RESEARCH ARTICLE SUMMARY
◥
SYNTHETIC BIOLOGY
Designer membraneless organelles
enable codon reassignment of
selected mRNAs in eukaryotes
Christopher D. Reinkemeier*, Gemma Estrada Girona*, Edward A. Lemke†
INTRODUCTION: The ability to engineer
translation of noncanonical (unnatural) amino
acids (ncAAs) site-specifically into proteins in
living cells greatly expands the chemical space
that can be used to control, tailor, and study
cellular function. However, translation is a
complex multistep process in which at least
20 different aminoacylated tRNAs, their cognate
tRNA synthetases, ribosomes, and other
factors need to act in concert to synthesize a
polypeptide chain encoded by an mRNA transcript.
To minimize interference with the host
machinery, we aimed to engineer fully orthogonal
translation into eukaryotes: to encode a
new functionality in response to a specific
codon in only one targeted mRNA, leading to
site-specific ncAA incorporation only into the
selected protein of choice. Although codon
specificity can be achieved with genetic code
expansion (GCE), this technology relies on
using an orthogonal tRNA/tRNA synthetase
pair (one that does not cross-react with any
of the endogenous pairs) to reprogram a stop
codon. Most commonly, the Amber (UAG) stop
codon is used (20% abundance in human
cells), and in principle, stop codon suppression
can happen for every cytoplasmic mRNA
that terminates naturally on this codon. Here,
we present a strategy to generate a distinctly
expanded genetic code for only selected mRNAs.
RATIONALE: We hypothesized that it should
be possible to create an orthogonal translation
system by spatially enriching the key
components of the GCE machinery in an orthogonally
translating (OT) synthetic designer
organelle and by targeting a specific mRNA
to it. In order to perform protein translation,
such an OT organelle would need to be readily
accessible to the entire translational machinery
of the host, thus precluding membrane encapsulation.
Inspired by the concept of phase
separation, which is used by cells to concentrate
specific proteins and RNA locally, we
hypothesized that it might be possible to use
this principle to create such membraneless
OT organelles. In our design, only a spatially
distinct set of ribosomes associated with OT
organelles can use the aminoacylated suppressor
tRNA and thus will decode Amber codons
only in the selected mRNA translated by the
OT organelle, leading to a protein containing
the ncAA.
RESULTS: To bring the modified suppressor
tRNA and the translated mRNA of choice in
close proximity to each other, we used different
strategies to generate highly concentrated
assemblies and spatial separation inside cells:
(i) proteins undergoing phase separation in
cells [fused-in sarcoma (FUS), Ewing sarcoma
breakpoint region 1 (EWSR1), and spindledefective
protein 5 (SPD5), which contain
long intrinsically disordered domains] and
(ii) kinesin motor proteins, which spatially
enrich at microtubule plus ends (KIF13A and
KIF16B). We fused each of these to the suppressor
tRNA synthetase as well as an RNAbinding
domain major capsid protein (MCP)
that binds to a specific RNA motif (ms2 loops)
engineered into the untranslated region of
the mRNA of choice, forming an ms2-MCP
complex. Each of these approaches yielded
the desired local enrichment and preferential
stop codon suppression of the mRNA
tagged with ms2 loops. However, by far the
best performing system was a combination
of phase and spatial separation,
which typically
formed a micrometer-sized
organelle-like structure per
cell. Cells that contained
this organelle efficiently
and selectively performed
Amber suppression of only the targeted mRNA.
We were able to demonstrate the utility and
robustness of these OT organelles by selectively
decoding any of the three stop codons
in a variety of proteins with different ncAA
functionalities in two different mammalian
cell lines.
CONCLUSION: Our results show how to
combine phase and spatial separation inside
cells to allow the concentration of a custom
designed task into a distinct specially designed
membraneless organelle. We successfully demonstrated
that specific and selective protein
translation could be achieved within these OT
organelles, which allowed the introduction of
noncanonical functionalities into proteins in a
codon-specific and mRNA-selective manner.
The system only requires engineering five components
into the cell and can be reprogrammed
to other stop codons in a single step. We expect
this concept to be a scalable platform for further
organelle engineering and to provide
a route toward generation of semisynthetic
eukaryotic cells and organisms.
▪
RESEARCH
Reinkemeier et al., Science 363, 1415 (2019) 29 March 2019 1 of 1
The list of author affiliations is available in the full article online.
*These authors contributed equally to this work.
†Corresponding author. Email: edlemke@uni-mainz.de
Cite this article as C. D. Reinkemeier et al., Science 363,
eaaw2644 (2019). DOI: 10.1126/science.aaw2644
Membraneless OT
organelles enable
mRNA-specific GCE in
eukaryotes. OT organelles
are designed organelles
enriched in a suppressor
tRNA/tRNA synthetase
pair and a specific
mRNA binding domain
(MCP) by means of using
an assembler protein
(such as FUS and/or
KIFs). A spatially distinct set of ribosomes associated with the OT organelle preferentially translates recruited mRNAs tagged with ms2 loops to
yield the selected protein with the targeted site-specific noncanonical functionality.
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at http://dx.doi.
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RESEARCH ARTICLE
◥
SYNTHETIC BIOLOGY
Designer membraneless organelles
enable codon reassignment of
selected mRNAs in eukaryotes
Christopher D. Reinkemeier1,2,3
*, Gemma Estrada Girona3
*, Edward A. Lemke1,2,3
†
Nature regulates interference between cellular processes—allowing more complexity of life—by
confining specific functions to organelles. Inspired by this concept, we designed an artificial
organelle dedicated to protein engineering. We generated a membraneless organelle to
translate only one type of messenger RNA—by recruiting an RNA-targeting system, stop
codon–suppression machinery, and ribosomes—by means of phase separation and spatial
targeting.This enables site-specific protein engineering with a tailored noncanonical function in
response to one specific codon in the entire genome only in the protein of choice. Our results
demonstrate a simple yet effective approach to the generation of artificial organelles that
provides a route toward customized orthogonal translation and protein engineering in
semisynthetic eukaryotic cells.
T
he ability to engineer orthogonal (non–
cross-reactive) translation site-specifically
into living cells enables the introduction of
new functionalities into proteins. However,
this is a herculean task because translation
is a complex multistep process in which at least
20 different aminoacylated tRNAs, their cognate
tRNA synthetases (RSs), ribosomes, and diverse
other factors work in concert to synthesize a polypeptide
chain from the RNA transcript. An ideal
orthogonal system would show no cross-reactivity
with factors of the host machinery, minimizing
its impact on the housekeeping translational activity
and normal physiology of the cell.
