Cell-Penetrating Streptavidin: A General Tool for Bifunctional Delivery with Spatiotemporal Control, Mediated by Transport Systems Such as Adaptive Benzopolysulfane Networks

Article abstract of DOI:10.1021/jacs.9b13621

In this report, cell-penetrating streptavidin (CPS) is introduced to exploit the full power of streptavidin-biotin biotechnology in cellular uptake. For this purpose, transporters, here cyclic oligochalcogenides (COCs), are covalently attached to lysines of wild-type streptavidin. This leaves all four biotin binding sites free for at least bifunctional delivery. To maximize the standards of the quantitative evaluation of cytosolic delivery, the recent chloroalkane penetration assay (CAPA) is coupled with automated high content (HC) imaging, a technique that combines the advantages of fluorescence microscopy and flow cytometry. According to the resulting HC-CAPA, cytosolic delivery of CPS equipped with four benzopolysulfanes was the best among all tested CPSs, also better than the much smaller TAT peptide, the original cell-penetrating peptide from HIV. HaloTag-GFP fusion proteins expressed on mitochondria were successfully targeted using CPS carrying two different biotinylated ligands, HaloTag substrates or anti-GFP nanobodies, interfaced with peptide nucleic acids, flipper force probes, or fluorescent substrates. The delivered substrates could be released from CPS into the cytosol through desthiobiotin-biotin exchange. These results validate CPS as a general tool which enables unrestricted use of streptavidin-biotin biotechnology in cellular uptake.

Full text of DOI:10.1021/jacs.9b13621

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Article
Cell-Penetrating Streptavidin: A General Tool for Bifunctional
Delivery with Spatiotemporal Control, Mediated by Transport
Systems Such as Adaptive Benzopolysulfane Networks
́
Javier Lopez-Andarias,* Jacques Saarbach, Dimitri Moreau, Yangyang Cheng, Emmanuel Derivery,
́
́
Quentin Laurent, Marcos Gonzalez-Gaitan, Nicolas Winssinger, Naomi Sakai, and Stefan Matile*
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ABSTRACT: In this report, cell-penetrating streptavidin (CPS) is
introduced to exploit the full power of streptavidin−biotin
biotechnology in cellular uptake. For this purpose, transporters,
here cyclic oligochalcogenides (COCs), are covalently attached to
lysines of wild-type streptavidin. This leaves all four biotin binding
sites free for at least bifunctional delivery. To maximize the
standards of the quantitative evaluation of cytosolic delivery, the
recent chloroalkane penetration assay (CAPA) is coupled with
automated high content (HC) imaging, a technique that combines
the advantages of fluorescence microscopy and flow cytometry.
According to the resulting HC-CAPA, cytosolic delivery of CPS
equipped with four benzopolysulfanes was the best among all tested
CPSs, also better than the much smaller TAT peptide, the original cell-penetrating peptide from HIV. HaloTag-GFP fusion proteins
expressed on mitochondria were successfully targeted using CPS carrying two different biotinylated ligands, HaloTag substrates or
anti-GFP nanobodies, interfaced with peptide nucleic acids, flipper force probes, or fluorescent substrates. The delivered substrates
could be released from CPS into the cytosol through desthiobiotin−biotin exchange. These results validate CPS as a general tool
which enables unrestricted use of streptavidin−biotin biotechnology in cellular uptake.
Covalent protein modification has been used previously for
cellular uptake. Examples include supercharging of proteins by
either addition of positive or removal of negative charges17 or
simple covalent conjugation to possible new transporters such
as boronic acids or halogen-bond donors.5−10,18
To elaborate on the idea of CPS, we selected COCs as
transporters. COCs such as asparagusic acid (AA),11,12
epidiketodithiopiperazines,13 diselenolipoic acid (DSL),14 or
the most recent benzopolysulfanes (BPS)15 are currently being
explored to access increasingly unorthodox dynamic covalent
oligochalcogenide exchange chemistry on the way into the
cytosol. Such thiol-mediated uptake of COCs11−15 and related
transporters7−10 has allowed delivery of not only small
molecules but also larger substrates such as DNA,9b
antibodies,9c quantum dots,8 other nanoparticles,9c liposomes,
and polymersomes11d to the cytosol without significant capture
INTRODUCTION
■
Streptavidin1−4 is a 52 kDa β-barrel tetramer that binds one
biotin per monomer with exceptionally high affinity because
the barrels close upon binding (Figure 1A). The advantages of
such encapsulation combined with tetravalency, stability, and
ease of use empower the high versatility of biotin−streptavidin
technology. For cellular uptake, streptavidin has been used to
noncovalently couple biotinylated substrates to biotinylated
transporters such as cell-penetrating peptides (CPPs)5 and
other dynamic covalent systems,6 including cell-penetrating
poly(disulfide)s,7,8 related disulfide-containing systems,9,10 and
cyclic oligochalcogenides (COCs, Figure 1B).11−15 However,
this approach is limited to the reliable delivery of only one
functionality because it is difficult to control the interfacing of
more than two different ligands with the streptavidin tetramer.
