incubate in full medium for 30 min. Then the
cells were fixed and the membranes were permeabilized to
remove free Cy3-dUTP. Quantitative counterstaining of DNA with
DAPI followed with flow cytometry analysis revealed identical
3
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10.1002/anie.201801306
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distributions of cells within the cell cycle phases in both direct
incorporation and BrdU assays, respectively (Figure 5). However,
in contrast to the multi-step BrdU procedure, SNTT 1-based
approach requires notably fewer operations. Thus, it is not only
faster and simpler but more importantly, it significantly prevents
the overall loss and damage of cells (cf. Figures 5A and 5B)
allowing scale-down of the assay.
Krásný (Institute of Microbiology, CAS) for the assistance with E.
coli experiments, and Jena Bioscience GmbH for a donation of
NTPs. Financial support was provided by the IOCB CAS
(Targeted research groups) and by Grant Agency of the Czech
Republic (n. 17-14791S).
Keywords: anion transporters •DNA labelling • cell-penetrating
peptides • cyclodextrins
A
B
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Figure 5. Comparison of cell (U2-OS) proliferation assays: A) direct
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In conclusion, the new transporting system described herein
provides an operationally simple means for the selective, rapid
and gentle transport of NTPs (tripolyphosphate anions in general)
into cells without apparent damage to the plasma membrane and
drop in cell viability. The transported NTP is rapidly liberated from
the complex and available in nuclei for incorporation into DNA.
The SNTTs are thus expected to have numerous applications in
related scientific fields, where in cellulo incorporation of unnatural
nucleoside triphosphates into DNA or RNA is required.
Particularly, the immediate intracellular availability of
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enzymatic phosphorylation or cleavage of masking groups in
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direct
transport
of
DNA-incorporable
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alphabet[8a,8b,15] and construction of artificial DNA using
engineered polymerases.[16] In medicinal chemistry, SNTT-
mediated direct application of NTPs could be beneficial in
research on active antiviral and anticancer nucleotides. In a
broader perspective, the conceptually new design of the
molecular transporter is modular and allows tailoring of both the
receptor and the cell-penetrating agent to achieve particular
selectivity towards other anions and/or to specific receptors
present on the membranes of certain cell types.
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Acknowledgements
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We thank our colleagues from the Institute (IOCB CAS): E.
Kužmová, J. Kozák, M. Zavřel, M. Hájek, J. Günterová and J.
Starková for the assistance with cell culturing and cell biology
experiments; K. Nováková, E. Slabá and K. Kertisová for mass
spectrometry experiments; K. Stříšovský, M. Downey and R. Pelc
(Institute of Physiology) for reading the manuscript; M. Hocek
group for the support. We are also indebted to M. Šiková and L.
4
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COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
Nucleoside triphosphates (NTPs) can
be directly transported into eukaryotic
cells and bacteria by the action of
designed synthetic transporters. The
transport of NTPs proceeds rapidly –
within seconds to minutes – and
selectively even in the presence of
other organic anions. This
operationally simple and efficient
means of transport of fluorescently
labeled NTPs into cells can be used
for metabolic labeling of DNA in live
cells.
Zbigniew Zawada, Ameneh Tatar, Pavle
Mocilac, Miloš Buděšínský and Tomáš
Kraus*
Page No. – Page No.
Transport of Nucleoside
Triphosphates into Cells by
Synthetic Molecular Transporters
5
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