Toward this goal, genetic code expansion
(GCE) is a method that enables reprogramming
of a specific codon. With GCE, an orthogonal
suppressor RS can aminoacylate its cognate suppressor
tRNA with noncanonical amino acids
(ncAAs). These ncAAs are typically custom designed
and harbor chemical functionalities that
can, for example, enable protein function to be
photocontrolled, encode posttranslational modifications,
or allow the introduction of fluorescent
labels for microscopy studies by using click
chemistry. To introduce ncAAs site-specifically
into a protein of interest (POI), the anticodon
loop of the tRNA is chosen to decode and thus
suppress one of the stop codons [(1–3), reviews].
To minimize the impact on the host cell machinery,
the Amber stop codon (TAG) is often used,
owing to its particularly low abundance in
Escherichia coli, to terminate endogenous proteins
(<10%). Nevertheless, in principle any Amber
codon in the transcriptome can be suppressed,
potentially leading to unwanted modification of
nontargeted host proteins. If ncAA-modified proteins
are recombinantly produced for in vitro
applications, this background incorporation can
be largely ignored as long as the yields of purified
full-length protein are acceptable. However, the
challenge is different if the host is considered
more than just a bioreactor vessel that can be
sacrificed for its protein. In order to study the
function of a host-cell POI in situ, the physiological
condition of that host cell is an important factor.
In that context, minimization of background incorporation
of the ncAA is required to ensure
well-controlled experiments.
At least three elegant approaches have been
developed to enable orthogonal translation in
E. coli—that is, to decode a specific codon only
for the RNA of the POI and not the entire genome.
(i) Orthogonal ribosomes that recognize
RNA with a specific Shine-Dalgarno sequence
(4–6) have been developed to enhance codon
specificity, to site-specifically encode an ncAA
into a POI. (ii) Recently, genome engineering has
advanced to the stage that E. coli strains can be
depleted of selected native codons (7–10), providing
a genetically clean (such as Amber codon–
free) host background for selective decoding of
specific codons only in the POI. (iii) Distinct noncanonical
codons have been designed by using
an artificial base pair encoded only in the coding
sequence of the POI. This lowers the risk of nonspecific
decoding in other parts of the genome
(11). However, because of genome complexity, it
is not straightforward to transfer these orthogonal
translation approaches to eukaryotes [(12),
review], in which the Amber codon is highly
abundant (~20% in human cells).
We hypothesized that it is possible to create an
orthogonal translation system by spatially enriching
specific components of the GCE machinery
in an artificial orthogonally translating (OT)
organelle/droplet/aggregate/condensate/dense
phase. For an OT organelle to translate only the
mRNA of the POI, it should be readily accessible
to the translational machinery (>100 different biomolecules,
such as canonical aminoacylated
tRNAs, translation factors, and ribosomal subunits)
and thus cannot be further easily membraneencapsulated
inside the cell. Another requirement
is that the small cognate suppressor tRNA localizes
efficiently to the OT organelle and is depleted
from the rest of the cytoplasm.
The idea to create such an OT organelle was
inspired by the concept of phase separation,
which can generate high local concentrations of
proteins and RNAs in cells (13, 14). Recently, phase
separation has gained attention owing to the
discovery of its prevalence in cell biology and its
role in the formation of specialized organelles
such as nucleoli, stress/RNA granules, and Balbiani
bodies [(15), review]. Although our understanding
of the design and functional principles of these
organelles is emerging, it has been established
that they are membraneless and thus are in direct
contact and exchange with the surrounding cytoplasm
and/or nucleoplasm. Despite lacking a membrane,
these organelles can efficiently perform
complextasks,suchastranscriptioninthenucleolus.
We aimed to create a new OT organelle in a
living mammalian cell and envisioned use of a
strategy in which we selectively target the RS
and the mRNA of a POI to a spatially distinct
site in the cytoplasm. We found that a combination
of phase separation with spatial targeting
by using motor proteins yields an organelle-like
structure enriched in RS and mRNA, to which
the cognate suppressor tRNA and ribosomes
effectively copartition. This affords a set of spatially
distinct ribosomes, forming an OT system
that preferentially translates only our tagged
mRNA, which enables site-specific recoding of
a stop codon only in this mRNA. We show for a
variety of proteins, including membrane proteins,
that we can incorporate site-specific noncanonical
functions only into a POI, whereas other mRNAs
in the cytoplasm that contain the same stop
codon are not translated efficiently.
Design of OT organelles
Our synthetic designer OT organelle (Fig. 1) is
engineered with the following components.
(i) An mRNA-targeting system in which two
ms2 RNA stem loops are fused to the mRNA of
choice, creating an mRNA::ms2 fusion coding
for the POI. We denoted DNA in italics. The
ms2 loops bind specifically to the phage-derived
major capsid protein (MCP) (16), which will thus
form a stable and specific mRNA::ms2–MCP
complex in cells. The ms2 loops were always
fused to the 3′ untranslated region (3′UTR) of
the mRNA, which ensures translation to yield
a scarless final POI.
RESEARCH
Reinkemeier et al., Science 363, eaaw2644 (2019) 29 March 2019 1 of 9
1
Biocentre, Departments of Biology and Chemistry,
Pharmacy and Geosciences, Johannes Gutenberg–University
Mainz, Hanns-Dieter-Hüsch-Weg 15, 55128 Mainz, Germany.
2
Institute of Molecular Biology, Ackermannweg 4, 55128
Mainz, Germany. 3
Structural and Computational Biology Unit
and Cell Biology and Biophysics Unit, European Molecular
Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg,
Germany.
*These authors contributed equally to this work.
†Corresponding author. Email: edlemke@uni-mainz.de
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(ii) A tRNA/RS suppressor pair. We chose the
orthogonaltRNA/RSpairfromtheMethanosarcina
mazei pyrrolysyl system (tRNAPyl
/PylRS) because it
has enabled the encoding of more than 100 ncAAs
with diverse functionalities into proteins by using
GCE in a multitude of cell types and species, including
E. coli, mammalian cells, and even living
mice [(1–3), reviews].
(iii) The assembler, the key component required
to form an OT organelle. The purpose of the assembler
is to create a dense phase or condensate,
in which the mRNA::ms2–MCP complex is brought
into close proximity of the tRNAPyl
/PylRS pair.
The simplest assembler strategy we tested is
the bimolecular fusion of MCP::PylRS (termed
B) (Fig. 2A). In addition, we tested strategies in
which we expected to yield much larger assemblies.
All of those assembly systems are composed
of an assembler fusion to PylRS coexpressed with
an assembler fusion to MCP. We expected assembler::PylRS•assembler::MCP
to form large
aggregates (we denote coexpression with a center
dot, “•”). One tested assembly strategy was
based on phase separation of proteins, and one
was based on the assembly of kinesins, which we
abbreviate here as P and K, respectively (Fig. 2A).
Furthermore, for each P and K approach, we
tested two different molecular designs: P1, P2
and K1, K2, respectively.