This limitation is occasionally overcome by covalent linking of
fluorophores to proteins.14 A more powerful solution would be
cell-penetrating streptavidin (CPS) with covalently attached
transporters and all four binding sites free to harness the full
power of streptavidin−biotin technology for bifunctional
delivery (Figure 1C) with, for example, retention-using-
selective-hooks (RUSH)-like16 spatiotemporal control (Figure
1D).
Received: December 18, 2019
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Figure 1. (A) Molecular model of wild-type streptavidin tetramer
with bound biotins (yellow) and lysine residues (red, front view) used
to (C) covalently attach transporters. (B) Conventional use of
streptavidin to interface biotinylated transporters with one biotiny-
lated substrate. (C) Cell-penetrating streptavidin (CPS) with all
biotin binding sites free to interface with two different biotinylated
substrates S1 and S2 for bifunctional uptake with (D) spatiotemporal
control.
within endosomes. Mechanistic hypotheses envision COCs as
molecular walkers, walking along disulfide tracks in membrane
proteins12,14,19,20 through the transient micellar pores known
from CPPs but also from disulfide-rich scramblases or voltage-
gated ion channels (Figures 2B and 2C).14,20,21 Driving the
growing impact of dynamic covalent chemistry to cellular
uptake6−14 to the extreme, BPS have been hypothesized to act
by forming adaptive networks of rare sulfur species such as
macrocycles 1 containing up to 19 sulfur atoms for cells to
select from (Figure 2D).15 BPS are known to occur in marine
natural products,22 have attracted early attention in total
synthesis,23 and appeared top in recent library screens to
reverse cognitive defects in mouse models.24
The objective of this study was to create a general tool which
warrants unrestricted availability of streptavidin−biotin tech-
nology in cellular uptake. With COCs as a timely example of
freely variable transporters, bifunctional delivery with spatio-
temporal16 controllability is tackled as a functional challenge of
biological relevance (Figure 1D). Specifically, HC-CAPA, a
new combination of the chloroalkane penetration assay
(CAPA)25,26 with automated high content fluorescence
microscopy,27 is introduced first to secure direct quantitative
functional evidence for cytosolic delivery of CPS. On the basis
of these results, compatibility of CPS with targeted delivery,
the interfacing of nanobodies,8 peptide nucleic acids (PNAs),28
and mechanosensitive fluorescent flipper probes29 with
HaloTag-GFP fusion proteins,25,30 and their controlled release
through intracellular desthiobiotin−biotin exchange are
demonstrated.3,4,16,29a
Figure 2. (A) Synthesis of CPSs 3−6: (a) PBS, pH 7.4, rt, 2 h; (b)
CuSO4·5H2O, BTTAA, sodium ascorbate, aminoguanidine hydro-
chloride, PBS, pH 7.4, rt, 1 h; (c, d) ref 15. (B−D) Working
hypotheses for the modes of action of COCs as molecular walkers (B)
first exchanging with exofacial thiols and (C) then walking along
disulfide tracks and transient micellar pores into cells and of (D) BPS
as an adaptive network including macrocycles as large as 1.
rather than carboxylates to preclude misleading CPP-like
contributions5−8,21 to the uptake activity of CPS due to
increased net positive charge.26,17 The synthesis of CPSs 3−6
is shown as an example (Figures 2A and 3, Schemes S7 and
S8). The reaction of 2 and a controlled amount of activated
ester 7 gave modified streptavidin 8 with up to ca. eight azide
groups. The degree of functionalization was estimated using a
colorimetric amine assay (Figure S1). The obtained key
intermediate 8 underwent copper-catalyzed alkyne−azide
cycloaddition reaction (CuAAC) quantitatively with alkyny-
lated transporter 9 under mild conditions to give CPSs with ca.
one (3, BPS1), two (4, BPS2), four (5, BPS4) or eight
covalently linked BPSs (6, BPS8, Figure 2A). BPS 9 was
prepared by multistep synthesis from catechol 10 following
previously reported procedures.15 CuAAC between pre-CPS 8
and other alkynylated transporters and controls afforded CPSs
covalently linked with four or eight AAs (11, AA4; 12, AA8),
four AA-arginines (13, RAA4), four or eight DSLs (14, DSL4;
15, DSL8), four DSL-arginines (16, RDSL4), and four or eight
myristic acids (17, MA4; 18, MA8, Figure 3, Schemes S1, S7,
and S8). An overview of CPS with different transporters, CPS
complexes with different substrates, and controls described in
the following is provided in Figure 3. A comprehensive
summary of all structures used can be found in Figure S39,
Supporting Information.
RESULTS AND DISCUSSION
■
Design and Synthesis of CPS. Wild-type streptavidin
tetramer 2 contains 16 lysine residues (Figures 1A and 2A),1−4
which were partially derivatized to give CPS with different
numbers of COCs. We chose to chemically modify amines
Cytosolic Delivery of CPS Evidenced by HC-CAPA.
CAPA has been introduced recently by the Kritzer group to
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Figure 3. Structure of monomers, CPS with n transporters T, CPS complexes with n substrates S1 and S2, and controls made and used in this study
with representative assembly of 39 and indication of pertinent figures (Fig) reporting on activity. n is the stoichiometry used for complex
preparation (35: n = 2−4, see text); 47 and 49 are a mixture of m/p-carboxyfluorescein isomers.
quantify cytosolic delivery reliably based on function.25 This is
important because the commonly used fluorescence intensity-
based assays are often significantly affected by the environ-
ment2,11−15 and, in general, demonstrate neither cytosolic
location nor intact functionality.25,26 CAPA operates with
HGM cells, e.g., HeLa cells that have a fusion protein of
HaloTag and GFP (Figure 4F, green) expressed on the outer
surface of mitochondria (Figure 4A).25,30 Delivered chlor-
oalkanes, such as 19, react in cytosol with HaloTags (Figure
4B) and thus inhibit reaction with the subsequently added
fluorescent chloroalkane 20 (Figure 4C and 4H vs 4I, red).
Thus, the delivery of chloroalkanes can be quantified from the
fluorescence intensity of 20 on mitochondria. To secure
quantitative data of the highest possible quality, we adapted
CAPA to an image-based assay using automated high-content
fluorescence microscopy (referred to also as high-content
screening (HCS) or high-content analysis (HCA)),27 preserv-
ing the power of flow cytometry to record and analyze
thousands of cells in a short period of time without losing the
subcellular spatial resolution of fluorescence imaging. For HC-
CAPA, the mitochondria with HaloTags were first segmented
applying a top-hat transform to the unmodifiable fluorescence
of the GFP (Figure 4F). Then this mask was used to collect the
intensity of 20 only in the region of mitochondria, thus
excluding any possible false positives from off-target signals
(Figure 4G, Figures S2−S5).
is the CPS concentration needed for 50% inhibition of
HaloTagging by rhodamine 20. The best CP50 = 2.1 0.3 μM
was found for BPS4 23, with the onset of activity detectable
already in the nanomolar range (Figures 4D (●), 4E, and 4I).
This activity exceeded the CP50 = 3.1 0.5 μM reported for
chloroalkane-tagged Tat peptide, the original CPP from the
HIV virus.25 Although direct quantitative comparison of these
two values has to be made with due caution considering the
different methods used for data collection, it perfectly
illustrates the high efficiency of cytosolic delivery with BPS4
23. BPS together with a large 52 kDa protein outperforming
this small undecapeptide in cytosolic delivery is particularly
interesting considering that uptake efficiencies in general
decrease rapidly with increasing substrate size.5,8,9,11
The presence of fewer and more transporters in BPS1 21,
BPS2 22, and BPS8 24 reduced activity (Figure 4E). The
second best COC was AA with CP50 = 8 1 μM for AA8 26
and CP50 = 14.9 0.5 μM for AA4 25, followed by DSL4 28
with CP50 = 34 6 μM (Figures 4D−I). The presence of more
transporters in DSL8 29 rather deteriorated the poor
performance of DSL under these conditions. Insertion of
guanidinium cations between the protein and COCs in RAA4
27 and RDSL4 30 was also ineffective in improving the
transport activity, thus excluding contributions from CPP-like
mechanisms7−11 (CP50 = 20 1 μM for 27, >35 μM for 30).