P1
Previous studies have established the capacity of
the proteins fused-in sarcoma (FUS) and Ewing
sarcoma breakpoint region 1 (EWSR1) to form
mixed dropletlike structures by means of phase
separation. They both contain a prion-like disordered
domain that facilitates phase separation
into liquid, gel, and solid states (17, 18). In a phaseseparated
state, these proteins are locally highly
concentrated (approximately orders of magnitude)
compared with the remaining soluble fraction
in the cell. FUS was fused to PylRS, and EWSR1
fused to MCP, and we speculated that this would
lead to the formation of droplets in which MCP
and PylRS are highly enriched. P1 is denoted
FUS::PylRS•EWSR1::MCP.
P2
The Caenorhabditis elegans protein spindledefective
protein 5 (SPD5) has recently been
shown to phase-separate into particularly large
(several micrometer-sized) droplets (19). In a
phase-separated state, SPD5 is locally highly concentrated
compared with the remaining soluble
fraction in the cytoplasm (by orders of magnitude).
We speculate that a protein fused to SPD5
will condense into droplets. Similarly to FUSEWSR1
droplets, we speculated that PylRS
fused to SPD5 and MCP fused to SPD5 will be
highly enriched. P2 is denoted SPD5::PylRS•
SPD5::MCP.
K1
Certain kinesin truncations constitutively move
toward microtubule-plus ends in living cells (20).
One such truncated kinesin is KIF13A1–411,DP390,
and we speculated that PylRS and MCP respectively
fused to this kinesin truncation and coexpressed
would be locally enriched, owing to
spatial targeting to microtubule-plus ends. K1 is
denoted KIF13A1-411,DP390::PylRS•KIF13A1-411,DP390::
MCP. (Single-letter abbreviations for the amino
acid residues are as follows: A, Ala; C, Cys;
F, Phe; N, Asn; P, Pro; and Y, Tyr. In the mutants,
other amino acids were substituted at
certain locations; for example, Y306A indicates
that tyrosine at position 306 was replaced by
alanine).
Reinkemeier et al., Science 363, eaaw2644 (2019) 29 March 2019 2 of 9
Fig. 1. Spatial separation of the necessary components to enable
orthogonal translation to decode a specific stop codon in a specifically
tagged mRNA. (A) Expression of the synthetase PylRS leads to aminoacylation
of its cognate stop codon suppressor tRNAPyl
with a custom
designed ncAA. This leads to site-specific ncAA incorporation whenever the
respective stop codon occurs in the mRNA of the POI. Given that many
endogenous mRNAs terminate on the same stop codon, using this approach
in the cytoplasm potentially leads to misincorporation of the ncAA into
unwanted proteins. (B) To avoid this, we propose to spatially enrich all
components to an OT organelle, including the mRNA of the POI, the
aminoacyl-tRNA synthetase, the tRNA, and ribosomes through the use of
“assemblers.” Aminoacylated tRNAPyl
should only be available in direct
proximity of the OT organelle, so that only here stop codon suppression can
occur. The corresponding stop codon in mRNAs that are not targeted to the
OT organelle should not get translated. Whereas in (A) GCE is stop codon
specific, in (B), it is stop codon– and mRNA-specific.
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Reinkemeier et al., Science 363, eaaw2644 (2019) 29 March 2019 3 of 9
Fig. 2. Local enrichment
by means of phase
separation is a means
to generate OTorganelles.
(A) Schematic
representation of different
assembler classes. B,
bimolecular MCP::PylRS
fusion; P1, fusions to FUS
and EWSR1; P2, SPD5;
K1, truncation of kinesin
KIF13A (KIF13A1-411,DP390);
K2, truncation of kinesin
KIF16B (KIF16B1-400)
and combinations thereof
(K1::P1, K1::P2, K2::P1, and
K2::P2). (B) Schematic
representation of the dualcolor
reporter. mRNAs
encoding the
fluorescent proteins GFP
and mCherry, containing
stop codons at permissive
sites, are expressed from
one plasmid, each with
its own CMV promoter,
ensuring a constant ratio of
mRNA throughout each
experiment.The mRNA of
the mCherry reporter is
tagged with two ms2
stem loops, mCherry::
ms2. In the presence of
ncAA and tRNAPyl
, in the
case of cytoplasmic PylRS,
both GFP39STOP
and
mCherry185STOP
are
produced, leading to a
diagonal (schematically
drawn in orange) in FFC
analysis. However, under
the same conditions,
orthogonal translation in
OTorganelles should
enable selective stop
codon suppression of
mCherry::ms2 mRNA,
resulting in an mCherrypositive
and GFP-negative
population (drawn schematically
as a red vertical
population). In both
schemes, nontransfected
HEK293Tcells, which are
also detected with FFC, are
represented by a gray
circle. (C) For all experiments, the indicated constructs were coexpressed with
tRNAPyl
(anticodon corresponding to the indicated codon) and the dual reporter
(GFP39STOP
, mCherry185STOP
::ms2). GCE was performed in presence of the
indicated ncAAs, and cells were analyzed by means of FFC.The dark gray bars
(normalized to cytoplasmic PylRS) represent the fold change in the ratios r of the
mean fluorescence intensities of mCherry versus GFP (derived from FFC) (Fig. 2, D
and E, and fig. S1) for all the systems tested in this study.The light gray bars
represent the relative efficiency as defined by the mean fluorescence intensity of
mCherry for each condition divided by cytoplasmic PylRS control (derived from
FFC) (Fig. 2, D and E, and fig. S1). Shown are the mean values of at least three
independent experiments; error bars represent the SEM.The red box highlights the
best performing OTorganelle (OTK2::P1
). (D) FFC analysis of the dual-color reporter
expressed with the four indicated systems in transfected HEK293Tcells and
tRNAPyl
in the presence of the ncAA SCO, a lysine derivative with a cyclooctyne
side chain. Highly selective and efficient orthogonal translation was observed for
the OTorganelle (the black arrow indicates a bright, highly mCherry-positive
population). Shown in the dot plots are the sums of at least three independent
experiments. Axes indicate fluorescence intensity in arbitrary units (all FFC plots
are summarized in fig. S1). (E) FFC plots for the OTorganelle selectively
translating Opal and Ochre codons only of recruited mCherry185TGA
::ms2 and
mCherry185TAA
::ms2 mRNA, respectively (corresponding cytoplasmic PylRS
controls for those stop codons are provided in fig. S1).
GFP
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K2
By analogy to K1, we also tested the truncated
kinesin KIF16B1–400. K2 is denoted KIF16B1-400::
PylRS•KIF16B1-400::MCP.