Decreasing activities from BPS4 23 to BPS8 24 but not from
AA4 25 to AA8 26 suggested that the higher hydrophobicity of
BPS starts to hinder thiol-mediated uptake in the presence of
more than four transporters per streptavidin, possibly due to
decreasing solubility in water, aggregation, and even precip-
itation (Figure 4E). The best activity found with an
intermediate number of BPS, or “Goldilocks” behavior,31
implied that the right balance between sufficient hydrophilicity
The newly devised HC-CAPA was then used to assess
cytosolic delivery of CPS-chloroalkane complexes 21−31,
prepared by mixing the corresponding CPS with biotinylated
chloroalkane 19 (Figure 3, Scheme S9). The absence of
unbound 19 was assured using a substoichiometric 3 equiv of
ligand per CPS and by meticulous purification using centrifugal
filters. Results from HC-CAPA were quantified as CP50, which
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mechanistic studies on BPS-mediated uptake was less relevant
and off topic. Nevertheless, retention on and thiol-meditated
release from thiol-affinity columns, arguably the most
compelling test for thiol-mediated uptake,14a,15 was confirmed
for intact BPS4 23 (Figure S7). As previously noted with AA,12
the presence of 10% serum in Leibovitz’s media did not affect
the activity much, resulting in a less than doubled CP50 of BPS4
23.
Targetless Delivery of CPS into Various Cells. The
delivery of four biotinylated fluoresceins 3214 in BPS4 33 into
unmodified HeLa cells afforded confocal laser scanning
microscopy (CLSM) images with diffusely emitting cells,
including their nuclei (Figures 5B and S8).15 Similar images
Figure 4. (A−C) Schematic representation of CAPA: (A) HGM cells
were incubated with (B) 21−31 and then (C) 20. (D) HC-CAPA
■
●
▼
dose−response curves for 23 ( ), 25 ( ), and 28 ( ). (E) CP50
values of 21−26 and 28. Statistical significance (**** p < 0.0001)
was estimated by the t test using GraphPad Prism. (F−I)
Representative images for HC-CAPA: (F) top-hat transform (white)
of the image generated from the emission of GFP (green); (G) cell
body (light blue) and mitochondria (yellow) masks applied to
selectively extract from the mitochondria the emission intensity in the
rhodamine channel; (H) rhodamine channel (red) from labeling with
20 without incubation with transporters and (I) after incubation with
20 μM 23 (nuclei in blue; Hoechst 33342, poststaining, scale bars 50
μm).
Figure 5. CLSM images of (A) C2C12 cells after incubation with
fluorescently labeled BPS4 35 (n34 = 2, 10 μM, 2 h), of HeLa cells
after incubation with (B) BPS4 33, (C) AA4 36, and (D) RAA4 37 (all
10 μM, 8 h), and of HGM cells after incubation with (E) BPS4 41
(emission top, 40; bottom, GFP; 5 μM, 2 h), (F) 39 (top, 34;
bottom, 34 (red) + GFP (green), 5 μM, 2 h), (G) 45 (top, 34;
bottom, 34 (red) + GFP (green), 2.5 μM, 2 h), and (H) 46 (top, 40;
bottom, 40 (red) + GFP (green), 5 μM, 2 h) in Leibovitz’s media;
complex stoichiometry indicates the ratios of preparation. (F′−H′)
Design of experiments F−H. Scale bars: 10 μm.
and number of transporters is rather subtle. Goldilocks
behavior further supported that simple passive diffusion does
not account for the top activity of BPS4 23. Corroborative
experimental support for this important conclusion was
secured with myristyl control MA4 31, which is as hydrophobic
as BPS but completely inactive (Figure S6, Table S3, and
Scheme S9).
We previously reported the mechanistic insights in support
of thiol-mediated uptake of BPS, including partial inhibition
with Ellman’s reagent which inactivates cell surface thiols,
insensitivity toward endocytosis inhibitors (cytochalasin B,
wortmannin, chlorpromazine, or methyl-β-cyclodextrin), the
formation of complex adaptive networks of extreme sulfur
species in the presence of thiols and disulfides (up to
heptamers 1, Figure 1D), and stability in PBS buffer for >2
weeks.15 In the current context focusing on cell-penetrating
streptavidin, which is a general and unrestricted availability of
biotin−streptavidin biotechnology for cellular uptake inde-
pendent of the transporter used, repetition of these
were obtained with biotinylated rhodamine 342 in BPS4 35
inside C2C12, HeLa, MCF7, or MDCK cells (Figures 5A and
S9; n34 = 2). Weaker fluorescence was observed in CLSM
images of AA4 36 in HeLa cells (Figure 5C). The added
cations in RAA4 37 and RDSL4 38 did not help to improve the
activity significantly, confirming that CPP-like mechanisms7−11
are negligible for COC-mediated uptake (Figures 5D and S8).
Contrary to BPS4 33, images of AA4 36 and RAA4 37 contain
bright punctate patterns on a diffuse background, which usually
indicate contributions from endolysosomes and perhaps also
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precipitating aggregates besides emission from the cytosol.
With flow cytometry, such contributions from endosomal
capture are recorded as false positives. The here introduced
HC-CAPA is powerful because all eventual false positives25,26
are eliminated without losses in fast acquisition of statistically
relevant high-content data.
fluorescein as a responsive model substrate was equipped with
desthiobiotin 47, N-ethylbiotin 48, and biotin 49 (Figures 3
and 6, Schemes S4 and S13). Desthiobiotin (Kd = 10−11 M)3
Targeted Delivery of Fluorophores, Nanobodies, and
PNA. Using the best CPS, that is, BPS4 5, we explored the
simultaneous delivery of two functions, here a reporter and a
targeting unit. BPS4 5 was loaded with biotinylated
chloroalkane 19 and rhodamine 34 (Figures 3 and 5F′,
Scheme S12). Incubation of HeLa cells expressing HaloTags
on mitochondria with the resulting complex 39 afforded
CLSM images with fluorescently labeled mitochondria (Figure
5F, top). The diffuse emission from cytosol and nuclei
observed without targeting (Figures 5A and 5B) disappeared,
thus confirming that delivery of complex 39 to the HaloTag on
the surface of mitochondria is as specific as expected. A good
colocalization with GFP emission demonstrated the targeted
delivery of the complex to HaloTags (Figures 5F, bottom,
S10A, and S10B). HaloTag targeting was substrate independ-
ent; the substitution of rhodamine 34 with flipper 4029 gave
similar images for delivery of the respective complex 41 to
HaloTags on mitochondria (Figures 3, 5E, and S11). This
result was of interest for the fluorescence imaging of precisely
localized membrane tension within living cells.29 Targeted
delivery of biotinylated and Cy5-labeled 18-mer PNA 4228 in
complex 43 was also accomplished in HGM cells (vide infra,
Figure S13).
Biotinylated anti-GFP nanobodies8 44 were probed as
alternative targeting units in complex 45, recognizing GFP
instead of HaloTag of the fusion protein on mitochondria. The
resulting images showed good colocalization with GFP
together with less important endolysosomal capture and/or
precipitates (red dots, Figures 5G and S12). These off-
targeted, endolysosomal capture and/or precipitates probably
originate from the larger oligomers of complex 45 cross-linked
through the multivalent anti-GFP nanobodies8 44 with more
than one biotin (Figures 3 and 5G′). Shifts in uptake
mechanism from cytosolic delivery to endosomal capture
have been observed frequently with increasing substrate size,
although much more for CPPs5 than for thiol-mediated
uptake.8,9,11 To minimize these side effects, complex 45 was
prepared by incubation of BPS4 5 with 3 equiv of fluorophores
34 before incubation with the multivalent nanobodies 44, a
procedure that should in turn increase formation of the
targetless CPS side product 35 loaded with four fluorophores
34 only (Figure 3, n34 = 4). However, eventual contributions
from off-targeted delivery with CPS 35 were very minor
(diffuse red as in Figures 5A, 5G, and S12). Considering this
situation, the targeted delivery mediated by anti-GFP nano-
bodies8 44 to GFP expressed on the surface of mitochondria
was remarkably efficient and supported, with red fluorescence
originating from ligand 34, that the majority of complex 45
reaches the cytosol in intact functional form (yellow, Figures
5G, 5G′, and S12). The control complex 46 without COCs
was unable to penetrate cells and showed no colocalization
with GFP (Figures 5H, S10C, and S10D).