We also tested whether combinations of these
systems would lead to efficient OT organelles:
K1::P1 = KIF13A1–411,DP390::FUS::
PylRS•KIF13A1–411,DP390::EWSR1::MCP
K2::P1 = KIF16B1–400::FUS::
PylRS•KIF16B1–400::EWSR1::MCP
K1::P2 = KIF13A1–411,DP390::SPD5::
PylRS•KIF13A1–411,DP390::SPD5::MCP
K2::P2 = KIF16B1–400::SPD5::
PylRS•KIF16B1–400::SPD5::MCP
In order to evaluate these assemblers for
facilitating functional orthogonal translation
of an ms2-tagged mRNA, we designed a dualreporter
construct, in which green fluorescent
protein (GFP) and mCherry mutants are simultaneously
expressed from two different expression
cassettes from one plasmid, ensuring that
the mRNA ratio between them is constant across
all experiments. Stop codons were introduced
at permissive sites into GFP at position 39
(GFP39STOP
) and into mCherry at position 185
(mCherry185STOP
) (Fig. 2B). Only if stop codon
suppression is successful will the corresponding
GFP or mCherry be produced. Transfected cells
were analyzed by means of fluorescence flow
cytometry (FFC); settings were adjusted so that
an approximate diagonal results in the FFC plots
if GFP and mCherry are expressed from this
plasmid by using the conventional cytoplasmic
PylRS system, which cannot differentiate mRNAs.
A selective and functional OT organelle should
selectively express mCherry only if the ms2 loops
are fused to the 3′UTR of the mCherry mRNA,
leading to appearance of a vertical line in the
cytometryplot(Fig.2B).Unlessotherwisereported,
all experiments were performed in the presence
of tRNAPyl
and the ncAA SCO, a widely used and
well-characterized lysine derivative, the side chain
of which carries a cyclooctyne that can be used
in a variety of click-chemistry reactions to install
diverse chemical groups onto the protein. As
previously reported, this ncAA is efficiently encoded
by a Y306A, Y384F double mutant of
PylRS (for simplicity we refer to this PylRSAF
mutant
as PylRS unless otherwise specified) (21–23).
Omission of the ncAA serves as a standard negative
control and leads to no expression of GFP
or mCherry (fig. S1).
We evaluated the performance of each OT system
according to their selectivity and relative
efficiency. We define selectivity as the ratio r of
the mean mCherry FFC signal divided by the
mean GFP signal. Final values are expressed as
fold selectivity relative to that of cytoplasmic
PylRS. We define relative efficiency as the mean
mCherry signal of each system divided by the
mean mCherry signal of the cytoplasmic PylRS
system, which serves as the reference (here defined
as 100%). All results on selectivity (Fig. 2C,
dark gray positive bars) and efficiency (Fig. 2C,
light gray negative bars) are summarized in
the bar plot in Fig. 2C, and all corresponding
FFC dot plots are summarized in fig. S1, whereas
selected FFC data are also shown in Fig. 2,
D and E.
Combining two assembler strategies
yields highly selective and efficient
OT systems
The conceptually simplest assembly strategy B
(MCP fused to PylRS) showed only a minor selectivity
gain of about 1.5-fold, which is concomitant
with a 60% decrease in efficiency (Fig. 2C
and fig. S1). The OT system P1 (based on phase
separation of FUS/EWSR1) performed similarly
in terms of selectivity gain. In addition, a 50%
decrease in efficiency was measured (Fig. 2, C
and D). The P2 system (based on SPD5) showed
an approximate twofold selectivity gain accompanied
by an almost 90% decrease in efficiency
(Fig. 2C and fig. S1). Analogously, we tested the
kinesin-based assembly strategy and observed
for K1 a twofold selectivity increase, with an
efficiency decrease of ~90% (Fig. 2C and fig.
S1). The K2 system behaved similarly (Fig. 2, C
and D). In total, the selectivity gains were small
but robustly detected, indicating that bringing
the ncAA aminoacylation activity (the tRNAPyl
/
PylRS in the presence of ncAA) in direct proximity
of the target mRNA represents a pathway
to more selective codon suppression.
Next, we tested the assembler combination
strategies (K1::P1, K2::P1, K1::P2, and K2::
P2). For all combinations, we observed at least
fivefold selectivity gain, indicating orthogonal
translation [the observed selectivity effect is
robust across a titration of Amber suppression
efficiencies (fig. S2)]. The best performing system
on the basis of the fusion of FUS/EWSR1 with
KIF16B1-400, K2::P1, exhibited a selectivity of
eightfold and 40% efficiency (Fig. 2C, red box).
This was also directly obvious from the FFC data,
in which the bright, mCherry-positive cell population
was clearly retained, whereas GFP expression
was minimal (Fig. 2D, black arrow).
To validate that the observed selectivity gain is
specific to the ms2-MCP interaction, we further
characterized OT organelles by expressing the RS
assembler fusion of each OT system without
MCP (figs. S1 and S3). As expected, no selective
orthogonal translation of ms2-tagged mRNA was
observedin those cases. Additionally, we performed
a reporter inversion by moving the ms2 loops from
the mCherry to the GFP cassette in the dual-color
reporter, which as expected inverted selectivity
of the system toward dominant GFP expression
(fig. S3B). This establishes that the OT system
acts selectively on the ms2-tagged RNA.
GCE can also be used to introduce multiple
ncAAs into the same POI [(1–3), reviews]. However,
only very few publications report on more
than one—that is, two- or three-codon suppression
in the same protein in eukaryotes—because
yields typically suffer compared with single-codon
suppression (24–26). Even dual- and triple-Amber
proteins were still suppressed with the OT organelle
(fig. S4).
To ensure that other ncAAs also can be incorporated
by the OT system, we tested another
structurally different ncAA (3-iodophenylalanine),
which is a phenylalanine derivative instead of
a lysine derivative and is encoded by a different
PylRS mutant (N346A and C348A) (27). Consistent
results were also observed for this system (Fig. 2C
and fig. S1).
Because Opal (TGA) and Ochre (TAA) codons
are highly abundant in eukaryotic genomes
(~52% Opal, ~28% Ochre in the human genome),
the Amber codon is by far the most used for GCE
in eukaryotes. In addition, genomic approaches
to orthogonal translation by removing these
codons in the entire eukaryotic genome would
be even more challenging than for the Amber
codon and are currently beyond the state of the
art. However, in the OT system, a simple mutation
in the anticodon loop of the tRNAPyl
, as well
as in the respective codon in the mRNA::ms2,
should allow orthogonal translation of these
codons. FFC analysis revealed that the OT organelles
provide freedom of choice with respect
to the stop codon (Fig. 2, C and E, and fig. S1).
In fact, Opal suppression showed an 11-fold selectivity
increase at 50% efficiency (slightly
outperforming Amber suppression). Ochre suppression
still showed fivefold selectivity increase,
with 20% efficiency.
The OTK2::P1
organelle enables
orthogonal translation of proteins
of various cellular compartments
To visualize the power of the OTK2::P1
organelle
(our best performing Amber suppression OT organelle
in terms of selectivity and efficiency)
beyond “simple” reporters, we next aimed to
show differential expression of human nucleoporin
153 (Nup153) versus cytoskeletal vimentin.
Nup153 locates to the nuclear pore complex and
is more than 1500 amino acids long. Hence, its
mRNA is approximately sixfold larger than those
of the fluorescent protein reporters used above.
We used a previously described C-terminal
GFP fusion, with an Amber mutation (Nup153::
GFP149TAG
) that gave rise to a characteristic
nuclear envelope stain in confocal images only if
Amber suppression was successful (28). Nup153::
GFP149TAG
was then tagged with two ms2 loops
(NUP153::GFP149TAG
::ms2) and coexpressed from
the same plasmid with vimentin containing an
Amber codon at position 116 fused to mOrange
(VIM116TAG
::mOrange). Expression in human embryonic
kidney (HEK) 293T cells resulted in production
of both proteins in the presence of the
cytoplasmic PylRS showing the characteristic
nuclear envelope and cytoskeletal staining, respectively.
In the presence of the OTK2::P1
organelle,
only Nup153::GFP was visible (Fig. 3A, selective
nuclear rim stain). Consistent results were also
observed in Cercopithecus aethiops kidney (COS-7)
cells (fig. S5). Swapping the ms2 loops to vimentin
inverted the effect, so that only Vimentin116TAG
::
mOrange was visible (further experiments for
COS-7 and for HEK293T cell experiments are
shown in Fig. 3B and fig. S5). This showed that
the OTK2::P1
worked for dramatically different
mRNAs.
Next, we asked whether it would be possible to
selectively express transmembrane proteins by
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using our synthetic OTK2::P1
organelle. Membrane
protein expression represents another layer of
translational complexity because ribosomes need
to bind the endoplasmic reticulum (ER) during
translation, where the proteins are cotranslationally
inserted into the membrane. To this end,
we used a fusion of insulin receptor 1 with an
Amber codon at position 676 with mOrange
(INSR676TAG
::mOrange), which locates to the
plasma membrane and gives rise to a characteristic
plasma membrane stain in HEK293T
cells (21). This construct was tagged with ms2
loops in the 3′UTR and cloned with Nup153::
GFP149TAG
into one dual-cassette plasmid. We
then expressed it in HEK293T cells either in the
presence of the cytoplasmic PylRS system or in
the presence of the OTK2::P1
organelle (Fig. 3C).
In the presence of the synthetic OTK2::P1
organelle,
we observed selective expression of the
ms2-tagged protein and the expected plasma
membrane localization of INSR676TAG
::mOrange,
indicating the potential of our organelle to participate
in evenmorecomplex membrane-associated
translational processes.
Reinkemeier et al., Science 363, eaaw2644 (2019) 29 March 2019 5 of 9
Fig. 3. A versatile OTorganelle for selective and efficient orthogonal
translation. (A to C) Confocal images of cells transfected with constructs
encoding PylRS (left column) or the OTK2::P1
organelle (right column) for
different protein pairs. SCO and tRNAPyl
were present in all cases. (A)
HEK293T cells were transfected with NUP153::GFP149TAG
::ms2 and
VIM116TAG
::mOrange. (B) VIM116TAG
::mOrange::ms2 and NUP153::GFP149TAG
transfected in COS-7 cells. More representative examples for (A) using COS-7
cells and for (B) using HEK293T cells are shown in fig. S5. Shown from top
to bottom are Vimentin116TAG
::mOrange (magenta, characteristic cytoskeletal
stain), Nup153::GFP149TAG
(yellow, characteristic nuclear envelope stain),
overlay with Hoechst (cyan, nuclear stain), magnified images of representative
cells (red boxes), and line profiles for the mOrange and GFP channel
(red line, magenta and yellow curves, respectively), to highlight that only the
ms2-tagged mRNA yields its respective expressed protein if the OTK2::P1
organelle is present. Scale bars, 20 mm. (C) HEK293T cells were transfected
with INSR676TAG
::mOrange::ms2 and NUP153::GFP149TAG
. Shown from top
to bottom are INSR676TAG
::mOrange (magenta, characteristic plasma membrane
stain), Nup153::GFP149TAG
(yellow), overlay with Hoechst (cyan),
magnified images of representative cells (red boxes), and line profiles for the
mOrange and GFP channel (red line, magenta and yellow profiles, respectively),
demonstrating selective translation of insr676TAG
::mOrange::ms2
mRNA by the OTK2::P1
organelle. Scale bars, 20 mm.
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The OT organelle functions by recruiting
ribosomes and tRNAPyl
The above experiments demonstrated the functionality
of our synthetic OTK2::P1
organelle.
Next, we aimed to study the spatial distribution
of the different systems in the cell to understand
the basic working mechanism of the OT
organelle.
To assess the spatial distribution of PylRS in
cells, we used immunofluorescence (IF). We also
used fluorescence in situ hybridization (FISH)
against tRNAPyl
. In contrast to the dual-color
reporter used in the FFC experiments above, in
all IF/FISH experiments we used a single-color
NLS::GFP39TAG
reporter fused to ms2 loops to
identify cells active in Amber suppression (this
yields a green nucleus if Amber suppression is
successful). IF and FISH stainings showed that
in contrast to cytoplasmic PylRS, the P1 system
formed small, intracellular assembler::PylRS
droplets (Fig. 4A). This indicated the occurrence
of phase separation. The tRNAPyl
colocalized
well with assembler::PylRS droplets (Fig. 4A),
indicating that it could nicely partition into the
assembler::PylRS phase. Additional stainings show
further colocalization with assembler::MCP (fig.
S6). Compared with P1, the P2 system (figs. S6
and S7) showed larger but still multiple dispersed
dropletlike structures. Phase separation of proteins
is based on exceeding the critical concentration
up to which proteins are fully soluble in
the cytoplasm. However, a soluble species coexists
with the phase-separated species (29) that
can contribute to stop codon suppression outside
the droplet. The components of the K1 (fig. S7)
and K2 (Fig. 4A and fig. S6) systems were mostly
observed distributed across the cytoplasm, likely
because of binding to the microtubule cytoskeleton,
which appears rather distributed throughout the
cytoplasm (fig. S8). Because small tRNAs can diffuse
rapidly, we believe that a critical factor for the
design of an OT organelle is how well the tRNAPyl
is confined to few sites in the cell and thus spatially
separated and sequestered from the rest of
the cytoplasm. None of the P1, P2, K1, or K2
systems displaying high selectivity (Fig. 2C) is
consistent with the observation that the systems
showed a rather dispersed distribution in the cell.