Figure 6. (A) Delivery of 47 with BPS4 5 followed by addition of
biotin 53 to release 47 from complex 50 in the cytosol. (B−D)
Original image of the multiwell plate to monitor the time course of
substrate release in HeLa cells by automated high-content
fluorescence microscopy. Thousands of cells preincubated with (B)
51, (C) 50, or (D) 52 (10 μM, 6 h in Leibovitz’s medium) were
treated with 53 (middle), 54 (bottom; 40 μM each), or none (top)
and imaged every 12 min (left to right; shown are every other
images). (E−G) Spatiotemporal control of (F) the delivery of 58 with
5 and 59 to the mitochondria of HGM cells (5 μM, 2 h incubation in
Leibovitz’s medium) and (G) the release of 58 after addition of 53
(58 (red) + GFP (green), 40 μM). Scale bars: 10 μm.
and N-ethylbiotin (Kd = 10−9 M)4 bind to streptavidin but
with reduced affinity compared to biotin (Kd = 10−15 M).3,4
The corresponding complexes 50−52 were readily accessible
by incubation with BPS4 5 (Figures 3 and 6).
Desthiobiotin 47 was first delivered in complex 50; then
biotin 53, a nontoxic vitamin, or its methyl ester 54 was added
at a 40 μM concentration as recommended by the RUSH
technology16 to diffuse into the cell, exchange, and release
substrate 47 in the cytosol (Figure 6A). As streptavidin
strongly quenches the fluorescence of bound carboxyfluor-
escein,2 intracellular desthiobiotin−biotin exchange was
detectable by fluorescence recovery of the released substrate
47, whereas the biotin−streptavidin side product 55 or 56
passes undetected.
Controlled Intracellular Release of Substrates from
CPS. Once delivered in the cells, the substrate would ideally be
detached from transporter and target at a well-defined time16
to ensure and follow its proper function on demand (Figure
1D). To elaborate on the timed release of substrates from CPS,
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Quantitative release kinetics were obtained by automated
high-content fluorescence microscopy of wells containing
thousands of cells with an increasing incubation time with
biotin 53 and its methyl ester 54 (Figures 6B−D and S14).
Identical t50 = 17 min obtained for controlled release of
desthiobiotin 47 from CPS 50 with 53 and 54 demonstrated
that passive diffusion of the protonatable carboxylate 53 into
the cell is not rate limiting (Figures 6C and S14). In clear
contrast, the release of N-ethylbiotin 48 from complex 51 was
spontaneous and not affected by the presence of either 53 or
54 (Figure 6B). The lower fluorescence intensity observed
with 48 (Figure 6B) compared with 47 (Figure 6C) suggested
that 48 dissociated from CPS already during incubation time,
diffused out of cells, and was washed away before the start of
the acquisition (Figure S14C). Similar spontaneous release of
desthiobiotin 47 was slow on the time scale of biotin-
controlled release (Figures 6C and S14B). This difference in
spontaneous release was in agreement with the poorer binding
affinity of N-ethylbiotin 48 (Kd = 10−9 M)4 compared to
desthiobiotin 47 (Kd = 10−11 M).3 On the other hand, the
controlled release of 49 from 52 by biotin−biotin exchange
was negligible even after an extended incubation period
(Figure 6D), consistent with much stronger binding of biotin
49 compared to desthiobiotin 47. Insufficient release of biotin
49 and excessive release of N-ethylbiotin 48 added up again in
Goldilocks behavior,31 identifying desthiobiotin 47 with
neither too strong nor too weak binding as the ligand of
choice for controlled release within cells.