However, if we combined both assembler strategies
(K1::P1, K2::P1, K1::P2, and K2::P2),
we observed the formation of large micrometersized,
organelle-like structures in the cytoplasm,
which were in most cases localized to few or even
a single position per cell. Association of the OT
organelle with the microtubule cytoskeleton was
also observed (fig. S8). As shown for K2::P1 in
Fig. 4A and figs. S6 and S8, mRNA::ms2, tRNAPyl
,
assembler::PylRS, and assembler::MCP all colocalize
to organelle-like structures (other combined
assemblers are shown in figs. S6 and S7).
The combination of the two assembler strategies—
that is, phase separation paired with spatial targeting
by kinesin truncations—yielded the best
confinement as determined with FISH and IF
and the highest selectivity increase.
This is consistent with our hypothesis that
the higher spatial segregation and thus higher
local concentration of aminoacylated tRNAPyl
and mRNA correlates with higher selectivity.
This effectively translates into a higher partition
coefficient of tRNAPyl
into the droplet and thus
depletion of tRNAPyl
from the cytoplasm, yielding
a high concentration gradient between cytoplasm
and OT organelle (fig. S7).
We next performed staining for ribosomes
to see whether they colocalize to the OTK2::P1
organelle. IF staining of the ribosomal protein
RPL26L1 showed its distribution throughout the
cell but also an enrichment at the OTK2::P1
organelle
[Fig. 4, B and C, two-dimensional (2D)
projection and a 3D reconstruction; movie S1;
and fig. S8), demonstrating partial ribosome recruitment,
tentatively owing to binding to mRNA::
ms2 during translation. We conclude that only
ribosomes sufficiently immersed into the tRNAPyl
gradient can perform codon suppression efficiently.
This also visualizes the mobility of a set
of ribosomes in the cell. High mobility can also
explain why we were even able to express the
membrane protein INSR (Fig. 3C) because for
orthogonal translation of a membrane protein,
two things must happen either sequentially or
at the same time: The translating ribosome needs
to interact with the ER and with the OT organelle.
Together, this body of evidence strongly suggests
that selective orthogonal translation happens
within close proximity of the OT organelles, potentially
even inside the organelle, by a set of
recruited ribosomes that are near or fully immersed
into a concentrated pool of tRNAPyl
.
tRNAPyl
itself is recruited to the OTK2::P1
organelle
because of its affinity for assembler::PylRS
and can readily copartition into the droplet to be
aminoacylated with its cognate ncAA, whereas
assembler::MCP recruits ms2-tagged mRNA. This
in turn attracts ribosomes to migrate to the dense
phase formed by the dual assembler system
(K2::P1 = KIF16B::FUS::PylRS and KIF16B::
EWSR1::MCP), which maintains access to other
translation factors for translation to function
(Fig. 4D). Ribosomes elsewhere in the cytoplasm
that are not exposed to tRNAPyl
perform their
canonical function to terminate translation whenever
they encounter a stop codon.
Our route to enable orthogonal translation
required only five extra components and represents
an important step toward generating semisynthetic
eukaryotic organisms that can potentially
follow what has been dubbed the “orthogonal
central dogma” (30). The OT organelle also represents
a general strategy for tailoring complex
functions in eukaryotes by mimicking the evolutionary
concept to build distinct, but membraneless,
organelles inside eukaryotic cells. Proteins
such as FUS, EWSR1, and SPD5 have many vital
functions in the cell, and we cannot exclude the
possibility that the lower expression yield (half,
in many cases) we observed is also due to overexpression
of components of the OT organelle.
The need to combine two different assembly strategies
(phase separation–based assemblers with
spatial targeting by using kinesin truncations)
puts potentially an additional burden on the cell.
However, our understanding of phase separation
is developing dramatically, such as the identification
of amino acid sequences that can be used
to make layered droplets (31). We can thus expect
future versions of this technology to afford even
better selectivity, efficiency, and the ability to
bestow additional functions on the OT organelle
or for constructing other novel organelles with
new functions.
Materials and methods
Cell culture
HEK293T cells (ATCC CRL-3216) and COS-7 cells
(Sigma-Aldrich 87021302) were maintained in
Dulbecco's modified Eagle's medium (DMEM,
Gibco 41965-039) supplemented with 1% penicillinstreptomycin(Sigma-Aldrich
P0781), 1% L-Glutamine
(Sigma-Aldrich G7513), 1% sodium pyruvate (Life
Technologies 11360), and 10% FBS (Sigma-Aldrich
F7524). Cells were cultured at 37°C in a 5% CO2
atmosphere and passaged every 2-3 days up to
20 passages.
In all cases, cells were seeded 15-20 hours prior
to transfection at a density resulting in 70-80%
confluency at the time of transfection. Flow cytometry
was performed using 24-well plates
with plastic bottom (Nunclon Delta Surface
ThermoScientific). IF labeling and FISH were
performed on 24-well plates with glass bottom
(Greiner Bio-One) or four-well chambered LabTek
#1.0 borosilicate coverglass (ThermoFisher).
Constructs, cloning, and mutagenesis
Dual-color reporters: The dual fluorescent protein
reporters were cloned in a pBI-CMV1 vector
(Clontech 631630), with ms2 tagged fluorescence
protein (mRNA) version in one multiple cloning
site and ms2 free version in the other. GFP39TAGI
or mCherry185TAG
were used as N-terminal fusions
with nuclear localization sequences (NLS). Similar
reporters for Ochre and Opal suppression were
prepared (with GFP39TAA
, mCherry185TAA
and
GFP39TGA
, mCherry185TGA
, respectively).
NLS::GFP39TAG
::ms2 reporter: NLS::GFP39TAG
was cloned with two copies of ms2 loops into
the pBI-CMV1 vector as a reporter for successful
Amber suppression for imaging experiments.
Double and triple Amber GFP: Position 149
and subsequently position 182 of GFP were
mutated to TAG to obtain pBI-CMV constructs
with multiple amber codons, these constructs
did not have mCherry in the second multiple
cloning site.
OT organelle constructs: Pyrrolysyl tRNA was
cloned under the control of a human U6 (hU6)
promoter, and all other constructs were under
CMV (cytomegalovirus) promoters cloned in the
pcDNA3.1 (Invitrogen V86020) vector. MCP was
cloned from the Addgene plasmid #31230 and
FUS from the Addgene plasmid #26374. In all
FUS fusions, amino acids 1-478 were used, replacing
the C-terminal NLS region by a Flag-tag.
In all pyrrolysine synthetase fusions the previously
reported efficient NES::PylRSAF
(Y306A,
Y384F) sequence was used (21, 28). NES::PylRSAA
(N346A, C348A) was cloned via site-directed
mutagenesis starting from NES::PylRSWT
. The
SPD5 gene was ordered from Genewiz and fused
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to MCP and PylRSAF
via restriction cloning.
KIF13A1-411 and KIF16B1-400 were cloned from
human cDNA and inserted into pcDNA3.1 via
restriction cloning. P390 of KIF13A1-411 was removed
via site-directed mutagenesis. KIF13A1-411,DP390 and
KIF16B1-400 fusions with MCP, PylRSAF
, EWSR1::
MCP, FUS::PylRSAF
, FUS::PylRSAA
, SPD5::MCP,
and SPD5::PylRSAF
were assembled via Gibson
assembly (32).
Constructs for differential imaging experiments
(Fig. 3): To selectively express Nup153::
GFP149TAG
and Vimentin116TAG
::mOrange, one
gene was first inserted with ms2 loops into
pBI-CMV1 (21). Subsequently, the other gene
was inserted without ms2 loops. INSR676TAG
::
mOrange was fused to ms2 loops by replacing
VIM116TAG
::mOrange in the pBI vector bearing
NUP153::GFP149TAG
and VIM116TAG
::mOrange::ms2
Reinkemeier et al., Science 363, eaaw2644 (2019) 29 March 2019 7 of 9
Fig. 4. OT organelles enrich tRNAPyl
and
ribosomes for orthogonal translation. (A) IF and
FISH imaging of HEK293T cells expressing
tRNAPyl
and the indicated system. For simplicity
and to direct the eye, in all imaging experiments, a
simple NLS::GFP39TAG
::ms2 (nuclear staining)
was used instead of the dual-color reporter.
Green-colored nuclei report on faithful
Amber suppression of the cells (shown only
in overlay). Shown from left to right are
IF against PylRS in magenta, FISH against
tRNAPyl
in yellow, overlay, and magnified images of
representative cells (red boxes and red arrows
highlight representative structures). Scale
bars, 20 mm. (B) Maximum intensity Z-projection
of IF image Z-stacks of HEK293T cells transfected with
constructs encoding OTK2::P1
organelle and
NLS::GFP39TAG
::ms2 in the presence of SCO and
tRNAPyl
. Shown from left to right are IF against
PylRS (magenta), IF against RPL26L1 (cyan),
merge (NLS::GFP39TAG
::ms2 is shown in green in all
images), and line profiles for the PylRS and
RPL26L1 channels (red line, magenta and cyan
curves, respectively). Scale bar, 20 mm. (C) 3D
reconstructions of IF images corresponding to those in
(B) and movie S1. Shown from left to right are IF
against PylRS (magenta), IF against RPL26L1
(cyan), and merge [NLS::GFP39TAG
::ms2 in green; gray
dashed lines highlight approximate cell boundaries in
(B) and (C)]. RPL26L1 staining of the OT organelle
demonstrates partial recruitment of ribosomes,
which appear to be immersed into the organelle.
(D) Working model of OTK2::P1
-organelle–enabled
orthogonal translation. KIF16B::FUS::PylRS and
KIF16B::EWSR1::MCP form a spatially separated
organelle inside a living cell. PylRS recruits tRNAPyl
and
largely depletes its availability in the cytoplasm,
whereas MCP recruits ms2-tagged mRNA.
Ribosomes and other translation factors are
recruited to the organelle for orthogonal translation.
Because the charged tRNAPyl
is now in close
proximity to only the recruited mRNA of the
POI and the spatially distinct set of ribosomes, the
selected stop codon can only be translated in the
immediate vicinity of the synthetic OT organelle.
Meanwhile, all other mRNAs that are not
recruited to the OT organelle are subject to
normal translational processing of the host
machinery and available ribosomes in the
remaining cytoplasm—that is, a stop codon
will terminate translation. The assembly order
of the OT organelle is unknown and is only
shown here with arrows for illustrative purposes.
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to yield a bicistronic vector with INSR676TAG
::
mOrange in one and NUP153::GFP149TAG
in the
other cassette.
Multicistronic Amber suppression vectors:
For ease of experiments with the OTK2::P1
organelle
we generated multicistronic vectors harboring
all necessary components. To assemble
multicistronic Amber suppression vectors, first
one copy of tRNAPyl
under the control of a hU6
promoter was inserted into the pBI-CMV1 vector
via Gibson assembly. Subsequently, first
KIF16B::FUS::PylRSAF
and finally KIF16B::
EWSR1::MCP were inserted via Gibson assembly.
Alternatively, a previously published pcDNA3.1
based construct (21) expressing NES::PylRSAF
under a CMV promoter and tRNAPyl
under a hU6
promoter was used. Alternatively, hU6-tRNAPyl
and NES::PylRSAF
or the components of OTK2::P1
were inserted into a pDonor vector (GeneCopoeia).
These constructs were used for all experiments
in COS-7 and for ribosome/tubulin imaging
experiments.
OTK2::P1
construct tagging with iRFP (fig. S8):
To exclude the possibility of staining artefacts
we replaced the KIF16B::FUS::PylRSAF
in the
multicistronic pBI-CMV1 vector with a KIF16B::
iRFP::FUS::PylRSAF
fusion via Gibson assembly.
The final construct additionally encodes KIF16B::
EWSR1::MCP and tRNAPyl
.
Transfections and used ncAAs
Transfections of HEK293T cells were performed
with polyethylenimine (PEI, SigmaAldrich
408727) using 3 mg PEI per 1 mg DNA
(1200 ng total DNA, diluted in DMEM without
Phenol Red, Gibco11880-028). COS-7 cells were
transfected using the JetPrime reagent (PeqLab)
according to the manufacturer’s recommendations
at a ratio of 1:2 (1000 ng total DNA).
For Amber suppression system tests, cells were
transfected at a ratio of a 1:1:1:1 with POITAG
vectors,
tRNAPyl
, PylRS assembler fusions and MCP
assembler fusions or mock constructs. 4-6 hours
after transfection, the medium was swapped to
fresh one containing ncAA. HEK293T cells were
analyzed one day after transfection, while COS-7
cells were processed after two days.
Stock and working solutions for all the used
ncAAs were prepared as described in previous
work (33). SCO (cyclooctyne lysine, SiChem SC-
8000) was used at a final concentration of 250 mM,
while 3-Iodophenylalanine (Chem-Impex International
Inc.) was used at a final concentration of
1 mM. SCO is efficiently recognized by PylRSAF
(Y306A, Y384F) (23) while 3-Iodophenylalanine
is recognized by PylRSAA
(C346A, N348A) (27).
Flow cytometry
HEK293T cells were harvested 1 day after transfection
by removing the medium, resuspending
the cells in 1xPBS (phosphate buffered saline)
and passing them through 100 mm nylon mesh.
Data acquisition was performed on an
LSRFortessa SORP Cell Analyzer (BD). Analysis
was done using the FlowJo software (FlowJo).