To finally combine targeted delivery and controlled release,
PNA was selected as a prototypical example of a useful reagent
that has been limited in scope by its cellular permeability.28 In
complex 57, CPS 5 was loaded with desthiobiotinylated 18-
mer PNA 58 and chloroalkane 59 (Figures 3 and 6E, Schemes
S5, S6, and S13). The good colocalization (yellow) of Cy5
attached to PNA 58 (red) and GFP (green) demonstrated
targeted delivery of complex 57 to HaloTags on the
mitochondria of HGM cells in intact form and with excellent
selectivity (Figures 6F and S15). Subsequent addition of biotin
53 induced PNA release, detectable as weakened emission of
Cy5 on the mitochondria due to dilution of the liberated PNA
58 into the cytosol (Figures 6G, 6E, and S15). These results
thus supported the compatibility of CPS delivery with
spatiotemporal control using biotin analogs.16
Materials and methods; small-molecule synthesis; cell
lines and plasmids; CPS synthesis; combining CAPA
with automated HC microscopy; cellular uptake of CPS;
targeted delivery; supporting references; NMR spectra;
characterization of PNA derivatives; summary of
structures (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
́
Javier Lopez-Andarias − School of Chemistry and Biochemistry
and National Centre of Competence in Research (NCCR)
Chemical Biology, University of Geneva, Geneva 1211,
Switzerland; Email: javier.lopez@unige.ch
Stefan Matile − School of Chemistry and Biochemistry and
National Centre of Competence in Research (NCCR) Chemical
Biology, University of Geneva, Geneva 1211, Switzerland;
orcid.org/0000-0002-8537-8349; Email: stefan.matile@
unige.ch
Authors
Jacques Saarbach − School of Chemistry and Biochemistry and
National Centre of Competence in Research (NCCR) Chemical
Biology, University of Geneva, Geneva 1211, Switzerland
Dimitri Moreau − School of Chemistry and Biochemistry and
National Centre of Competence in Research (NCCR) Chemical
Biology, University of Geneva, Geneva 1211, Switzerland
Yangyang Cheng − School of Chemistry and Biochemistry and
National Centre of Competence in Research (NCCR) Chemical
Biology, University of Geneva, Geneva 1211, Switzerland
Emmanuel Derivery − MRC Laboratory of Molecular Biology,
Cambridge CB2 0QH, United Kingdom; orcid.org/0000-
0003-3927-5944
Quentin Laurent − School of Chemistry and Biochemistry and
National Centre of Competence in Research (NCCR) Chemical
Biology, University of Geneva, Geneva 1211, Switzerland
́
́
Marcos Gonzalez-Gaitan − School of Chemistry and
Biochemistry and National Centre of Competence in Research
(NCCR) Chemical Biology, University of Geneva, Geneva
1211, Switzerland
Nicolas Winssinger − School of Chemistry and Biochemistry
and National Centre of Competence in Research (NCCR)
Chemical Biology, University of Geneva, Geneva 1211,
Switzerland; orcid.org/0000-0003-1636-7766
Naomi Sakai − School of Chemistry and Biochemistry and
National Centre of Competence in Research (NCCR) Chemical
Biology, University of Geneva, Geneva 1211, Switzerland
CONCLUSION
■
This study introduces CPS as a privileged scaffold for cellular
uptake, simple and robust, with biotin−streptavidin technology
made fully available for general bifunctional delivery with
spatiotemporal control. HC-CAPA, the recent CAPA25
combined with automated high-content fluorescence micros-
copy27 is devised as the method of choice to quantify cytosolic
delivery; the currently emerging benzopolysulfanes15 are
validated as COCs of choice for cytosolic delivery. Their
performance supports the integration of increasingly unortho-
dox32 oligochalcogenide exchange chemistry to find new ways
to enter into cells and encourages studies on mechanistic
hypotheses, i.e., molecular walkers and dynamic-covalent
adaptive networks.14,15,19−22
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.9b13621
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This paper is dedicated to George Whitesides on the occasion
of his 80th birthday. We thank E. Bartolami, A. Goujon, R.
Martinent, and K. Strakova for contributions to CPS synthesis,
́
H. Riezman, N. Jimenez-Rojo, and S. Vossio for help with
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.9b13621.
plasmid amplification, J. A. Kritzer (Tufts) for providing
materials, the NMR, the MS, and the Bioimaging platforms for
services, and the University of Geneva, the NCCR Chemical
Biology, the NCCR Molecular Systems Engineering, and the
■
sı
F
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J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
pubs.acs.org/JACS
Article
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Ward, T. R. A Cell-Penetrating Artificial Metalloenzyme Regulates a
Gene Switch in a Designer Mammalian Cell. Nat. Commun. 2018, 9,
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(8) Derivery, E.; Bartolami, E.; Matile, S.; Gonzalez-Gaitan, M.
Efficient Delivery of Quantum Dots into the Cytosol of Cells Using
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Swiss NSF for financial support. J.L.A. acknowledges a Curie
fellowship (740288).
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