Cells were first gated by cell type (using FSC-A x
SSC-A parameters) and then by single cell (SSCA
x SSC-W). The workflow of cell gating is shown
in Fig. S9. Each shown FFC plot is the sum of
three independent biological replicates from
which the mean and SEM were calculated. At
least 130000 single cells were analyzed per condition.
Lastly, fluorescence was acquired in the
488-530/30 channel for GFP signal and in the
561-610/20 channel for mCherry signal.
IF labeling, FISH, and confocal imaging
IF: For immunolabeling experiments, cells were
rinsed with PBS, fixed in 2% paraformaldehyde
in 1xPBS at room temperature (RT) for 10 min.
Alternatively, if cells were stained for a-Tubulin,
they were rinsed with DMEM without Phenol
Red and subsequently fixed in a buffer to
preserve microtubule structures (100 mM 1,4Piperazinediethanesulfonic
acid (PIPES), 1 mM
MgCl2, 0.1 mM CaCl2, 0.1% Triton-x-100 and
2% PFA; pH = 7) for 10 min at RT.
Subsequently, cells were rinsed with PBS and
permeabilized with 0.5% Triton-x-100 solution in
1xPBS for 15 min at RT and rinsed twice prior to
blocking. Samples were blocked in 3% BSA in
1xPBS for 90 min at RT, after which incubation
with the primary antibody was done overnight at
4°C in blocking solution (AbPylRS (1 mg/mL (21)),
AbMCP (Merck ABE76-I, 1:333), Aba-Tubulin (SigmaAldrich
T6199, 2 mg/mL) and/or AbRPL26L1 (Abcam
ab137046, 1:200)). The next day, cells were rinsed
with PBS and incubated with secondary antibody
(ThermoFisher A-21471, A-31553 and/or
A-21246, at 2 or 4 mg/mL in blocking solution)
for 60 min at RT. Then, cells were rinsed with
PBS and fresh PBS was added for imaging.
If only DNA was stained, cells were fixed and
permeabilized the same way prior to staining
with Hoechst 33342 (Sigma-Aldrich B2261) at
1 mg/mL in 1xPBS for 10 min at RT. Subsequently,
cells were rinsed with PBS and fresh PBS was
added for imaging.
FISH: FISH experiments were performed
one day after transfection analogously to described
previously (21). Briefly, the hybridization
protocol was adapted for 24-well plates from
Pierce et al. (34). In general, IF stainings appear
crisper then FISH stainings.
For imaging of only tRNAPyl,
the hybridization
probe (5′-(Cy5)-CTAACCCGGCTGAACGGATTTAGAGTCCATTCGATC-3′)
was used at 0.25 mM (hybridization
at 37°C, overnight). After four washes
with saline sodium citrate buffer (SSC) and one
wash with Tris-HCl•NaCl buffer (TN), cells were
incubated for 1 hour at RT in 3% BSA prior to IF
labeling. Cells were incubated with primary antibodies
for 2 hours at RT, rinsed with PBS and
incubated with secondary antibodies for 2 hours
at RT (antibodies described above). Finally, cells
were rinsed with PBS and fresh PBS was added
for imaging.
For imaging of both tRNAPyl
and mRNA::ms2,
the hybridization probe for tRNAPyl
(5′-(DIG)-
CTAACCCGGCTGAACGGATTTAGAGTCCATTCGATC-3′)
was used at 0.16 mM, and the probe for
ms2 (5′-(Alexa647)-CTGCAGACATGGGTGATCCTCATGTTTTCTA)
was used at 0.75 mM. After the SSC
washes, cells were incubated for 1 hour at RT in
blocking buffer (0.1 M TrisHCl, 150 mM NaCl, 1x
blocking reagent (Sigma-Aldrich 11096176001).
Then, cells were incubated with anti-digoxigeninfluorescein
antibody (Sigma-Aldrich 11207741910)
at a 1:200 dilution in blocking buffer overnight at
4°C. The next day, 3 washes of 5 min were done in
Tween buffer (0.1 M TrisHCl, 150 mM NaCl, 0.5%
Tween20), before cells were rinsed with PBS and
fresh PBS was added for imaging.
Imaging: Confocal images were acquired on a
Leica SP8 STED 3X microscope using the 405 nm
(for Hoechst), 488 nm (for fluorescein, GFP),
548 nm (mOrange), 594 nm (for Alexa594) and
647 nm (for Alexa647, Cy5) laser lines for excitation.
For HEK293T and COS-7 cells a 63x/1.40 oil
immersion objective was used. IF images with
ribosomes, microtubules, and/or iRFP were taken
on an Olympus Fluoroview FV3000 microscope
using 405 nm (Alexa405), 488 nm (GFP), 594 nm
(for Alexa594), and 640 nm (for Alexa 647 and
iRFP) lasers for excitation with a 60x/1.40 oil
immersion objective for acquisition. Images were
processed using FIJI software.
3D Reconstruction
3D reconstructions in Fig. 4C and corresponding
movie S1 were made by using the arivis Vision4D
software (arivis AG).
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ACKNOWLEDGMENTS
We thank I. Schneider as well as all members of the Lemke
laboratory, in particular C. Koehler and J. Caria, for helpful
discussions. We thank the European Molecular Biology
Laboratory (EMBL) flow cytometry core facility and the
advanced light microscopy facility for expert assistance.
Funding: The Lemke laboratory acknowledges generous support
from European Research Council (ERC) SMPFv2.0, SPP1623,
and SFB1129 (Projektnummer 240245660 funded by Deutsche
Forschungsgemeinschaft) and the Gutenberg Research College
(GRC). Author contributions: E.A.L. conceived the project.
C.D.R. and G.E.G. designed and performed all experiments.
C.D.R., G.E.G., and E.A.L. analyzed all of the data and cowrote
the manuscript. Competing interests: C.D.R., G.E.G., and
E.A.L. have filed a patent application on OT organelle technology
(EP 19157257.7). Data and materials availability:
All data are available in the main text or the supplementary
materials. All plasmids can be obtained upon reasonable
request via an EMBL materials transfer agreement (free of
charge for noncommercial purposes).
SUPPLEMENTARY MATERIALS
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eukaryotes
Designer membraneless organelles enable codon reassignment of selected mRNAs in
Christopher D. Reinkemeier, Gemma Estrada Girona and Edward A. Lemke
DOI: 10.1126/science.aaw2644
(6434), eaaw2644.363Science
, this issue p. eaaw2644Science
This approach opens possibilities in synthetic cell engineering and biomedical research.
organelle were able to introduce chemical functionalities site-specifically, expanding the canonical set of amino acids.
orthogonal translation. In response to a specific codon in a selected messenger RNA, ribosomes confined to this
designed an artificial, membraneless organelle into mammalian cells to performet al.organelles. Reinkemeier
A key step in the evolution of complex organisms like eukaryotes was the organization of specific tasks into
How to make an organelle in eukaryotes
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REFERENCES
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